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Biomineralization From Molecular and Nano-structural Analyses to Environmental Science: From Molecular and Nano-structural Analyses to Environmental Science

January 2018

DOI:10.1007/978-981-13-1002-7

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Kazuyoshi Endo

The University of Tokyo

Hiromichi Nagasawa

The University of Tokyo

This open access book is the proceedings of the 14th International Symposium on Biomineralization (BIOMIN XIV) held in 2017 at Tsukuba. Over the past 45 years, biomineralization research has unveiled details of the characteristics of the nano-structure of various biominerals; the formation mechanism of this nano-structure, including the initial stage of crystallization; and the function of organic matrices in biominerals, and this knowledge has been applied to dental, medical, pharmaceutical, materials, agricultural and environmental sciences and paleontology. As such, biomineralization is an important interdisciplinary research area, and further advances are expected in both fundamental and applied research.

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KazuyoshiEndo· ToshihiroKogure

HiromichiNagasawa Editors

Biomineralization

From Molecular and Nano-structural

Analyses to Environmental Science

Biomineralization

The 14th International Symposium on Biomineralization was held during October 9–13, 2017, at

Tsukuba International Congress Center in Tsukuba, Japan

Kazuyoshi Endo • Toshihiro Kogure

Hiromichi Nagasawa

Editors

Biomineralization

From Molecular and Nano-structural

Analyses to Environmental Science

ISBN 978-981-13-1001-0 ISBN 978-981-13-1002-7 (eBook)

https://doi.org/10.1007/978-981-13-1002-7

Library of Congress Control Number: 2018952998

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Singapore

Editors

Kazuyoshi Endo

Department of Earth and Planetary Science

The University of Tokyo Graduate School

of Science

Tokyo, Japan

Hiromichi Nagasawa

Department of Applied Biological Chemistry

Graduate School of Agricultural and Life

Sciences

The University of Tokyo

Tokyo, Japan

Toshihiro Kogure

Department of Earth and Planetary Science

The University of Tokyo Graduate School

of Science

Tokyo, Japan

corrected publication 2018. This book is an

open access publication.

Preface

Biomineralization is a process by which organisms form mineral-like inorganic sub-

stances inside or outside their bodies. Biominerals are diverse in terms of structure,

composition, morphology and role, depending on organisms. The research area of

biomineralization is really interdisciplinary in methodology, ranging from micro-

scopic observation to molecular biology. It includes not only basic researches

mainly focusing on the mechanisms and evolutionary processes of biomineral for-

mation but also applied researches including medical, dental, agricultural, environ-

mental and materials sciences. Therefore, the society of biomineralization research

constitutes a group of researchers with a variety of background. It is a good stimula-

tion for us to get together and exchange ideas for new developments.

The International Symposium on Biomineralization began in 1970 and was held

irregularly up to the 10th symposium but thereafter regularly every 2 years. This

volume was planned as a record for the proceedings of the 14th International

Symposium on Biomineralization (BIOMIN XIV): From Molecular and Nano-

structural Analyses to Environmental Science. The symposium was held during

October 9–13, 2017, at Tsukuba International Congress Center in Tsukuba, Japan.

A total of 210 participants, including four accompanying persons, from 18 countries

and areas attended this symposium. The participants included 157 regular research-

ers and 49 students, who contributed to the 106 oral presentations, including 10

keynote lectures, and 84 poster presentations. The presentations were classied into

the following 8 topics:

1. Structure and analysis of biominerals

2. Molecular and cellular regulation of biomineralization

3. Genome-based analysis of biomineralization

4. Evolution in biomineralization

5. Biomineralization in medical and dental sciences

6. Bio-inspired materials science and engineering

7. Biominerals for environmental and paleoenvironmental sciences

8. Mollusk shell formation

This volume consists of 42 articles which are arranged in the order of the above

topics. Most of them are original articles, and a few are reviews. The contributors

were those who chose to submit their manuscript. All articles were peer-reviewed.

Although the volume does not necessarily represent the whole contents of the sym-

posium, it contains articles from all 8 topics. Thus, we are able to understand the

present status of the cutting-edge of various aspects in biomineralization research

from this volume. The volume has an appendix, which comprises valuable SEM and

TEM images taken and left unpublished by late Dr. Hiroshi Nakahara, a distin-

guished researcher majoring in electron microscopy of biominerals. This appendix

was arranged by Dr. Mitsuo Kakei, who selected the photographs from about 160

slides, which were shown on the screen during the lunch times in the symposium.

The symposium and the publication of this volume were supported by a number

of Japanese scientic societies, to which we extend our gratitude. We express our

sincere thanks to the Naito Foundation, Tokyo Ouka Foundation for the Promotion

of Science and Technology, Kato Memorial Bioscience Foundation, Inoue

Foundation for Science, Suntory Foundation for Life Sciences, Life Science

Foundation of Japan, Tsukuba Tourism and Convention Association, and Tsukuba

City for their nancial support. Thanks are also due to Japanese Fossil Museum,

Kouchiken Nihonkei Hozonkai, K. MIKIMOTO & Co. Ltd., Mikimoto

Pharmaceutical Co., Ltd., Kao Corporation, LOTTE Co., Ltd., and Kotegawa

Sangyo Co., Ltd., for their kind donations.

Tokyo Japan KazuyoshiEndo

ToshihiroKogure

HiromichiNagasawa

April, 2018

Preface

vii

Contents

Part I Structure and Analysis of Biominerals

1 On theTransition Temperature toCalcite andCell Lengths

forVarious Biogenic Aragonites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Taiga Okumura, Masahiro Yoshimura, and Toshihiro Kogure

2 TEM Study oftheRadular Teeth oftheChiton

Acanthopleura japonica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Mitsuo Kakei, Masayoshi Yoshikawa, and Hiroyuki Mishima

3 Experimental Cremation ofBone: Crystallite Size andLattice

Parameter Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Martina Greiner, Balazs Kocsis, Mario F. Heinig, Katrin Mayer,

Anita Toncala, Gisela Grupe, and Wolfgang W. Schmahl

4 Effect ofCarbonic Anhydrase Immobilized onEggshell

Membranes onCalcium Carbonate Crystallization InVitro . . . . . . . 31

M. Soledad Fernández, Betzabe Montt, Liliana Ortiz,

Andrónico Neira-

Carrillo, and José Luis Arias

Proteomic Analysis ofVenomous Fang Matrix Proteins

ofProtobothrops flavoviridis (Habu) Snake . . . . . . . . . . . . . . . . . . . . . . 39

Tomohisa Ogawa, Asa Sekikawa, Hajime Sato, Koji Muramoto,

Hiroki Shibata, and Shosaku Hattori

Characterization ofGoldfish Scales byVibrational

Spectroscopic Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Masayuki Nara, Yusuke Maruyama, and Atsuhiko Hattori

Relationship Between Bone Morphology andBone Quality

inFemale Femurs: Implication forAdditive Risk ofAlternative

Forced Molting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Natsuko Ishikawa, Chihiro Nishii, Koh-en Yamauchi,

Hiroyuki Mishima, and Yoshiki Matsumoto

viii

8 Spectroscopic Investigation ofShell Pigments fromtheFamily

Neritidae (Mollusca: Gastropoda) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Toshiyuki Komura, Hiroyuki Kagi, Makiko Ishikawa, Mana Yasui,

and Takenori Sasaki

9 3D Visualization ofCalcified andNon-calcified Molluscan

Tissues Using Computed Tomography . . . . . . . . . . . . . . . . . . . . . . . . . 83

Takenori Sasaki, Yu Maekawa, Yusuke Takeda, Maki Atsushiba,

Chong Chen, Koji Noshita, Kentaro Uesugi, and Masato Hoshino

Part II Molecular and Cellular Regulation of Biomineralization

10 Calcium Ion andMineral Pathways inBiomineralization:

APerspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Gal Mor Khalifa, Keren Kahil, Lia Addadi, and Steve Weiner

11 Identification ofBarnacle Shell Proteins byTranscriptome

andProteomic Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Yue Him Wong, Noriaki Ozaki, Wei-Pang Zhang, Jin Sun,

Erina Yoshimura, Mieko Oguro-Okano, Yasuyuki Nogata,

Hsiu-Chin Lin, Benny K. K. Chan, Pei-Yuan Qian, and Keiju Okano

12 The Optical Characteristics ofCultured Akoya Pearl

Are Influenced by Both Donor andRecipient Oysters . . . . . . . . . . . . 113

Toshiharu Iwai, Masaharu Takahashi, Chiemi Miura,

and Takeshi Miura

13 Influence ofB Vitamins onProliferation andDifferentiation

ofOsteoblastic Bovine Cell Cultures: AnInVitro Study . . . . . . . . . . 121

Kent Urban, Julia Auer, Sebastian Bürklein, and Ulrich Plate

Rice Plant Biomineralization: Electron Microscopic Study

onPlant Opals andExploration ofOrganic Matrices

Involved inBiosilica Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Noriaki Ozaki, Takuya Ishida, Akiyoshi Osawa, Yumi Sasaki,

Hiromi Sato, Michio Suzuki, Keiju Okano, and Yuko Yoshizawa

DMP1 Binds Specifically toType ICollagen andRegulates

Mineral Nucleation andGrowth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Anne George, Elizabeth Guirado, and Yinghua Chen

Exploration ofGenes Associated withSponge Silicon

Biomineralization intheWhole Genome Sequence

oftheHexactinellid Euplectella curvistellata . . . . . . . . . . . . . . . . . . . . 147

Katsuhiko Shimizu, Hiroki Kobayashi, Michika Nishi,

Masatoshi Tsukahara, Tomohiro Bito, and Jiro Arima

Contents

Part III Genome-Based Analysis of Biomineralization

17 The Origin andEarly Evolution ofSCPP Genes andTissue

Mineralization inVertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Kazuhiko Kawasaki

Part IV Evolution in Biomineralization

18 Immunolocalization ofEnamel Matrix Protein-Like Proteins

intheTooth Enameloid ofActinopterygian Bony Fish . . . . . . . . . . . . 167

Ichiro Sasagawa, Shunya Oka, Masato Mikami, Hiroyuki Yokosuka,

and Mikio Ishiyama

19 Geographical andSeasonal Variations oftheShell

Microstructures intheBivalve Scapharca broughtonii . . . . . . . . . . . . 177

Kozue Nishida and Takenori Sasaki

Part V Biomineralization in Medical and Dental Sciences

20 Enhancement ofBone Tissue Repair byOctacalcium Phosphate

Crystallizing into Hydroxyapatite InSitu . . . . . . . . . . . . . . . . . . . . . . 189

Osamu Suzuki and Takahisa Anada

21 The Relationship Between theStructure andCalcification

ofDentin andtheRole ofMelatonin . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

Hiroyuki Mishima, Saki Tanabe, Atsuhiko Hattori, Nobuo Suzuki,

Mitsuo Kakei, Takashi Matsumoto, Mika Ikegame, Yasuo Miake,

Natsuko Ishikawa, and Yoshiki Matsumoto

22 Fabrication ofHydroxyapatite Nanofibers withHigh Aspect

Ratio via Low-Temperature Wet Precipitation Methods Under

Acidic Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

Masahiro Okada, Emilio Satoshi Hara, and Takuya Matsumoto

Physico-chemical Characterisation oftheProcesses Involved

inEnamel Remineralisation by CPP-ACP . . . . . . . . . . . . . . . . . . . . . . 219

Keith J. Cross, N.Laila Huq, Boon Loh, Li-Ming Bhutta,

Bill Madytianos, Sarah Peterson, David P. Stanton, Yi Yuan,

Coralie Reynolds, Glen Walker, Peiyan Shen, and Eric C. Reynolds

24 Molecular Interactions ofPeptide Encapsulated Calcium

Phosphate Delivery Vehicle at Enamel Surfaces . . . . . . . . . . . . . . . . . 229

Noorjahan Laila Huq, Keith John Cross, Helen Myroforidis,

David Phillip Stanton, Yu-Yen Chen, Brent Robert Ward,

and Eric Charles Reynolds

Preparation ofRandom andAligned Polycaprolactone Fiber

asTemplate forClassical Calcium Oxalate Through

Electrocrystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

Lazy Farias, Nicole Butto, and Andrónico Neira-Carrillo

Contents

Part VI Bio-inspired Materials Science and Engineering

26 Dysprosium Biomineralization by Penidiella sp. Strain T9 . . . . . . . . . 251

Takumi Horiike, Hajime Kiyono, and Mitsuo Yamashita

27 Various Shapes ofGold Nanoparticles Synthesized by Glycolipids

Extracted fromLactobacillus casei . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

Yugo Kato, Fumiya Kikuchi, Yuki Imura, Etsuro Yoshimura,

and Michio Suzuki

28 Octacalcium Phosphate Overgrowth onβ-Tricalcium Phosphate

Substrate inMetastable Calcium Phosphate Solution . . . . . . . . . . . . 267

Mayumi Iijima and Kazuo Onuma

Part VII Biominerals for Environmental and Paleoenvironmental

Sciences

29 Coral-Based Approaches toPaleoclimate Studies, Future Ocean

Environment Assessment, andDisaster Research . . . . . . . . . . . . . . . . 275

Atsushi Suzuki

30 An Elemental Fractionation Mechanism Common toBiogenic

Calcium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283

Kotaro Shirai

31 Biomineralization ofMetallic Tellurium byBacteria Isolated

FromMarine Sediment Off Niigata Japan . . . . . . . . . . . . . . . . . . . . . . 291

Madison Pascual Munar, Tadaaki Matsuo, Hiromi Kimura,

Hirokazu Takahashi, and Yoshiko Okamura

32 Calcium Oxalate Crystals inPlant Communities oftheSoutheast

ofthePampean Plain, Argentina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Stella Maris Altamirano, Natalia Borrelli, María Laura Benvenuto,

Mariana Fernández Honaine, and Margarita Osterrieth

33 Iron andCalcium Biomineralizations inthePampean Coastal Plains,

Argentina: Their Role intheEnvironmental Reconstruction

oftheHolocene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

Margarita Osterrieth, Celia Frayssinet, and Lucrecia Frayssinet

Part VIII Mollusk Shell Formation

Skeletal Organic Matrices inMolluscs: Origin, Evolution,

Diagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

Frédéric Marin, Aurélien Chmiel, Takeshi Takeuchi, Irina

Bundeleva, Christophe Durlet, Elias Samankassou, and Davorin

Medakovic

Contents

35 Functional Analysis onShelk2 ofPacific Oyster . . . . . . . . . . . . . . . . . 333

Jun Takahashi, Chieko Yamashita, Kenji Kanasaki,

and Haruhiko Toyohara

36 Mollusk Shells: Does theNacro-prismatic “Model” Exist? . . . . . . . . 341

Yannicke Dauphin and Jean-Pierre Cuif

37 The Marsh’s Membrane: AKey-Role foraForgotten Structure . . . . 349

Jean-Pierre Cuif and Yannicke Dauphin

38 Pearl Production by Implantation ofOuter Epithelial Cells

Isolated fromtheMantle ofPinctada fucata andtheEffects

ofBlending ofEpithelial Cells withDifferent Genetic

Backgrounds onPearl Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

Masahiko Awaji, Takashi Yamamoto, Yasunori Iwahashi,

Kiyohito Nagai, Fumihiro Hattori, Kaoru Maeyama,

Makoto Kakinuma, Shigeharu Kinoshita, and Shugo Watabe

39 Functional Analyses ofMMP Genes intheLigament

ofPinctada fucata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

Kazuki Kubota, Yasushi Tsuchihashi, Toshihiro Kogure,

Kaoru Maeyama, Fumihiro Hattori, Shigeharu Kinoshita,

Shohei Sakuda, Hiromichi Nagasawa, Etsuro Yoshimura,

and Michio Suzuki

40 Chitin Degraded by Chitinolytic Enzymes Induces Crystal

Defects ofCalcites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375

Hiroyuki Kintsu, Taiga Okumura, Lumi Negishi, Shinsuke Ifuku,

Toshihiro Kogure, Shohei Sakuda, and Michio Suzuki

Screening forGenes Participating intheFormation

ofPrismatic andNacreous Layers oftheJapanese Pearl Oyster

Pinctada fucata by RNA Interference Knockdown . . . . . . . . . . . . . . . 383

Daisuke Funabara, Fumito Ohmori, Shigeharu Kinoshita,

Kiyohito Nagai, Kaoru Maeyama, Kikuhiko Okamoto,

Satoshi Kanoh, Shuichi Asakawa, and Shugo Watabe

Gene Expression Patterns intheMantle andPearl Sac Tissues

ofthePearl Oyster Pinctada fucata . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

Shigeharu Kinoshita, Kaoru Maeyama, Kiyohito Nagai,

Shuichi Asakawa, and Shugo Watabe

Part IX Appendix

43 Selected SEM andTEM Images by Late Dr. Hiroshi Nakahara . . . . 399

Mitsuo Kakei

Correction to: TEM Study oftheRadular Teeth

oftheChiton Acanthopleura japonica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E1

Contents

Part I

Structure and Analysis of Biominerals

3© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_1

Chapter 1

On theTransition Temperature toCalcite

andCell Lengths forVarious Biogenic

Aragonites

TaigaOkumura, MasahiroYoshimura, andToshihiroKogure

Abstract In order to understand the mineralogical difference between biogenic

aragonites and their geological or synthetic ones, the transition temperature from

aragonite to calcite by heating and the cell lengths of a number of biogenic arago-

nites have been measured using conventional and high-temperature XRD, as well as

those of abiotic ones. Among 21 specimens, most biogenic aragonites showed a

transition temperature 60–100°C lower than that for abiotic ones. However, the

shells of land snails showed almost similar transition temperatures. The temperature

range from the beginning to the completion of the transition was also varied among

the biogenic aragonites. On the other hand, the axial ratios (a/b and c/b) of arago-

nites in marine molluscan species were considerably larger than those of abiotic

ones. However, aragonites in freshwater molluscan species and land snails showed

axial ratios similar to abiotic ones. X-ray microanalysis suggested that the origin of

such abnormal cell lengths was sodium incorporated in the aragonite crystals, not

due to lattice distortion induced by the intracrystalline organic molecules proposed

in previous researches.

Keywords Aragonite · Calcite · Transition temperature · Cell lengths · Axial ratio

· Sodium

1.1 Introduction

Aragonite is one of the polymorphs of anhydrous calcium carbonate (CaCO3) and

thermodynamically slightly less stable than calcite at the ambient temperature and

pressure in which organisms are alive. However, aragonite commonly occurs by

biomineralization processes. It has been often reported that biominerals possess

distinct characteristics and properties which are not observed in their geological or

T. Okumura · M. Yoshimura · T. Kogure (*)

Department of Earth and Planetary Science, The University of Tokyo, Tokyo, Japan

e-mail: okumura@eps.s.u-tokyo.ac.jp; Masahiro.Y@eps.s.u-tokyo.ac.jp;

kogure@eps.s.u-tokyo.ac.jp

synthetic counterparts. Biogenic aragonite is not the exception. For instance, Koga

and Nishikawa (2014) investigated the transition from coral aragonite to calcite by

heating in a thermogravimetric (TG)-differential thermal analysis (DTA) apparatus.

They found that when the temperature was increased at a certain rate, the tempera-

ture at which the aragonite-calcite phase transition occurred was around 100°C

lower for the coral aragonite than the geological ones. Please note that this tempera-

ture is not corresponding to that at which the thermodynamic stability between ara-

gonite and calcite is reversed. They ascribed such a low temperature for the transition

to the existence of interstitial water between aragonite crystals, which was released

during the transition.

On the other hand, the crystallographic parameters of biominerals have been also

reported to be specic, compared to abiotic minerals. Pokroy etal. (2004, 2007)

found that the cell lengths of aragonite in the shells of three molluscan species were

slightly different from those of abiotic minerals; the a- and c-lengths are longer, and

the b-length is shorter. They suggested that this “distortion” was induced by intrac-

rystalline organic molecules, namely, a biological effect.

These two examples as the specic characters of biogenic aragonite are interest-

ing to consider, for instance, the diagenetic effect on biominerals to form fossils.

However, it is not certain whether such characters can be observed ubiquitously in

biogenic aragonite formed by other species, families, etc. The present study investi-

gated more than 15 specimens for biogenic aragonite as well as geological and

synthetic ones, with respect to their temperatures for aragonite-calcite transition by

heating and cell lengths.

1.2 Materials andMethods

The aragonite specimens investigated are listed in Table1.1. In general, all speci-

mens except the synthetic ones were powdered using an agate mortar and pestle for

X-ray diffraction (XRD). The two synthetic aragonite specimens (Syn-PVA and

Syn-Mg) were prepared according to Kim etal. (2005) and Kitano (1962) using

polyvinyl alcohol (PVA) and MgCl2, respectively. In case of multilayered shells

with a calcite layer, the calcite layer was removed by grinding with a micro drill,

and then the remaining aragonite layer(s) was crushed into powder using an agate

mortar and pestle. They were washed with distilled water and ethanol and then dried

in an oven.

In the present study, we used an X-ray diffractometer (Rigaku SmartLab) with a

high-temperature specimen holder (Rigaku DHS 900), to measure the temperature

for the transition from aragonite to calcite. A copper X-ray tube was used and CuKα

was selected by Ni lter. X-ray was detected using a silicon strip detector (Rigaku

D/teX Ultra 2). The powdered specimens were processed into a disk of 3mm in

diameter and 0.5mm thick by a press and placed on the high-temperature specimen

holder. The temperature was raised at a rate of 5°C, and 2θ was scanned repeatedly

T. Okumura et al.

from 25.5° to 30.5° at a rate of 5°(2θ)/min. One hundred eleven reection of aragonite

and 104 reection of calcite were recorded in the 2θ range.

The XRD measurement to rene the cell lengths of aragonite was conducted

using a Rint-Ultima+ diffractometer (Rigaku) with CuKα radiation monochromated

with Ni lter and a silicon strip detector (Rigaku D/teX Ultra 2). The 2θ range was

10°–90° with continuous scan and a rate of 1° (2θ)/min. Data was collected at every

0.02° (2θ). The powder specimens were mounted in a shallow dimple on a non-

Table 1.1 Aragonite specimens investigated in this study

Geological aragonite

Specimen name Morphology Occurrence

Sefrou, Morocco Hexagonal prism Unknown

70087aLath Veins in serpentinite rock

70096aAciculum Deposit from hot spring

Synthetic aragonite

Syn-PVA Precipitated with PVA

Syn-Mg Precipitated with Mg

Molluscan shells (freshwater)

Microstructure Class Species

Nacre Bivalvia Anodonta cygnea

Hyriopsis schlegelii

Unio douglasiae

Prism Bivalvia Anodonta cygnea

Otolith

Oncorhynchus mykiss

Molluscan shells (terrestrial)

Microstructure Class Species

Cross-lamellar Gastropoda Coniglobus mercatorius

Acusta despecta

Zaptychopsis buschi

Molluscan shells (brackish water)

Microstructure Class Species

Cross-lamellar Gastropoda Pythia pantherina

Molluscan shells (salt water)

Microstructure Class Species

Cross-lamellar Gastropoda Murex pecten

Acanthopleura japonica

Lottia dorsuosa

Nacre Cephalopoda Nautilus pompilius

Bivalvia Pinctada fucata

Gastropoda Haliotis discus

Coral

Galaxea fascicularis

aCollection of The University Museum, The University of Tokyo

1 On theTransition Temperature toCalcite andCell Lengths forVarious Biogenic…

reective plate made of silicon. The cell lengths (a, b, and c) of aragonite were

calculated from the 2θ values of around 48 reections, using PDXL software

(Rigaku).

Finally, the chemical composition, particularly the concentration of sodium and

chlorine, in aragonite was analyzed using an electron-probe microanalyzer (EPMA,

JEOL JXA-8530F). The specimens for EPMA were prepared by embedding frag-

ments of the minerals or shells in epoxy resin, polishing with diamond paste and

colloidal silica. Finally amorphous carbon was coated by vacuum deposition for

electron conductivity.

1.3 Results andDiscussion

Figure 1.1 represents an example of the high-temperature XRD measurement. The

temperature at the right of each pattern indicates the temperature at the beginning of

the scanning. Please note that it took only 1min for the 2θ scan, and the rate to

increase temperature was 5 °C/min. The peaks from aragonite were gradually

decreased, and conversely 104 peak of calcite was increased, indicating the progress

of the phase transition. We estimated by extrapolation the beginning temperature at

which 104 peak of calcite had the integrated intensity and the ending temperature at

Fig. 1.1 An example of in situ high-temperature XRD pattern to determine the temperature for the

aragonite-calcite transition

T. Okumura et al.

which 111 peak of aragonite completely lost the intensity. The two temperatures for

all biogenic aragonites of several geological and synthetic aragonites are shown in

Fig.1.2. As reported in the previous work (Koga and Nishikawa 2014), biogenic

aragonite showed lower beginning and ending temperatures for the transition than

geological and synthetic aragonites. Particularly, the beginning temperature for the

coral was extremely low. However, the transition temperatures for terrestrial mol-

luscan shells, or land snails, were similar to those for abiotic aragonite, suggesting

that biogenic aragonites are not always less stable than abiogenic ones. The origin

of the difference of the temperature for the transition is not clear at present. Koga

and Nishikawa (2014) proposed that the origin is intercrystalline water in the coral

aragonite because water molecules were detected by mass spectroscopy at the tran-

sition. However, this is not convincing because the release of water may not be the

origin of the transition but the accompanied phenomenon of the transition. It should

be revealed by further investigations in the future.

The cell lengths of most of the samples are shown in Fig.1.3. In the gure, the

cell lengths are expressed with the ratio to those of geological aragonite from

Sefrou, Morocco (a=4.9629 (10) Å, b=7.9690 (15) Å, c=5.7430 (11) Å), accord-

ing to Pokroy etal. (2007). As Pokroy etal. (2007) reported, a- and c-lengths are

considerably larger, and b-length is shorter for some aragonites in molluscan shells,

Fig. 1.2 The beginning and ending temperatures of the transition for aragonite specimens of vari-

ous origins. PT indicates phase transition

1 On theTransition Temperature toCalcite andCell Lengths forVarious Biogenic…

but this trend is not true for all biogenic aragonites. Among all samples investigated,

the trend was distinctly observed only for marine molluscan shells. From this result,

we supposed that salinity or incorporation of sodium and/or chlorine in the arago-

nite structure may have resulted in such a systematic change of the cell lengths.

Hence, we conducted quantitative analysis of sodium/chlorine concentrations in the

samples using EPMA.It was revealed that the content of chlorine was extremely

low for all samples and not related to the cell lengths. On the other hand, the con-

centration of sodium was considerably varied, probably depending on the environ-

ments where the aragonites were formed. Considering the origin of the samples, the

relationship between the axial ratio (a/b and c/b) and the sodium concentration was

summarized as shown in Fig.1.4. From the gure, it is apparent that distinct cell

lengths or anisotropic lattice distortions observed in some biogenic aragonite com-

pared to geological and synthetic ones are originated from the incorporation of

sodium in the crystal structure of aragonite. We insist that the abnormality is not

owing to intracrystalline organic molecules as proposed in the previous works

(Pokroy etal. 2004, 2007).

Pokroy etal. (2004) considered Na-substitution in biogenic aragonite to change

the cell lengths, but they denied its possibility because the ionic radii of Na+ and

Ca2+ are so close that a small substitution of Na+ in aragonite cannot change its cell

lengths signicantly. It is probably true if we suspect only the isomorphic substitu-

tion, but the charges of the two cations are different, and some substitution for

anions must be accompanied. As stated above, chlorine was not sufciently detected

Fig. 1.3 The differences of the cell lengths of various aragonites from that of a geological sample

(Sefrou, Morocco). The specimens surrounded with the rectangle are marine molluscan shells

and coral

T. Okumura et al.

in the sodium-bearing aragonites. One possibility is the incorporation of proton or

hydroxyl (OH−) which substitutes O2− or HCO3− instead of CO32−. Probably Na+ and

OH− reside closely in the aragonite structure, and they locally modify the atomic

arrangement around the substitution, which may affect the cell lengths. Of course it

is not clear why the a- and c-lengths were elongated and b-length was shrunk by

such substitutions, but the idea of intracrystalline organic molecules also cannot

explain the anisotropic change. Pokroy etal. (2007) reported that when the biogenic

aragonites were heated to 350°C in air, the cell lengths became identical to those of

geological aragonite, which they proposed the evidence for organic molecules as the

origin of the lattice distortion. However, our preliminary experiment revealed that

when a marine molluscan shell (N. pompilius) was annealed at around 350°C, the

concentration of sodium was signicantly decreased, probably by diffusing away

from the aragonite structure. Accordingly, the Na-substitution can also explain the

annihilation of abnormality of the cell lengths by heating.

Acknowledgments We are grateful to Prof. A.Checa (Univ. Granada) and Prof. T.Sasaki (Univ.

Tokyo) for donating valuable shell samples, Prof. M.Suzuki (Univ. Tokyo) for instructing the

preparation of the synthetic aragonite, Mr. K.Fukawa (Univ. Tokyo) for assisting XRD measure-

ment, and Mr. K.Ichimura (Univ. Tokyo) for assisting EPMA analysis.

References

Kim W, Robertson RE, Zand R (2005) Effects of some nonionic polymeric additives on the crystal-

lization of calcium carbonate. Cryst Growth Des 5:513–522

Kitano Y (1962) The behavior of various inorganic ions in the separation of calcium carbonate

from a bicarbonate solution. Chem Soc Jpn 35:1973–1980

Fig. 1.4 The axial ratios (a/b in the upper and c/b in the lower gure) vs. concentration of Na in

the aragonites of various origins. The concentration of Na is expressed as wt.% of Na2CO3

1 On theTransition Temperature toCalcite andCell Lengths forVarious Biogenic…

Koga N, Nishikawa K (2014) Mutual relationship between solid-state aragonite−calcite trans-

formation and thermal dehydration of included water in coral aragonite. Cryst Growth Des

14:879–887

Pokroy B, Quintana JP, Caspi EN, Berner A, Zolotoyabko E (2004) Anisotropic lattice distortions

in biogenic aragonite. Nat Mater 3:900–902

Pokroy B, Fieramosca JS, Von Dreele RB, Fitch AN, Caspi EN, Zolotoyabko E (2007) Atomic

structure of biogenic aragonite. Chem Mater 19:3244–3251

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

T. Okumura et al.

11© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_2

Chapter 2

TEM Study oftheRadular Teeth

oftheChiton Acanthopleura japonica

MitsuoKakei, MasayoshiYoshikawa, andHiroyukiMishima

Abstract The radula chiton teeth, Acanthopleura japonica, were examined using

transmission electron microscopy (TEM). After cutting into segments correspond-

ing roughly to three developmental stages from the onset of tooth development, the

middle and the fully matured stages, toluidine blue staining has given the posterior

side three different color patterns, colorless, reddish-brown, and black colors,

respectively. At the colorless stage, the microvilli attached along the surface of the

tooth cusp appeared to be dissembled and convert into the lamellar structure in the

tooth interior. At the reddish-brown stage, the electron density between brous lay-

ers increased. A complex of tiny clusters of grains appeared along the brous layers.

They seemed to aggregate each other to become larger. At the black stage, multiple

layers consisting of irregular-shaped and various size of iron minerals were formed.

After treating with an aqua regia solution, organic substances have remained

between iron minerals, suggesting the abrasion-resistant role at the posterior side of

chiton teeth during feeding. In addition, these minerals were randomly arranged.

The lattice intervals of the ion minerals varied at an approximate range from 4.8 to

10.2Å. Also, we have conrmed clearly the lattice fringe of apatite crystal in the

core region.

Keywords Chiton · Transmission electron microscopy · Iron minerals · Apatite

crystal

M. Kakei (*)

Tokyo Nishinomori Dental Hygienist College, Tokyo, Japan

e-mail: mkakei@jcom.home.ne.jp

M. Yoshikawa

Division of Orthodontics, Meikai University School of Dentistry, Sakado, Japan

e-mail: ym-ortho@dent.meikai.ac.jp

H. Mishima

Department of Dental Engineering, Tsurumi University School of Dental Medicine,

Yokohama, Japan

e-mail: mishima-h@tsurumi-u.ac.jp

The original version of this chapter was revised. A correction to this chapter is available at

https://doi.org/10.1007/978-981-13-1002-7_44

2.1 Introduction

The lateral teeth of the radula exhibit a sequential series of tooth developments

along with the production of iron biominerals from the organic stage to the fully

mineralized stage. Regarding the iron biominerals in the radula teeth of the chiton,

the teeth were composed of multilayers of iron oxide, predominately in the form of

magnetite, but also in other forms of ferrihydrite and lepidocrocite (Lowenstam

1967). Therefore, many studies have focused mainly on these iron biominerals in

the tooth cusp (Lowenstam 1967; Kim etal. 1986; Weaver etal. 2010; van der Wal

1989; Martin etal. 2009; Han etal. 2011). The tooth cusps of posterior side are

mainly reinforced by the mineralization of magnetite at the matured stage. Due to

its hardness, TEM study conducted by making thin sections has not been available

in particular for the fully matured chiton teeth so far. On the other hand, the pres-

ence of a calcium phosphate mineral in the tooth core region has been examined

using various techniques for a considerable time (Lowenstam and Weiner 1985;

Kim et al. 1986; Evans et al. 1992; Evans and Alvarez 1999; Lee et al. 2000).

Although a study using electron microscopy has been tried to demonstrate the pres-

ence of apatite crystal (Evans etal. 1992), the precise structure of apatite crystal has

not yet fully elucidated. In this study, we have conducted the present study to dem-

onstrate the unique iron mineral deposits and verify the detailed structure of apatite

crystal mineral using an electron microscope.

2.2 Materials andMethods

Samples of the chiton Acanthopleura japonica were collected at Hachijojima’s

coastal area, Tokyo, Japan. Radulae were extracted from the chiton. After the

removal of the soft tissue, radulae were cut into segments corresponding roughly to

the three developmental stages of radula teeth from the onset of tooth development,

the middle, and the fully matured stages, by using a razor blade. Then samples were

subjected to examine using transmission electron microscope. They were xed with

2% glutaraldehyde in 0.1M cacodylate buffer at pH 7.4 for 1h at 5°C, post-xed

with 1% osmium tetroxide in the same buffer for 1h at 5°C, dehydrated by passage

through a series of ascending ethanol concentrations, and then embedded in Araldite

502. Thick sections were stained with toluidine blue solution. Based on different

color patterns at the posterior side of ralular teeth, these developmental stages ten-

tatively called the initial stage with colorless, the middle stage with reddish-brown,

and the fully matured stage with black color were examined. Thin sections were

obtained with a Porter Blum MT2 ultra-microtome (SORVALL) equipped with a

diamond knife. Sections were stained with saturated uranyl acetate and lead citrate,

and some were left unstained. Also, some treated with an aqua regia solution were

subjected to study. Then, they were examined under a JEM 100CX electron micro-

scope (JEOL) at an accelerating voltage of 80kV.

M. Kakei et al.

2.3 Results andDiscussion

Toluidine blue staining showed three different color patterns at the posterior side of

three developmental stages from the initial stage with colorless, the middle stage

with reddish-brown, and the fully matured stage with black color, respectively

(Fig.2.1).

At the colorless stage of the onset of tooth development, the internal structure of

tooth cusps was lled with brous layers arranging in relatively parallel to the tooth

surface at the anterior side (Fig.2.2a). On the other hand, at the posterior side,

brous layers bent upward adjacent to the inside of tooth cusp and run through

toward the tooth tip (Fig.2.2b).

At the reddish-brown stage of horizontal section, it was noted that higher magni-

cations of unstained sections have demonstrated that the deposits of ion minerals

appeared immediately and grew more quickly at the posterior side than at the ante-

rior side. So, it is suitable to examine the mineral deposits at the anterior side of the

tooth cusp. A comparison between stained and unstained sections showed that the

electron-dense zones were sandwiched by electron-lucent brous layers (Fig.2.3).

This suggests that the electron-dense zones might store mineral ions. At this stage,

brous layers were altered to be the plume structure (Fig.2.4a). Double-stained sec-

tion showed the small and discrete particles were developed along brous layers.

Also, it have been observed that a complex of tiny clusters of ne grains showing

different electron density, which looked like a bunch of grapes, developed associat-

ing with the plume structure (Fig.2.4b). The developed ne grains showing differ-

ent electron densities seemed to aggregate each other and increase its size to create

larger nonuniform grains.

Fig. 2.1 Toluidine blue staining of the radula chiton teeth at three different stages. The change of

color patterns at the posterior side reects the degree of iron mineralization process. (a) The initial

stage, (b) the middle stage, and (c) the fully matured stage. Toluidine blue-stained sections.

Bar=150μm

2 TEM Study oftheRadular Teeth oftheChiton Acanthopleura japonica

Fig. 2.2 TEM observations of the initial stage of chiton tooth. The arrangement of brous layer in

the anterior side is different from posterior side of tooth cup. Double-stained sections. Bar=0.5μm

Fig. 2.3 TEM observations of the middle stage. By comparing stained with unstained sections, the

space between organic layers shows a relatively high electron density. Double-stained (a) and

unstained (b) sections. Bar=1.0μm

M. Kakei et al.

At the black color stage of longitudinal sections, TEM study has demonstrated

that the multilayers of iron minerals at the posterior side showed a brick-like or

veneer-like wall structure (Weaver etal. 2010) (Fig.2.5a). Each iron layer seemed

to be comprised of relatively large minerals (Fig.2.5b). Also, it was noted that small

minerals were scattering in distribution. After treating with an aqua regia solution,

organic substances remained between iron minerals (Fig.2.6a, b). Although the role

of organic matrix is not fully elucidated, it has been considered that the organic

matrix may control the mineralization processes (van der Wal etal. 2000; Nemoto

etal. 2012) and contribute to resist crack and increase the tensile strength and ex-

ibility during the feeding by the teeth (Evans etal. 1990; van der Wal etal. 2000).

Iron minerals observed near the core (Fig.2.7a, b) and in the magnetite (Fig.2.7c,

d) regions are shown in Fig.2.7. Regarding the lattice fringes of iron minerals, the

estimate of lattice intervals of these iron minerals was ranging from 4.8 to 10.2Å,

approximately. On the basis of the observation at a high magnication of Fig.2.5b,

it has been considered that small iron minerals might gather together to create a

lump of iron minerals. It is also considered that random arrangement of iron miner-

als could prevent the radula teeth from becoming magnetic and attracting ion sand

which comes from the sandy beach.

TEM observation has clearly demonstrated the crystal fringe of apatite crystal in

the core region (Fig.2.8). Whether the crystals are uorapatite have been discussed

previously (Lowenstam 1967; Kim etal. 1986; Evans etal. 1992; Evans and Alvarez

1999; Lee etal. 2000). To our knowledge, the biologically induced apatite crystals

are divided into central dark line (CDL)-free and CDL-bearing types (Kakei etal.

2016). Viewing from the CDL-free type of crystal structure in the core region of the

chiton teeth, we assumed that uorapatite was formed.

Fig. 2.4 High magnication of the plume structure (a) and the development of iron mineral grains

(b) at the middle stage. The ne grains of iron minerals develop along the plume structure (b).

Double stain. Bars=100nm (a), 2.0μm (b)

2 TEM Study oftheRadular Teeth oftheChiton Acanthopleura japonica

Fig. 2.6 TEM observations of aqua regia solution-treated sections. Treating with an aqua regia

solution shows organic substances remained between iron minerals and suggesting a possible role

of glue. (a) 5min treatment, (b) 3min treatment. Arrows indicate organic substances. Double-

stained sections. Bar=100nm (a), 20nm (b)

Fig. 2.5 TEM observations of the posterior region at the fully matured stage. The mineral layers

consist of a brick-like wall or so-called veneer structure at the posterior side (a). Each mineral layer

consists of both large angular and small minerals (b). No stain. Bars=1.0μm (a), 200nm (b)

M. Kakei et al.

Fig. 2.7 TEM observations of iron minerals in the posterior region. Lattice fringes of iron miner-

als are recognized (a–d). Iron minerals are randomly arranged (d). No stain. Bar=10nm

2 TEM Study oftheRadular Teeth oftheChiton Acanthopleura japonica

References

Evans LA, Alvarez R (1999) Characterization of the calcium biomineral in the teeth of chiton

Pellisepentis. JBIC 4:166–170

Evans LA, Macey DJ, Webb J(1990) Characterization and structural organization of the organic

matrix of the radular teeth of the chiton Acanthopleura hirtosa. Philos Trans R Soc Lond B

329:87–96

Evans LA, Macey DJ, Webb J(1992) Calcium biomineralization in the radula teeth of the chiton,

Acanthopleura hirtosa. Calcif Tissue Int 51:78–82

Han Y, Liu C, Zhou D, Li F, Wang Y, Han X (2011) Magnetic and structural properties of magne-

tite in radular teeth of chiton. Bioelectromagnetics 32:226–233

Kakei M, Yoshikawa M, Mishima H (2016) Aspects of the apatite crystal: two pathways for apatite

formation, the mechanisms underlying crystal structure defects, and the pathological calcica-

tion event. JFossil Res 48(2):53–65

Kim K-S, Webb J, Macey DJ, Cohen DD (1986) Compositional changes during biomineralization

of the radula of the chiton Clavarizona hirtosa. JInorg Biochem 28:337–345

Lee AP, Brooker DJ, Macey DJ, van Bronswijk W, Webb J(2000) Apatite menralization in teeth of

the chiton Acanthopleura echinata. Apatite mineralization in teeth of the chiton Acanthopleura

echinata. Calcif Tissue Int 67:408–415

Lowenstam HA (1967) Lepidocrocite, an apatite mineral, and megnetite in teeth of chiton

(Polyplacophora). Science 156:1373–1375

Lowenstam HA, Weiner S (1985) Transformation of amorphous calcium phosphate to crystalline

dahllite in radular teeth of chitons. Science 227:51–53

Martin S, Kong C, Shaw JA, Macey DJ, Clode P (2009) Characterization of biominerals in the

teeth of the chiton, Acanthopleura hirtosa. JStruct Biol 167:55–61

Nemoto M, Wang Q, Li D, Pan S, Matsunaga T, Kisailus D (2012) Proteomic analysis from

the mineralized radular teeth of the giant Pacic chiton, Cryptochiton stelleri (Mollusca).

Proteomics 12:2890–2894

Fig. 2.8 TEM observations of calcium phosphate mineral in the core region. Two apatite crystals

are conrmed. No stain. Bar=10nm

M. Kakei et al.

van der Wal P (1989) Structural and material design of mature mineralized radula teeth of Patella

vulgata (Gastropoda). JUltrastruct Mol Struct Res 102:147–161

van der Wal P, Gleasen HJ, Videler JJ (2000) Radular teeth as models for the improvement of

industrial cutting device. Mater Sci Eng C Biomim Supramol Syst 7:129–142

Weaver JC, Wang Q, Miserez A, Tantuccio A, Stromberg R, Bozhilov KN, Maxwell P, Nay R,

Heier ST, DiMasi E, Kisailus D (2010) Analysis of an ultra hard magnetic biomineral in chiton

radular teeth. Materialstoday 13 (1–2):42–52

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

2 TEM Study oftheRadular Teeth oftheChiton Acanthopleura japonica

21© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_3

Chapter 3

Experimental Cremation ofBone:

Crystallite Size andLattice Parameter

Evolution

MartinaGreiner, BalazsKocsis, MarioF.Heinig, KatrinMayer,

AnitaToncala, GiselaGrupe, andWolfgangW.Schmahl

Abstract In this study we investigate pristine and experimentally incinerated

bovine bone material with differing annealing times and temperatures from 100 to

1000°C to analyse the crystallographic change of natural bone mineral during cre-

mation. We used X-ray powder diffraction (XRPD) and Fourier transform infrared

(FTIR) spectroscopy as complementary methods. We observe a structural change of

bone mineral during cremation. Our study highlights that there are only few or even

no hydroxyl ions in pristine bone mineral (bioapatite), which is a carbonate-hydro-

apatite rather than a hydroxyapatite. A signicant recrystallization reaction from

bioapatite to hydroxyapatite takes place at elevated temperatures from 700°C (after

30min cremation time). This process is associated with a signicant increase of

crystallite size, and it involves an increase of hydroxyl in the apatite lattice that goes

along with a depletion of water and carbonate contents during cremation. Our rst

results highlight the importance of both time and temperature on the recrystalliza-

tion reaction during cremation.

Keywords Carbonated apatite · Calcium phosphate · Bioapatite · Bone · X-ray

diffraction · FTIR · Rietveld renement

M. Greiner (*) · B. Kocsis · M. F. Heinig · W. W. Schmahl

Department für Geo- und Umweltwissenschaften, Ludwig-Maximilians-Universität,

Munich, Germany

e-mail: martina.greiner@lrz.uni-muenchen.de; wolfgang.schmahl@lrz.uni-muenchen.de

K. Mayer · A. Toncala · G. Grupe

Fakultät für Biologie, Anthropologie und Humangenetik, Ludwig-Maximilians-Universität,

Martinsried, Germany

e-mail: katrin.mayer@mnet-online.de; G.Grupe@lrz.uni-muenchen.de

3.1 Introduction

The Forschergruppe FOR 1670 project on human transalpine mobility in the Late

Bronze Age to Early Roman times performs isotope studies on archaeological bone

nds. During that age, cremating the deceased was the primary burial custom

(Grupe etal. 2015). To understand bone alteration by cremation, we study the evolu-

tion of bone crystallography and crystallite size as a function of cremation tempera-

ture and annealing time for bovine bone by FTIR and X-ray diffraction. Mammal

bone mineral is a nanocrystalline material consisting of an apatite mineral that is

chemically far more complex than hydroxyapatite and can be approximated as

(Ca,Mg,Na)10-x((PO4)6-x(CO3)x)(OH1-y-z, (CO3)y, (H2O)z)2 (Elliott 2002; Rey etal.

2007). It comprises between 5 and 8wt% carbonate, which substitutes in the [OH]−

site (A-type substitution) as well as the [PO]43- site (B-type substitution) of the apa-

tite structure (LeGeros etal. 1969; Wopenka and Pasteris 2005; Pasteris etal. 2012;

Yi etal. 2013). Previous studies showed that recrystallization of the bioapatite and

crystallite growth mainly sets in from about 600°C (Piga etal. 2009; Schmahl etal.

2017), but small changes are already apparent at low temperatures of 100 °C

(Harbeck etal. 2011). In this study, we want to dene more precisely the cremation

temperature and annealing time where hydroxyapatite crystallization sets in.

3.2 Materials andMethods

Pieces of compact bone were cut from the tubular part of bovine femur; the endos-

teal and periosteal surfaces were mechanically removed. Samples were ultrasoni-

cally washed in deionized H2O.After air-drying, the bones were defatted for 5days

with diethyl ether in a Soxhlet and air-dried. Finally, the samples were homogenized

to a ne powder and passed through a 100μm sieve.

Cremation experiments were carried out between 100 and 1000°C in air (oxidiz-

ing conditions) in steps of 100°C with 150min annealing time. After data analysis

of the initial results, we focussed on the temperatures of 650 and 700°C and used

shorter exposure times between 10 and 60min in intervals of 10min.

X-ray diffractograms were collected on a General Electric 3003 powder diffrac-

tometer in Bragg-Brentano reection geometry. Cu-Kα1 radiation was selected with

an exposure time of 1000s.

All samples were mixed with NIST 660b LaB6 as internal standard (2–5wt%).

The FULLPROF code (Rodríguez-Carvajal 1993; Rodríguez-Carvajal and Roisnel

2004) was applied for data evaluation and Rietveld renement (Rietveld 1969).

The Thompson-Cox-Hastings method for convolution of instrumental resolution

with size and isotropic microstrain broadening (Thompson etal. 1987) was applied.

For renement, a hexagonal symmetry model for carbonated apatite was used

(Wilson etal. 2004).

M. Greiner et al.

Infrared spectra were measured on a Bruker Equinox FTIR instrument with a

resolution of 4cm−1 with 128 scans.

3.3 Results

3.3.1

X-Ray Diffraction: 100°C Intervals

Figure 3.1a shows X-ray powder diffraction patterns of untreated and cremated

bones up to 1000°C. The untreated bone mineral displays extremely broadened

peaks. The broadening decreases only little with 150min heat treatment up until

600°C, whereas the diffraction pattern sharpens considerably from 700°C upwards.

Exemplary Rietveld renements of samples cremated at 600°C and 700°C for

150min annealing time are shown in Fig.3.1b, c. Enlarged images of the 31–35° 2θ

section show overlapping apatite diffraction peaks in Fig.3.1b, whereas peaks in

Fig.3.1c can clearly be distinguished from each other. The lattice parameters and

crystallite sizes obtained by Rietveld renement of diffractograms measured at

room temperature for the 150min heat-treated samples are shown in Fig.3.1d, e.

Note the sharp rise of the crystallite size setting in at 700°C.The lattice parameters

show an initial increase with annealing treatment and then sharply drop at tempera-

tures where the grain size sharply increases.

3.3.2 X-Ray Diffraction: 10Min Intervals

The X-ray diffractograms of the bovine bone cremated at 650°C from 10 to 60min

depict a steady narrowing of the diffraction peaks with increasing annealing time.

Nevertheless, a broad peak shape remains after 60min annealing (Fig.3.2a). The

unit cell parameters shrink (Fig.3.2b, c) already after 20min cremation. The crys-

tallite size increases more or less steadily with elapsed annealing time, whereas the

biggest change is between 30 and 40min with an increase of the crystallite size of

99.4(2) Å to 126.6(3) Å (Fig.3.2c).

X-ray diffractograms of bovine bone cremated at 700°C from 10 to 60min show

a broad diffraction peak shape from 10 to 30min annealing time. Samples annealed

for 40min or longer depict sharper peaks. Bovine bone incinerated at 700°C shows

a steady increase of the crystallite size up to 30min experimental cremation. From

30 to 40min elapsing time, we observe a signicant jump from 117.85(2) Å to

294.8(3) Å (2.5 times larger) (Fig.3.2f). Moreover, we observe a decrease of lattice

parameters a and c until 40min annealing times. This trend is reversed after 50min

annealing (Fig.3.2e, f).

3 Experimental Cremation ofBone: Crystallite Size andLattice Parameter Evolution

Fig. 3.1 (a) Comparison of the 10–60° 2θ range of X-ray diffractograms (Cu-Kα1) of cremated

bones from 100 to 1000°C; (b) Rietveld renement of bovine bone cremated at 600°C, 150min

annealing. Red dots, observed data points; black line, calculated XRD prole; bottom blue line,

difference of observed and calculated data; green vertical bars, positions of diffraction peaks; top

row, bone apatite; bottom row, LaB6 standard. Enlarged region shows 31–35° 2θ range with over-

lapping 121, 112, 030 and 022 peaks; (c) Rietveld renement of bovine bone cremated at 700°C

after 150min annealing. Enlarged region shows 30–35° 2θ range with clearly distinct 121, 112,

030 and 022 peaks; (d) Lattice parameters a (=b) and c of untreated and annealed bone material at

temperatures from 100 to 1000°C after 150min annealing; (e) Unit cell volume and crystallite size

of untreated and cremated bones from 100 to 1000°C after 150min annealing

3.3.3 FTIR

Figure 3.3a shows a comparison of IR spectra for different annealing temperatures.

Characteristic phosphate group absorption bands at 470–480cm−1 (ν2PO43−), 500–

750cm−1 (ν4PO43−), ~962cm−1 (ν1PO43−) and 980–1120cm−1 (ν3PO43−) were identi-

ed according to Destainville etal. (2003) and Raynaud etal. (2002). Annealed

bone at 1000°C shows a well-differentiated hydroxyl libration peak at ~632cm−1

which is absent in the FTIR spectra of untreated bovine bone (Fig.3.3b).

Absorption bands at 873–879 cm−1 and 1400–1458 cm−1 were attributed to

ν2CO32− and ν3CO32− (Fleet 2009; Grunenwald etal. 2014; Rey etal. 1989). The

intensity of carbonate bands begins to decrease from 400°C with increasing anneal-

ing temperatures and can barely be observed for the sample cremated at 1000°C

(see Fig.3.3a). For untreated bovine bone, we observe broad H2O absorption bands

from ~3000 to 3600cm−1 (Brubach etal. 2005). These peaks lose intensity with heat

treatment and disappear for the 700°C and higher heat treatments (Fig.3.3c).

Fig. 3.2 (a) Comparison of the 10–80° 2θ range of X-ray diffractograms (Cu-Kα1) of untreated

and cremated bones at 650 °C, 10–60 min annealing; (b) Lattice parameters a (=b) and c of

untreated and cremated bones at 650°C, 10–60min annealing; (c) Unit cell volume and crystallite

size of untreated and cremated bones at 650°C, 10–60min annealing; (d) 10–80° 2θ range of

X-ray diffractograms of untreated and cremated bones at 700°C, 10–60min annealing; (e) Lattice

parameters a(=b) and c of untreated and cremated bones at 700°C, 10–60min annealing; (f) Unit

cell volume and crystallite size of untreated and cremated bones at 700°C, 10–60min annealing

3 Experimental Cremation ofBone: Crystallite Size andLattice Parameter Evolution

The OH- stretch vibration gives a sharp peak at 3570cm−1 (González-Díaz and

Hidalgo 1976; González-Díaz and Santos 1977; Vandecandelaere etal. 2012). The

band at ~632 cm−1 was assigned to the OH− libration mode (Destainville et al.

2003). In the untreated bone spectra and for low annealing temperatures, neither the

OH− libration peak near 630cm−1 nor the sharp and distinct OH− stretching vibration

Fig. 3.3 (a) Comparison of the 400–1800cm−1 FTIR spectra of untreated and cremated bovine

bone at 400, 500, 600, 700 and 1000°C (annealing time 150min); (b) Enlargement of the 450–

750cm−1 region of Fig.3.2a) with ν2PO43− and ν4PO43− vibration bands and emerging OH− libra-

tion band with increasing cremation temperature; (c) 2600–3800cm−1 region of untreated and

cremated bone (400, 500, 600, 700 and 1000°C) indicating decreasing H2O absorption bands and

increasing OH− stretching mode with increasing cremation temperature

M. Greiner et al.

peak at 3570cm−1 is present. However, these OH− signals emerge with annealing at

400 °C and signicantly increase and become well-differentiated for samples

annealed at temperatures of 700°C or higher (see Fig.3.3b, c).

3.4 Discussion

In the original bone, we observe broad diffraction peaks (Figs.3.1a and 3.2a, b) due

to the nanoscale dimension of the bone apatite crystallites. Further, the high inten-

sity of the carbonate and H2O vibration bands and absence of OH− bands quite

clearly indicate that bone apatite is a carbonate-hydro-apatite rather than a hydroxy-

apatite (Fig. 3.3a–c). Loong et al. (2000) using inelastic neutron scattering and

Pasteris etal. (2004) using Raman spectroscopy came to similar conclusions.

With extended annealing treatment, the diffraction peaks of the bone mineral get

sharper; the (CO3)2− infrared signals decrease, while the OH− infrared peaks

increase. Within the broad water band, one can clearly see the rising of the OH−

stretching is a function of increasing annealing temperature (Fig.3.3c). This indi-

cates a reaction from bioapatite (carbonate-hydro-apatite) to hydroxyapatite with

heat treatment, as the material approaches stoichiometric chemistry. This reaction is

associated with growth of the crystallites; the growth becomes more rapid at tem-

peratures from 700°C and higher (Figs.3.1a and 3.2d). At the same time, loss of

carbonate and water and their replacement by OH− in the structure lead to a decrease

of unit cell parameters (Figs.3.1d, e and 3.2b–f).

While observing these structural changes occurring, it cannot be simply con-

cluded that the process occurs homogeneously within the apatite lattice. It is just as

likely that the bioapatite decomposes and that hydroxyapatite is formed in a hetero-

geneous reaction at the expense of the decomposing bioapatite. It must be borne in

mind that our techniques integrate over the whole volume of the sample, and the

diffraction peak positions as well as IR frequencies of both mineral phases (bioapa-

tite and hydroxyapatite) are very close and initially severely broadened. Thus our

techniques might well record a superposition of the signals of both phases coexist-

ing. Here it is important to note that the distinct increase of the crystallite size for

temperatures of and above 700°C sets in after 30min of annealing time (Fig.3.2e,

f). This may be indicative of a heterogeneous rather than a homogeneous reaction

from bioapatite to hydroxyapatite. For an increase of crystallite size of only one

homogeneous apatite phase by Ostwald ripening, the evolution of crystallite size

with time (Fig.3.2f) should have the opposite curvature than observed.

The time dependence (Fig.3.2) of the reaction process becomes important when

concluding from bone crystallinity to cremation temperature. Up to 40min crema-

tion time, we see the same trend for lattice parameters when comparing 650 and

700°C: a decrease of lattice parameters a and c and therefore a shrinkage of the unit

cell coupled with an increase of the crystallite size (Fig.3.2b–f). After more than

30min annealing at 700°C, we see more rapidly increasing lattice parameters, an

effect which is not observable in the 650°C temperature experiments. Moreover, at

3 Experimental Cremation ofBone: Crystallite Size andLattice Parameter Evolution

700°C after 30min annealing, we observe also a signicant change in crystallite

size which we attribute to a progressive recrystallization reaction of bioapatite to

hydroxyapatite. At that time, the decomposition products (essentially carbon) of

organics such as collagen, which comprise about 35wt% of the original bone mate-

rial (Rogers and Zioupos 1999), are almost completely gone, and the hydroxyapa-

tite crystallites can grow without being impeded by lms of organics or their

residues which separate the crystallites.

Note that bone annealed at 400°C for 150 min displays essentially the same

crystallite sizes as bone treated for 20min at 700°C (~97Å), and bone annealed at

600°C for 150min displays similar crystallite sizes as bone treated at 650°C for

50min (~133Å). Our experiments reveal that it is important to consider the inu-

ence of time when concluding from material state on cremation conditions.

Acknowledgement We thank the Deutsche Forschungsgemeinschaft (DFG) for funding the

project in Forschergruppe FOR1670 under Schm930/12-1 and Gr 959/20-1,2.

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3 Experimental Cremation ofBone: Crystallite Size andLattice Parameter Evolution

31© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_4

Chapter 4

Effect ofCarbonic Anhydrase Immobilized

onEggshell Membranes onCalcium

Carbonate Crystallization InVitro

M.SoledadFernández, BetzabeMontt, LilianaOrtiz,

AndrónicoNeira- Carrillo, andJoséLuisArias

Abstract The eggshell membranes (ESM) serve as the rst interface with the inor-

ganic phase during eggshell formation. During mineral growth, crystals nucleate on

the outer side of the ESM at specialized sites called mammillae, mainly consisting

of mammillan, a keratan sulfate proteoglycan together with the activity of carbonic

anhydrase (CA).

In order to get insight into the mechanisms of chicken eggshell mineralization,

ESM was used as a biotemplate for immobilizing carbonic anhydrase (CA) and

study invitro calcite crystallization. Here, we showed that when the eggshell mem-

brane supplemented with immobilized or dissolved carbonic anhydrase is located at

the gas-liquid interface, calcite nucleation and growth are sequestered by the ESM

scaffold from solution, thus affecting the morphology and size of the crystals formed.

Keywords Eggshell membrane · Carbonic anhydrase · Biomineralization

4.1 Introduction

Biomineralization is a widespread phenomenon in nature leading to the formation

of a variety of solid inorganic structures by living organisms, such as intracellular

crystals in prokaryotes; exoskeletons in protozoa, algae, and invertebrates; spicules

and lenses; bone, teeth, statoliths, and otoliths; eggshells; plant mineral structures;

and also pathological biominerals such as gall stones, kidney stones, and oyster

pearls (Lowenstam and Weiner 1989; Mann etal. 1989; Simkiss and Wilbur 1989;

Heuer et al. 1992; Arias and Fernandez 2008). By this process living organisms

precipitate inorganic minerals on organic matrices. The resulting biominerals are

deposited in elaborated shapes and hierarchical structures by interaction at the

organic-inorganic interface, where the rate of crystal formation is regulated by the

M. S. Fernández (*) · B. Montt · L. Ortiz · A. Neira-Carrillo · J. L. Arias

Faculty of Veterinary Sciences, University of Chile, Santiago, Chile

e-mail: sofernan@uchile.cl; lbortizmendez@ug.uchile.cl; aneira@uchile.cl; jarias@uchile.cl

control of the microenvironment in which such mineralization events take place.

One of the main biomineralization resulting mineral minerals is calcium carbonate

(CaCO3), especially the more stable form, calcite. The avian eggshell is a good

example of multifunctional biomineral where an organic scaffold (or matrix), the

eggshell membrane, plays a crucial role in regulating mineral nucleation and growth

by incorporating inorganic precursors, such as ions, ion clusters, and amorphous

phases (Rao etal. 2017); other organic matrices complete the formation of a calci-

ed layer (i.e., palisade) composed of calcite columns (Panheleux et al. 1999).

Structurally, the eggshell is a multilayered calcitic bioceramic. As the egg migrates

through the oviduct, the biomineral matures under the inuence of biomolecular

additives and an extracellular matrix (Nys etal. 1999; Fernandez etal. 2001). The

matrix, primarily composed of collagen bers, constitutes the eggshell membranes

(ESM) (Arias et al. 1991). Each ber exhibits a core surrounded by a glyco-

proteinous material termed as the mantle. This serves as the rst interface with the

inorganic phase. During mineral growth, crystals nucleate on the outer side of the

ESM at specialized sites called mammillae, mainly consisting of mammillan, a

keratan sulfate proteoglycan together with the activity of carbonic anhydrase (CA).

This metalloenzyme catalyzes the reversible hydration of carbonic dioxide to bicar-

bonate and a proton. In fact, it has been possible to mimic eggshell formation

invitro by adding the main organic components including CA, where an increase in

calcium carbonate crystals growth and fusion was observed (Fernandez etal. 2004).

However, the use of enzymes invitro is difcult, because of instability and the

complexity for maintaining the catalytic function in chemical reactions (Lu etal.

2013). For that reason enzymatic immobilization on a solid substrate appears as a

solution for this problem (Wanjari etal. 2013).

For invitro biomineralization, a specic conned environment is needed, that

means an inert scaffold which generates an almost two dimensional interface where

the crystal nucleation takes place. Currently, many kinds of surfaces and/or supports

are used for enzyme immobilization, and the ESM meets all the requirements to be

used as a natural support for CA immobilization and in vitro biomineralization

experiments.

In order to get insight into the mechanisms of chicken eggshell mineralization,

ESM was used as a biotemplate for immobilizing carbonic anhydrase (CA).

4.2 Material andMethods

4.2.1 Carbonic Anhydrase (EC 4.2.1.1) Immobilization

onESM

ESM were obtained after 30min incubation of an empty egg in 1% acetic acid to

detach the membrane from de shell, and then ESM was incubated for another 48h

in 1% acetic acid to eliminate any remaining calcium carbonate crystals and then

washed in deionized water three times (Arias etal. 2008).

M. S. Fernández et al.

For enzyme immobilization 1mg of carbonic anhydrase (2500 units/mg, Sigma,

St. Louis, MO, USA) in 1mL deionized water was used; membranes were incu-

bated for 1h with 100μL of this solution; then 20μL of 2.5% glutaraldehyde for

30min was used as cross-linking agent (Tembe etal. 2008). Then, membranes were

washed with TRIS buffer solution pH 9 at 4°C.

4.2.2 Crystallization Experiments

The crystallization assays were based on a variation of the sitting drop method

developed elsewhere (Dominguez-Vera etal. 2000). Briey, it consists of a chamber

built with a 85mm plastic petri dish having 18mm in diameter central hole in its

bottom, glued to a plastic cylindrical vessel (50mm in diameter and 30 mm in

height) (Fig.4.1). The bottom of the petri dish was divided in 16 radii to assure an

equidistant settling of odd number of polystyrene microbridges (Hampon Res.,

Laguna Niguel, CA). The microbridges were lled with 35μL of 200mM dihydrate

calcium chloride solution in 200 mM Tris buffer, pH 9.0. The cylindrical vessel

contained 3ml of 25 mM ammonium carbonate. One strip of eggshell membrane

with or without immobilized CA or with CA in solution was deposited on the bot-

tom or on the top of each microbridge with the mammillary side facing up or upside

down, respectively. Five replicates of each experiment were carried out inside the

chamber at 20°C for 24h. After the experiments, eggshell strips were taken out of

the microbridges, air-dried at room temperature, mounted on aluminum stubs with

scotch double-sided tape, and coated with gold. Crystal morphology was observed

and size estimated in an Hitachi TM 3000 scanning electron microscope.

ESM WITHOUT TREATMENT

ESM WITH IMMOBILIZED CA

ESM PLUS CA IN SOLUTION

ESM on bottom

9 cm

5 cm

NH4HCO3

CO3

Ca+Ca–

NH3

ESM on top

Fig. 4.1 Experimental setup used from growing invitro CaCO3 crystals showing the location of

eggshell membrane strips either on the bottom or the top of the microbridges containers

4 Effect ofCarbonic Anhydrase Immobilized onEggshell Membranes onCalcium…

4.3 Results

4.3.1 ESM onBottom oftheMicrobridge 24H Incubation

After 24h incubation using ESM without treatment, rounded calcite crystals were

observed deposited on the ESM (Fig.4.2a), with an average size of 21.33±1.97μm

(Table 4.1). By contrast, when ESM with immobilized CA was used, regular

Fig. 4.2 Eggshell membrane (ESM) on bottom of the microbridge after 24h incubation: (a) ESM

without treatment, 1000X; (b) ESM with immobilized CA, 1000X; (c) ESM without treatment but

with CA dissolved in the calcication medium 1000X

Table 4.1 Size of calcite crystals deposited on eggshell membrane (ESM) measured under

different conditions

Crystal size

μm ± S.D

ESM at the bottom of the microbridge Control 21.33 ± 1.97

CA immobilized 31.0 ± 2.08

CA in solution 20.0 ± 3.68

ESM at the top of the microbridge facing calcication

solution

Control 22.1 ± 1.91

CA immobilized 35.83 ± 3.53

CA in solution 27.83 ± 2.47

M. S. Fernández et al.

rhombohedral calcite crystals (Fig. 4.2b), 31 ± 2.08 μm in average size were

obtained (Table4.1). When intact ESM was used in combination with CA dissolved

in the calcication medium, rounded calcite crystals (Fig.4.2c) with an average size

of 20± 3.68 μm (Table4.1), similar to those observed in the rst condition were

obtained.

4.3.2 ESM onTop oftheMicrobridge Facing theCalcication

Medium After24H ofIncubation

After 24 h incubation using ESM without treatment, polyhedral calcite crystals

were obtained (Fig.4.3a), with 22.1 ± 1.91μm in average size (Table4.1). But when

ESM with immobilized CA was used, many polyhedral fused calcite crystals of

35.83 ± 3.13 μm in average size (Table 4.1) with symmetrical smooth edges

(Fig.4.3b) were observed. However, after using intact ESM with CA in solution,

less polyhedral fused calcite crystals with curved edges (Fig. 4.3c) of 27.83 ±

2.47μm in average size were observed (Table4.1).

Fig. 4.3 Eggshell membrane (ESM) on top of the microbridge facing calcication medium after

24h incubation: (a) ESM without treatment, 1000X; (b) ESM with immobilized CA, 1000X; (c)

ESM with CA in solution in the calcication medium 1000X

4 Effect ofCarbonic Anhydrase Immobilized onEggshell Membranes onCalcium…

4.4 Discussion

When invitro CaCO3 crystallization experiments are done in a gas-liquid interface

diffusion environment, the favorite place for crystal nucleation and growth must be

considered. In fact, in this chamber-mediated experiment, CO2 comes from the gas

phase, while carbonate ions are in aqueous solution. If the reaction is done without

any additional scaffold, such as the eggshell membrane, it is expected that nucle-

ation of CaCO3 crystals occurs close to the gas-liquid interface, and then, after a

determined period of growth, crystals formed precipitate reaching the bottom of the

reaction vessel (microbridge). However, here we showed that when an active het-

erogeneous nucleator scaffold, such as the eggshell membrane supplemented with

immobilized or dissolved carbonic anhydrase, is located upside down at the gas-

liquid interface for avoiding gravity effect, calcite nucleation and growth are seques-

tered by the scaffold from solution, thus affecting the morphology and size of the

crystals formed. The morphology changes, including calcite aggregations occurs in

a way that resembles the calcite column aggregation and fusion observed during

natural eggshell formation.

Acknowledgment Work supported by Fondecyt project 1150681 from CONICYT.

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

4 Effect ofCarbonic Anhydrase Immobilized onEggshell Membranes onCalcium…

39© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_5

Chapter 5

Proteomic Analysis ofVenomous Fang

Matrix Proteins ofProtobothrops avoviridis

(Habu) Snake

TomohisaOgawa, AsaSekikawa, HajimeSato, KojiMuramoto,

HirokiShibata, andShosakuHattori

Abstract Venomous animals have specialized venom delivery apparatus such as

nematocysts, stings, and fangs in addition to the poisonous organs consisting venom

gland or sac, which produce and stock the venom. Snake is one of the major venom-

ous animals, of which fangs are connected to the venom gland to inject the venom

into prey. Snake’s venomous fangs showed the unique characteristics including

mechanical strength and chemical stability. Especially, Protobothrops avoviridis

(habu) snake fangs showed the resistance against its venom digestive proteases,

whereas the bones and teeth of mouse were completely digested in the gastrointes-

tinal tract, although habu fangs were also drawn into the body with the prey. These

observations suggest that structural differences exist between venomous fangs and

mammalian bones and teeth.

In this study, to reveal the molecular properties of venomous snake fangs, the

matrix proteins of P. avoviridis (habu) snake venom fang were analyzed by using

proteomics experiments using 2D-PAGE and TOF MS/MS analyses. As a result,

several biomineralization-related proteins such as vimentin, tectorin, adaptin, and

collagen were identied in the venomous fang matrix proteins. Interestingly, the

inhibitory proteins against venomous proteins such as metalloproteinase and PLA2

were also identied in fang’s matrix proteins.

T. Ogawa (*) · A. Sekikawa · H. Sato · K. Muramoto

Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University,

Sendai, Japan

e-mail: tomohisa.ogawa.c3@tohoku.ac.jp; b1bm1012@s.tohoku.ac.jp; koji.muramoto.d5@

tohoku.ac.jp

H. Shibata

Division of Genomics, Medical Institute of Bioregulation, Kyushu University,

Fukuoka, Japan

e-mail: hshibata@gen.kyushu-u.ac.jp

S. Hattori

Amami Laboratory of Injurious Animals, The Institute of Medical Science,

The University of Tokyo, Kagoshima, Japan

e-mail: shattori@ims.u-tokyo.ac.jp

Keywords Biomineralization · Matrix protein · Proteome · Snake · Venomous

fang

5.1 Introduction

Venomous animals such as sea anemone, jellysh, lizards, scorpion, sh, arachnids,

bees, and snakes produce chemical weapon, toxic proteins, and peptides cocktail to

kill and capture pray. They deliver the toxins as venom into prey through the sophis-

ticated venom delivery systems consisting of an exocrine gland, a lumen, venom

duct, and also injector such as nematocyst, sting, fangs, harpoon-like sting, and

spine. These venomous apparatuses are thought to have evolved from the general

biological organs, namely, an ovipositor, a tooth, radula, and dorsal n, respectively.

Snake is one of the major venomous animals, of which fangs are connected to the

venom gland to inject the venom into prey. Venomous snakes can be classied into

two groups according to the fang systems, front fanged (elapid and vipers) and rear

fanged (grass snakes), and frontal fangs are further divided into two types, grooves

and tubes (Kardong 1979; Savitzky 1980; Jackson 2002; Kuch etal. 2006). Vonk

et al. (2008) reported the evolutionary origin and development of snake fangs,

showing that front fangs develop from the posterior end of the upper jaw and are

strikingly similar in morphogenesis to rear fangs. In the anterior part of the maxilla

of front-fanged snakes, gene expression of sonic hedgehog, which is responsible

among other things for the formation of the teeth, is suppressed. Despite such exten-

sive studies and the recent genome sequence analyses for two venomous snakes, the

king cobra (Ophiophagus hannah) (Vonk et al. 2013) and the ve-pacer viper

(Deinagkistrodon acutus) (Yin etal. 2016), the matrix proteins of venomous fangs,

their evolutionary origins, and the biomineralization mechanisms of venomous

fangs are still poorly understood.

Protobothrops avoviridis (habu) snake that inhabits Ryukyu (Okinawa,

Tokunoshima, and Amami) Islands are dangerous snakes having various toxic pep-

tides and proteins (multiple protein families) as venom. Their venomous fangs are

frequently lost and drawn into their own body with the prey after injection of the

venom. Interestingly, venomous fangs are excreted with no change and no diges-

tion, whereas the bones and teeth of the mouse (prey) are completely digested.

These observations suggest that structural differences between venomous fangs and

mammalian bones and teeth exist. In addition, it is conceivable that the adaptive

evolution of the venomous organ and venomous fang bestowed them to have resis-

tance to digestive juices. Thus, the snake fangs show the unique characteristics

including mechanical strength and chemical stability.

In this study, to reveal the characteristics of habu snake fangs such as chemical

stability, and their molecular evolution, proteomic analyses of fang matrix proteins

were conducted by using 2D-PAGE and MALDI-TOF MS/MS.

T. Ogawa et al.

5.2 Materials andMethods

5.2.1 Materials

The crude venomous fangs of Protobothrops avoviridis (habu) snakes captured in

Amami Island, Kagoshima Prefecture, Japan, were collected by dissection of the

head from sacrice. Subsequently, fangs and tissues were separately rinsed with

phosphate-buffered saline and stored at −80°C until use. Immobiline DryStrip for

two-dimensional electrophoresis and the IPG buffer (pH 3–11) were obtained from

GE Healthcare UK Ltd. (Buckinghamshire, England). Silver Stain MS kit was pur-

chased from Wako Pure Chemical Industry, Ltd. (Osaka, Japan). Achromobacter

protease I and Staphylococcus aureus V8 protease were obtained from Wako Pure

Chemicals (Osaka, Japan) and Sigma-Aldrich Co. (St. Louis, MO, USA), respec-

tively. ZipTip C18 was purchased from Millipore (Massachusetts, USA). All other

reagents were of the best commercially available grade from Wako Pure Chemicals

(Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).

5.2.2 Isolation andCharacterization oftheMatrix Proteins

fromtheVenomous Fang

Venomous fangs of habu snakes were decalcied with 50% formic acid at room

temperature for 2days. Then, the decalcied matrix proteins were dissolved in 6M

guanidinium hydrochloride in 50mM Tris-HCl buffer (pH 8.8) containing 200mM

NaCl at 60°C.After TCA-acetone precipitation, the pellet was dissolved in 8M

urea in 100mM Tris-HCl buffer (pH 8.2) at 60°C.For two-dimensional polyacryl-

amide gel electrophoresis (2D-PAGE), fang matrix proteins were directly dissolved

in 400μl of rehydration buffer (8M urea, 4% CHAPS, 2% immobilized pH gradient

(IPG) buffer (pH 3-11NL), DeStreak reagent (15mg/ml), and 0.002% bromophenol

blue) and were loaded onto IPG strips. After rehydration for 12h, isoelectric focus-

ing (IEF) was performed at 20°C using the running conditions of the following

focusing program: 500V for 1h, a gradient to 1000V for 1h, a gradient to 8000V

for 3h, and 8000V for 1.5h (3225V, 50μA, 19,742Vhs). After running IEF, IPG

strips were equilibrated in a reducing equilibration buffer for 15min and subse-

quently alkylated with iodoacetamide. Then, IPG strips were transferred onto 15%

polyacrylamide gel (18 × 16cm) and embedded with 0.5% agarose and electropho-

resed. Gels were stained using Silver Stain MS kit or Coomassie Brilliant Blue.

5 Proteomic Analysis ofVenomous Fang Matrix Proteins ofProtobothrops…

5.2.3 Proteome Analysis

The spots on 2D gel were cut into pieces and washed with Milli-Q water. After the

gels were dehydrated by acetonitrile with gentle agitation and completely dried in

vacuo, gel samples were reduced by 10mM DTT for 1h at 56°C.After cooling and

washing by 25mM ammonium bicarbonate buffer for 10min, the gel samples were

treated with 55mM iodoacetamide in 25 mM ammonium bicarbonate solution in

the dark. After removal of the solvent to be completely dried, gel particles were

digested by Achromobacter protease I (Lys-C) or V8 protease at 37°C for one night.

After concentrating the digest in speed vacuum, samples were desalted on ZipTip

C18 (Millipore). Samples were separated by using a DiNa Nano LC system equipped

with a DiNa MALDI spotting device (KYA Technologies Co., Tokyo, Japan) and

applied to MALDI-TOF MS and tandem MS/MS analysis using TOF/TOF™ 5800

Analyzer (AB SCIEX). Enzyme-digested matrix proteins without 2D-PAGE were

also analyzed by nanoLC-MALDI-TOF MS/MS.Molecular masses were calibrated

using the Sequazyme Peptide Mass Standards Kit (Applied Biosystems). Protein

identication was performed by searching of each MS/MS spectrum against the

protein sequence databases derived from the RNA-seq data of P. avoviridis snake

fang-forming tissues by using ProteinPilot software (version 3.0; AB Sciex) with

the Paragon method.

5.3 Results andDiscussion

5.3.1 Isolation andCharacterization oftheMatrix Proteins

fromP. avoviridis Venomous Fangs

First, the decalcication conditions of venomous fangs were investigated by using

hydrochloric acid and formic acid, respectively. The complete decalcication of the

venomous fang without protein degradation was achieved by 50% formic acid at

30°C for 2days, resulting typical yield of 5.6mg from 1.0g of P. avoviridis fangs,

while the decalcication of fang by 10% HCl treatment caused the degradation of

proteins (Fig.5.1a). Then, the matrix proteins were subjected to the proteome analy-

sis, in-gel enzymatic digestions for mass spectrometry characterization with

2D-PAGE and the shotgun proteomics of enzymatic digestions of total matrix pro-

teins by nanoLC-MS/MS, respectively, after dissolved in 6M guanidinium at 60°C

and concentrated by TCA-acetone precipitation.

T. Ogawa et al.

Fig. 5.1 Proteomic analysis of P. avoviridis fang matrix proteins

Brief explanation of procedure through decalcication and mass spectrometry analysis (a) and

typical proles of digested matrix proteins on nanoLC-MALDI-TOF MS/MS (b)

5 Proteomic Analysis ofVenomous Fang Matrix Proteins ofProtobothrops…

5.3.2 Proteome Analysis oftheFang Matrix Proteins

To identify the array of proteins in P. avoviridis venomous fangs, the extracted

matrix proteins were subjected to the 2D-PAGE (pH 3–11), resulting in identica-

tion in acidic region of around 20 appreciable major spots, of which pI values rang-

ing from 4 to 6 (Fig.5.2). These fang matrix proteins were roughly divided into ve

groups based on the molecular mass numbers: 55kDa (sample #1), 40kDa (#2),

35kDa (#3), 30kDa (#4), and 25kDa (#5) proteins. Preliminary proteomic analy-

ses of these protein spots allowed the identication of major components of fang

matrix proteins including type I collagens alpha-1 and alpha-2 and UV excision

repair protein RAD23-like protein (Table5.1). Interestingly, antihemorrhagic factor

HSF, which is a proteinaceous serum inhibitor against own venom metalloprotein-

ases, was also detected as a matrix protein. However, these proteomic data from

2D-PAGE could not provide satisfactory results.

To improve the proteomic data of P. avoviridis fang matrix proteins, a direct

shotgun proteomic analysis was conducted. As a result of 4 independent experi-

ments of shotgun proteomics, 36 proteins were identied as fang matrix proteins

(Table 5.2). In addition to the type I collagen alpha1 (isoform X1 and X2) and

alpha2 chains, the collagens type VI alpha2 and alpha3 chains and type XI alpha1

and alpha2 chains were identied. Because type I collagen has been reported to be

related to the formation of dentin and enamel, contributing to the nanoscale archi-

tecture in the teeth (Wallace etal. 2010), type I collagen seems to be an important

55 samle #1

samle #2

samle #3

samle #4

samle #5

34 567891

(kDa

)

Fig. 5.2 2D-PAGE prole of P. avoviridis venomous fang matrix proteins

Samples #1 to #5 were analyzed by nanoLC-MALDI-TOF MS/MS analysis after in-gel digestion,

respectively

T. Ogawa et al.

Table 5.1 Proteomic data for 2D-PAGE analysis of P. avoviridis fang matrix proteins

SampleaTotal %Cov Accession Representative RNA-seq data

Peptides

(95%) Identied proteins

#2–1 2 7.390999794 m.3684 g.3684 ORF comp179282_c0_seq1:3–695(+) 1 Antihemorrhagic factor HSF

#3–1 4 7.451999933 m.304793 g.304793

ORF comp195637_c1_seq80:3108–4358(+)

2 Collagen alpha-1(I) chain isoform X1

#3–2 2 7.390999794 m.3684 g.3684 ORF comp179282_c0_seq1:3–695(+) 1 Antihemorrhagic factor HSF

#3–3 1.3 12.8700003 m.32414 g.32414 ORF comp189831_c0_seq6:1773–2078(+) 1 UV excision repair protein RAD23-like B

2 12.8700003 m.32414 g.32414 ORF comp189831_c0_seq6:1773–2078(+) 1

#4–1 2 12.30999976 m.228287 g.228287

ORF comp194729_c4_seq50:4684–5076(+)

1 Collagen alpha-2(I) chain isoform X1

aSample numbers #2 to #4 correspond to the number of 2D-PAGE spots in Fig.5.2

5 Proteomic Analysis ofVenomous Fang Matrix Proteins ofProtobothrops…

Table 5.2 Protobothrops avoviridis fang matrix proteins identied by shotgun proteomic analyses

Trial/protein

numbers Total % Cov Accession Representative RNA-seq data

Peptides

(95%) Identied proteins Similarity (%) with mice homologs

1_1 8 9.855999798 m.304793 g.304793 ORF

comp195637_c1_seq80:3108–

4358(+)

4 Collagen alpha-1(I) 85

1_2 6 12.72999942 m.59430 g.59430

ORF

comp191065_c4_seq27:514–

1506(−)

3 Decorin 72

1_3 6 18.23000014 m.228281 g.228281 ORF

comp194729_c4_seq49:3624–

4235(+)

3 Collagen alpha-2(I) 75

1_4 4 3.852000087 m.380886 g.380886 ORF

comp196768_c0_seq1:1–

1872(+)

2 Serum albumin 32

1_5 4 10.79000011 m.280478 g.280478 ORF

comp195368_c6_seq17:648–

1679(−)

2 Osteonectin (SPARC) 83

1_6 2 4.098000005 m.229771 g.229771 ORF

comp194758_c6_seq15:710–

6349(+)

1 Collagen alpha-1(XI) 83

1_7 2 16.52999967 m.3685 g.3685

ORF

comp179282_c1_seq1:1–

366(+)

1 Antihemorrhagic factor

HSF-like

–

T. Ogawa et al.

Trial/protein

numbers Total % Cov Accession Representative RNA-seq data

Peptides

(95%) Identied proteins Similarity (%) with mice homologs

1_8 2 3.260999918 m.380873 g.380873 ORF

comp196660_c0_seq1:518–

1624(+)

1 Biglycan 83

1_9 2 6.086999923 m.3684 g.3684

ORF

comp179282_c0_seq1:3–

695(+)

1 Antihemorrhagic factor

HSF

–

1_10 1.7 12.8700003 m.32414 g.32414 ORF

comp189831_c0_seq6:1773–

2078(+)

1 UV excision repair

protein RAD23-like B

1_11 0.4 8.122000098 m.15495 g.15495

ORF

comp188251_c4_seq2:1–

594(+)

0 Dual specicity protein

phosphatase 3

1_12 0.17 2.26099994 m.302599 g.302599 ORF

comp195617_c0_seq11:1965–

3692(+)

0 Protein capicua homolog 66

1_13 0.14 8.653999865 m.361713 g.361713 ORF

comp196120_c6_seq13:2390–

3328(+)

0 L1-encoded reverse

transcriptase-like protein

–

1_14 0.09 5.152000114 m.137751 g.137751 ORF

comp193200_c4_seq25:1707–

2699(+)

0 SLIT and NTRK-like

protein 6/phospholipase

A2 inhibitor-like

1_15 0.08 5.05400002 m.190269 g.190269 ORF

comp194186_c4_seq5:195–

1028(−)

0 Insulin-like growth

factor binding protein 4

2_1 4 7.451999933 m.304793 g.304793 ORF

comp195637_c1_seq80:3108–

4358(+)

2 Collagen alpha-1(I) 85

(continued)

5 Proteomic Analysis ofVenomous Fang Matrix Proteins ofProtobothrops…

Table 5.2 (continued)

Trial/protein

numbers Total % Cov Accession Representative RNA-seq data

Peptides

(95%) Identied proteins Similarity (%) with mice homologs

2_2 2 7.390999794 m.3684 g.3684

ORF

comp179282_c0_seq1:3–

695(+)

1 Antihemorrhagic factor

HSF

–

2_3 2 12.30999976 m.228287 g.228287 ORF

comp194729_c4_seq50:4684–

5076(+)

1 Collagen alpha-2(I) 75

2_4 1.4 8.147999644 m.4964 g.4964

ORF

comp182948_c0_seq1:414–

821(+)

1 Uncharacterized protein/

SOGA3-like

–

2_5 0.8 12.8700003 m.32414 g.32414 ORF

comp189831_c0_seq6:1773–

2078(+)

0 UV excision repair

protein RAD23-like B

2_6 0.05 14.00000006 m.13350 g.13350 ORF

comp187844_c0_seq6:399–

701(+)

0 EF-hand domain-

containing family

member B

3_1 6 20.2000007 m.228281 g.228281 ORF

comp194729_c4_seq49:3624–

4235(+)

3 Collagen alpha-2(I) 75

3_2 4.11 14.35000002 m.3684 g.3684

ORF

comp179282_c0_seq1:3–

695(+)

2 Antihemorrhagic factor

HSF

–

3_3 4 3.60600017 m.304793 g.304793 ORF

comp195637_c1_seq80:3108–

4358(+)

2 Collagen alpha-1(I) 85

T. Ogawa et al.

Trial/protein

numbers Total % Cov Accession Representative RNA-seq data

Peptides

(95%) Identied proteins Similarity (%) with mice homologs

3_4 2 7.97900036 m.59432 g.59432

ORF

comp191065_c4_seq31:514–

1080(−)

1 Decorin 72

3_5 2 2.408000082 m.380886 g.380886 ORF

comp196768_c0_seq1:1–

1872(+)

1 Serum albumin 32

3_6 2 12.3999998 m.3685 g.3685

ORF

comp179282_c1_seq1:1–

366(+)

1 Antihemorrhagic factor

HSF-like

–

3_7 2 0.585399987 m.229771 g.229771 ORF

comp194758_c6_seq15:710–

6349(+)

1 Collagen alpha-1(XI) 83

3_8 0.8 3.78200002 m.2124 g.2124

ORF

comp168486_c1_seq1:1–

717(+)

0 Peroxisome proliferator-

activated receptor

gamma

3_9 0.37 0.608900003 m.103143 g.103143 ORF

comp192389_c5_seq5:3–

4439(+)

0 Receptor-type tyrosine-

protein phosphatase zeta

isoform X2

3_10 0.15 1.006999984 m.314611 g.314611 ORF

comp195716_c7_seq95:1655–

4339(−)

0 Probable

methyltransferase

TARBP1

3_11 0.13 1.692000031 m.177447 g.177447 ORF

comp193931_c1_seq1:2163–

10,676(−)

0 Adenomatous polyposis

coli protein

3_12 0.09 1.070000045 m.56612 g.56612 ORF

comp190997_c1_seq12:458–

4387(+)

0 BAH and coiled-coil

domain- containing

protein 1

(continued)

5 Proteomic Analysis ofVenomous Fang Matrix Proteins ofProtobothrops…

Table 5.2 (continued)

Trial/protein

numbers Total % Cov Accession Representative RNA-seq data

Peptides

(95%) Identied proteins Similarity (%) with mice homologs

3_13 0.09 2.153999917 m.41112 g.41112

ORF

comp190336_c0_seq6:152–

2104(−)

0 Leucine-rich repeat and

bronectin type III

domain-containing

protein 1

3_14 0.09 13.72999996 m.334963 g.334963 ORF

comp195890_c6_seq76:1–

309(+)

0 Unknown –

3_15 0.07 7.086999714 m.14110 g.14110 ORF

comp187994_c1_seq2:329–

712(+)

0 39S ribosomal protein

L55

4_1 10 11.0799998 m.380886 g.380886 ORF

comp196768_c0_seq1:1–

1872(+)

5 Serum albumin 32

4_2 10 25.65000057 m.3684 g.3684

ORF

comp179282_c0_seq1:3–

695(+)

5 Antihemorrhagic factor

HSF

–

4_3 8 18.35999936 m.120416 g.120416

ORF

comp192831_c6_seq13:1–

1683(−)

9 Collagen alpha-1(I)X2 85

4_4 6 15.76000005 m.380873 g.380873 ORF

comp196660_c0_seq1:518–

1624(+)

3 Biglycan 83

4_5 6 11.77999973 m.304793 g.304793 ORF

comp195637_c1_seq80:3108–

4358(+)

5 Collagen alpha-1(I)X1 85

T. Ogawa et al.

Trial/protein

numbers Total % Cov Accession Representative RNA-seq data

Peptides

(95%) Identied proteins Similarity (%) with mice homologs

4_6 6 13.96999955 m.228235 g.228235 ORF

comp194729_c4_seq37:1874–

3379(+)

3 Collagen alpha-2(I) 75

4_7 4 13.84000033 m.68810 g.68810 ORF

comp191390_c3_seq4:350–

1024(+)

2 Transferrin-like 26

4_8 4 9.329000115 m.280478 g.280478 ORF

comp195368_c6_seq17:648–

1679(−)

2 Osteonectin (SPARC) 83

4_9 2 7.213000208 m.237824 g.237824 ORF

comp194854_c3_seq3:504–

3542(+)

1 Collagen alpha-2(VI) 55

4_10 2 2.072999999 m.85240 g.85240 ORF

comp191877_c1_seq9:1401–

8060(+)

1 Collagen alpha-3(VI) 55

4_11 2 1.752999984 m.164214 g.164214 ORF

comp193681_c1_seq3:3–

4967(+)

1 Venom factor –

4_12 2 5.085000023 m.135289 g.135289

ORF

comp193175_c0_seq8:3–

1949(−)

1 Dentin matrix acidic

phosphoprotein 1-like

4_13 2 7.97900036 m.59432 g.59432

ORF

comp191065_c4_seq31:514–

1080(−)

1 Decorin 72

4_14 2 12.8700003 m.32414 g.32414 ORF

comp189831_c0_seq6:1773–

2078(+)

1 UV excision repair

protein RAD23-like B

(continued)

5 Proteomic Analysis ofVenomous Fang Matrix Proteins ofProtobothrops…

Table 5.2 (continued)

Trial/protein

numbers Total % Cov Accession Representative RNA-seq data

Peptides

(95%) Identied proteins Similarity (%) with mice homologs

4_15 2 6.25 m.209089 g.209089 ORF

comp194475_c7_seq8:944–

1474(+)

1 Galectin-9-like 61

4_16 1.7 13.0400002 m.1291 g.1291

ORF

comp137400_c0_seq1:563–

979(−)

1 Unconventional

myosin-Ie

–

4_17 1.52 13.24999928 m.148536 g.148536 ORF

comp193401_c3_seq19:317–

1474(+)

1 Actin, cytoplasmic 1 99

4_18 0.64 2.26099994 m.302599 g.302599 ORF

comp195617_c0_seq11:1965–

3692(+)

0 Protein capicua homolog 66

4_19 0.47 0.630899984 m.259829 g.259829 ORF

comp195117_c3_seq24:1–

5709(+)

0 Collagen alpha-2(XI) 66

4_20 0.06 2.785000019 m.198369 g.198369

ORF

comp194299_c9_seq1:2–

2371(−)

0 Titin-like 31

T. Ogawa et al.

component of venomous snake fang. Furthermore, the type VI collagen, which

forms microbrils and is primarily associated with the extracellular matrix of skel-

etal muscle and bone marrow, and type XI collagen, which is found in the cartilage

of the nose and external ears in human, were also identied as matrix proteins in

venomous fang, suggesting the unique distribution of type VI and type XI collagens

as part of the fang matrix. On the other hand, noncollagenous dentin matrix proteins

including proteoglycans (PGs), glycoproteins, serum proteins, enzymes, and growth

factors are deemed to play structural, metabolic, and functional roles as key compo-

nents in the mineralization process of dentin (Orsini etal. 2009). Shotgun proteomic

analysis showed the fang noncollagenous dentin matrix proteins include proteogly-

can such as decorin (1_2, 3_4, 4_13in Table5.2) and biglycan (1_8, 4_4), glyco-

proteins such as osteonectin (secreted protein acidic and rich in cysteine: SPARC)

(1_5, 4_8), the SIBLING proteins such as dentin matrix acidic phosphoprotein 1

(4_12), and serum proteins such as albumin (1_4, 3_5, 4_1), phospholipase A2

inhibitor (1_14) and antihemorrhagic factors, HSF (1_9, 2_2, 3_2, 4_2), and HSF-

like protein (1_7, 3_6). The coexistence of these serum inhibitors as fang matrix

proteins explains why venomous fang is stable against own venom enzymes com-

pared with mouse-derived teeth and bones. Compared with the homologous pro-

teins in mouse, several fang matrix proteins such as dentin matrix acidic

phosphoprotein 1 (36%), titin-like protein (31%), transferrin-like protein (26%),

and serum inhibitors including albumin (32%) and PLA2 inhibitor (29%) showed

lower sequence similarities, suggesting that these differences in matrix proteins

might be related to the functional differences and distinctive properties between

venomous fang and mouse’s teeth.

In this study, we identied 36 matrix proteins from P. avoviridis snake fangs by

proteomics analyses. They include proteinaceous inhibitor against own venom

enzymes in addition to several types of collagens (types I, VI, and XI) and noncol-

lagenous dentin matrix proteins. More recently, we have decoded the whole genome

sequence of P. avoviridis snakes (Shibata etal. 2018, in press). Further investiga-

tions are needed to elucidate the biomineralization mechanisms of venomous fang

and their biological functions.

Acknowledgments The authors thank Prof. Noriyuki Satoh, Drs. Shinichi Yamasaki and Kanako

Hisata, Okinawa Institute of Science and Technology Graduate University (OIST), Onna, Okinawa,

for providing RNA-seq data from P. avoviridis fang-forming tissues.

This study was partly supported by Grants-in-Aid of MEXT, Japan (#24651130 and #23107505

to TO). This study was also partly performed in the collaborative Research Project Program of the

Medical Institute of Bioregulation, Kyushu University.

References

Jackson K (2002) How tubular venom-conducting fangs are formed. J Morphol 252:291–297

Kardong KV (1979) Protovipers and the evolution of snake fangs. Evolution 33:433–443

Kuch U, Müller J, Mödden C, Mebs D (2006) Snake fangs from the Lower Miocene of Germany:

evolutionary stability of perfect weapons. Naturwissenschaften 93:84–87

5 Proteomic Analysis ofVenomous Fang Matrix Proteins ofProtobothrops…

Orsini G etal (2009) A review of the nature, role, and function of dentin non-collagenous proteins.

Part 1: Proteoglycans Glycoproteins 21:1–18

Savitzky AH (1980) The role of venom delivery strategies in snake evolution. Evolution

34:1194–1204

Shibata H., Chijiwa T., Oda-Ueda N., Nakamura H., Yamaguchi K., Hattori S., Matsubara K.,

Matsuda Y., Yamashita A., Isomoto A., Mori K., Tashiro K., Kuhara S., Yamasaki S., Fujie M.,

Goto H., Koyanagi R., Takeuchi T., Fukumaki Y., Ohno M., Shoguchi E., Hisata K., Satoh N.,

and Ogawa T (2018) The habu genome reveals accelerated evolution of venom protein genes.

Sci Rep (in press). https://doi.org/10.1038/s41598-018-28749-4

Vonk FJ, Admiraal JF, Jackson K, Reshef R, de Bakker MA, Vanderschoot K, van den Berge I, van

Atten M, Burgerhout E, Beck A (2008) Evolutionary origin and development of snake fangs.

Nature 454:630–633

Vonk FJ etal (2013) The king cobra genome reveals dynamic gene evolution and adaptation in the

snake venom system. Proc Natl Acad Sci U S A 110:20651–20656

Wallace JM etal (2010) Type I collagen exists as a distribution of nanoscale morphologies in teeth,

bones, and tendons. Langmuir 26:7349–7354

Yin W etal (2016) Evolutionary trajectories of snake genes and genomes revealed by compara-

tive analyses of ve-pacer viper. Nat Commun 7:13107. https://doi.org/10.1038/ncomms13107

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

T. Ogawa et al.

55© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_6

Chapter 6

Characterization ofGoldsh Scales

byVibrational Spectroscopic Analyses

MasayukiNara, YusukeMaruyama, andAtsuhikoHattori

Abstract Scales of bony shes are calcied tissues that contain osteoblasts, osteo-

clasts, and bone matrix, all of which are similar to those found in mammalian bone.

The scales are composed of hydroxylapatite (HAP) and extracellular matrix which

is mainly type I collagen bers. We investigated the scales from goldsh by Fourier

transform infrared (FTIR) and Raman spectroscopic analyses to characterize the

components in the scales. The attenuated total reection (ATR)-FTIR spectrum

obtained from the surface coat of the normal goldsh scale was quite different from

that of the backside coat of the same scale. The former showed a strong band at

1013cm−1, which was assignable to HAP, and weak bands at about 1643, 1415, and

870cm−1, whereas the latter showed a typical protein prole: strong bands at about

1631, 1550, and 1240cm−1, which were assignable to amide-I, amide-II, and amide-

III of collagen, respectively. We also investigated the local structure of goldsh

scales by using micro-Raman spectroscopy. The Raman spectrum of the normal

scale showed the amide-I band at about 1640cm−1 from the collagen and the PO43−

symmetric stretching band at about 961cm−1 from the HAP.We discuss the implica-

tion of Raman and FTIR proles for normal and regenerating goldsh scales.

Keywords Vibrational spectroscopy · Goldsh scale · Regeneration ·

Hydroxylapatite · Carbonate apatite

6.1 Introduction

Goldsh scale is a calcied tissue that contains osteoblasts, osteoclasts, and bone

matrix, all of which are similar to those found in mammalian bone (Azuma etal.

2007). The scale is composed of hydroxyapatite (HAP) and extracellular matrix

which is mainly type I collagen bers, forming a highly ordered three-dimensional

structure. It has been known that scales regenerate after being removed. Therefore,

M. Nara (*) · Y. Maruyama · A. Hattori

College of Liberal Arts and Sciences, Tokyo Medical and Dental University (TMDU),

Tokyo, Japan

e-mail: nara.las@tmd.ac.jp; ahattori.las@tmd.ac.jp

the goldsh scale is interesting from the viewpoint of the model system for the

regeneration of mammalian bone.

Vibrational spectroscopy is useful for characterizing developmental changes in

bone and other mineralized tissues. Infrared spectroscopy is used for examining the

mineral properties and collagen maturity of bones (Boskey and Mendelsohn 2005;

Paschalis etal. 2011) as well as protein structures such as collagen and osteocalcin

(Jackson etal. 1995; Barth 2007; Mizuguchi et al. 2001). Raman spectroscopy is

also available for bones and sh scales to provide information about protein and

mineral components (Ikoma etal. 2003a, b).

In the present study, FTIR and Raman spectroscopic analyses were applied to

scales from goldsh to characterize the components of the normal and regenerating

ones. In particular, we focused on the pocket side of them, since the epidermal side

of scales is more complicated because of the contribution of chromatophore.

6.2 Materials andMethods

We prepared normal scales and regenerating scales after 1 week and 2 weeks,

extracted from goldshes (Carassius auratus). As can be seen in Fig.6.1, the pic-

ture of the regenerating scale after 2weeks was morphologically different from that

of the normal scale. There were about ten circular ridges for regenerated scales after

2weeks, although a lot of circular ridges were observed for normal scales.

Attenuated total reection (ATR)-Fourier transform infrared (FTIR) measure-

ments were performed at 25°C on a PerkinElmer Spectrum-One FTIR spectrometer

equipped with a universal ATR unit and a liquid nitrogen-cooled MCT detector at

resolution 2cm−1. A scale sample was placed on a diamond/ZnSe 1-reection top-

plate (PerkinElmer). The sampling depth of the ATR method was approximately

1–2μm over the range of 2000–1000cm−1. We obtained the ATR-FTIR spectrum

for the spot (about 1.5mm in diameter) with the accumulations of 200 scans.

Fig. 6.1 Pictures of a normal scales (left) and a regenerating scale after 2weeks (right) of goldsh

(Carassius auratus)

M. Nara et al.

Raman spectra were collected at room temperature (25°C) with a Raman micro-

scope (Kaiser HoloLab 5000 of Kaiser Optical System Inc.) using 532nm Nd-YAG

laser (2–5mW at the sample surface), holographic transmission grating, and charge-

coupled device (CCD). The spectral resolution in the present system is approxi-

mately 4.8 cm−1. We obtained the Raman spectrum for a spot (about 2 μm in

diameter) with two accumulations of 30s each. The sampling depth of the Raman

measurement was about 100μm. A scale was set on a slide glass covered with alu-

minum foil, and the pocket sides of the surface were measured by Raman

spectroscopy.

6.3 Results andDiscussion

6.3.1 Characterization ofaNormal Scale fromGoldsh

byFTIR Spectra

Figure 6.2 shows ATR-FTIR spectra of the surface and backside coats of a normal

goldsh scale in the pocket side. Figure6.2a showed a strong band at 1013cm−1,

which was assignable to HAP, and weak bands at 1647, 1546, 1415, and 871cm−1.

The bands at 1647 and 1546cm−1 were probably assignable to proteins. The bands

Absorbance

2000 1800 1600 1400 1200 1000

800

Wavenumber / cm

-1

16311550

1453

1401 1240

10811033

1338 1203

1013

871

164715461415

0.05

Fig. 6.2 ATR-FTIR spectra from (a) the surface coat and (b) the backside coat of the normal

goldsh scale

6 Characterization ofGoldsh Scales byVibrational Spectroscopic Analyses

at 1415 and 871cm−1 were assigned to carbonate (CO32−). Figure6.2b showed a

protein prole of collagen: strong bands at 1631, 1550, and 1240cm−1, which are

undoubtedly assignable to amide-I, amide-II, and amide-III, respectively.

Figure 6.3a, d shows the ATR-FTIR second-derivative spectra of the correspond-

ing spectra in Fig.6.2. The second-derivative spectrum of surface coat showed the

bands at 1654, 1015, 981, and 781cm−1 in Fig.6.3a, whereas that of the backside

coat showed three bands at 1696, 1652, and 1625cm−1 in the region of amide-I, one

band at 1550cm−1 in the region of amide-II, and one band at 1240cm−1. Obviously,

the spectral prole for the surface coat reected the HAP as the main component of

the calcied layer, while the spectral prole for the back coat reected the collagen

as the main component of the brillary layer. The amide-I band observed from the

surface coat may be due to proteins other than collagen, since the prole was differ-

ent from that of collagen. The bands at 1415 and 871cm−1 are probably originated

from CO32− (Ikoma etal. 2003b).

-d

(Abs.) / d (Wave.)

2000 1800 1600 1400 1200 1000 80

Wavenumber / cm

-1

16541015 870

1017

984

1125

1630

1657

1014

874

1119

1627

1655 1553 1240

875

1692

Fig. 6.3 ATR-FTIR second-derivative spectra of (a–c) the surface coat and (d–f) the back coat in

the pocket side of three type of scales from goldsh: (a, d) a normal scale, (b, e) a regenerating

scale after 1week, and (c, f) a regenerating scale after 2weeks. The second derivatives were mul-

tiplied by −1

M. Nara et al.

6.3.2 Characterization ofRegenerating Scales fromGoldsh

byFTIR Spectra

Figure 6.3b, e shows the second-derivative spectra of the surface and back coats of

the regenerating scale after 1week in the pocket side, respectively. The band at

1017cm−1 caused by HAP in Fig.6.3b was quite sharper than the corresponding

band of normal scale in Fig.6.3a. The spectral prole of the amide-I region was

similar to that of the back coat, which suggested that the ATR spectra reected the

brillary layer as well as the calcied layer, since the thickness of the calcied one

was less than 2μm. The spectral prole of the back coat of regenerating scales after

1week (Fig.6.3e) was almost the same as that of the back coat of a normal scale

(Fig.6.3d).

Figure 6.3c, f shows the second-derivative spectra of the surface and back coats

of the regenerating scale after 2weeks in the pocket side, respectively. The absor-

bance of amide-I band was weaker than that of Fig.6.3b, which suggested that the

calcied layer became thicker. The spectral prole of the back coat was almost the

same as that of normal scale (Fig.6.3d).

It was difcult to obtain an infrared spectrum of sh scale by using transmission

mode, because the thickness of the normal scale is >100μm and the absorbance of

most interesting bands was saturated. ATR measurements have an advantage over

transmission measurement, because ATR technique makes it possible to obtain not

only the spectral proles of sh scales but also the information about the surface and

backside coats of the scales separately. Therefore, ATR-FTIR spectroscopy is prom-

ising for monitoring HAP and collagen components during the regeneration process

of goldsh scale.

6.3.3 Raman Spectra fromNormal andRegenerating Scales

ofGoldsh

Figure 6.4a, b shows Raman spectrum from the area between adjacent circular

ridges in a normal scale. The bands at about 1669cm−1 and 1242cm−1 were assign-

able to collagen amide-I and amide-III, respectively, and the intense band at about

961cm−1 was originated from the PO43− symmetric stretching band for HAP (Penel

etal. 1998). The intensity at 961cm−1 was quite weak in the most outer part of the

circular ridges, suggesting that HAP layer is not formed or thin on the collagen lay-

ers. The band at 1070 cm−1 was observed at the center of the scale, which was

caused by type-B carbonate apatite (Awonusi etal. 2007). This interpretation was

also conrmed by the FTIR bands at 1415 and 871cm−1 (Fig.6.2a). Furthermore,

we found that the HAP layer became slightly thicker along the ridge line than the

area between adjacent circular ridges (data not shown).

6 Characterization ofGoldsh Scales byVibrational Spectroscopic Analyses

Figure 6.4c–e shows the Raman spectra from the area between adjacent circular

ridges in regenerating scales after 2weeks. The spectral proles of the regenerating

scales were similar to those of the normal ones except for the band at 961cm−1,

which were weaker than that of normal scale (Fig.6.4a, b). The intensities of the

band at 961cm−1 were the order (c) < (d) < (e). This result demonstrated that the

HAP layers were formed from the center part to the outer part during the regenera-

tion process of the scale.

Micro-Raman spectroscopy is a useful method for understanding the local com-

ponents of goldsh scale because Raman spectroscopic method has higher resolu-

tion in the area compared with FTIR spectroscopic method. ATR-FTIR provides

information regarding the surface component, because the sampling depth of Raman

measurements was 100μm. Therefore, Raman spectroscopy in combination with

ATR-FTIR spectroscopy is powerful for analyzing HAP and carbonate apatite of the

regenerating scales.

References

Awonusi A, Morris MD, Tecklenburg MMJ (2007) Carbonate assignment and calibration in the

Raman spectrum of apatite. Calcif Tissue Int 81:46–52

Azuma K, Kobayashi M, Nakamura M, Suzuki N, Yashima S, Iwamuro S, Ikegame M, Yamamoto

T, Hattori A (2007) Two osteoclastic markers expressed in multinucleate osteoclasts of goldsh

scales. Biochem Biophys Res Commun 362:594–600

Barth A (2007) Infrared spectroscopy of proteins. Biochim Biophys Acta 1767:1073–1101

Intensity

1800 1600 1400 1200 1000 800

Raman shift / cm

-1

959

1242

1665

1666

1070

961

1669

958

1242

1240

Fig. 6.4 Raman spectra

from (a, b) the normal

goldsh scale and from

(c–e) the regenerating scale

after 2weeks. The spot

position for (a, c) were on

the outer part of the scale

in the pocket side, and the

spot positions for (b, d, e)

were on the inner part of

the scale in the pocket side.

The spot position on (d)

was in the middle of (c, e)

M. Nara et al.

Boskey AL, Mendelsohn R (2005) Infrared spectroscopic characterization of mineralized tissues.

Vib Spectrosc 38:107–114

Ikoma T, Kobayashi H, Tanaka J, Walsh D, Mann S (2003a) Microstructure, mechanical, and bio-

mimetic properties of sh scales from Pagrus major. JStruct Biol 142:327–333

Ikoma T, Kobayashi H, Tanaka J, Walsh D, Mann S (2003b) Physical properties of type I collagen

extracted from sh scales of Pagrus major and Oreochromis niloticus. Int JBiol Macromol

32:199–204

Jackson M, Choo LP, Watson PH, Halliday WC, Matsch HH (1995) Beware of connective tissue

proteins: assignment and implications of collagen absorptions in infrared spectra of human tis-

sues. Biochim Biophys Acta 1270:1–6

Mizuguchi M, Fujisawa R, Nara M, Kawano K, Nitta K (2001) Fourier-transform infrared spectro-

scopic study of Ca2+-binding to osteocalcin. Calcif Tissue Int 69:337–342

Paschalis EP, Mendelsoln R, Boskey AL (2011) Infrared assessment of bone quality: a review. Clin

Orthop Relat Res 469:2170–2178

Penel G, Leroy G, Rey C, Bres E (1998) Micro Raman spectral study of the PO4 and CO3 vibra-

tional modes in synthetic and biological apatites. Calcif Tissue Int 63:475–481

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

6 Characterization ofGoldsh Scales byVibrational Spectroscopic Analyses

63© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_7

Chapter 7

Relationship Between Bone Morphology

andBone Quality inFemale Femurs:

Implication forAdditive Risk ofAlternative

Forced Molting

NatsukoIshikawa, ChihiroNishii, Koh-enYamauchi, HiroyukiMishima,

andYoshikiMatsumoto

Abstract Calcium (Ca) storage in bone has a relationship with eggshell produc-

tion. Forced molting by feeding restriction for older hens improves eggshell quality

but leads to a decline in the bone quality. Dietary minerals improve the eggshell and

bone quality; however, the effect on bone quality post-molting has not been clari-

ed. This study evaluated the effects of dietary Ca and minerals on the eggshell and

bone quality during both pre- and post-molting periods. The bone quality was evalu-

ated by measurement of bone density and Fourier transform infrared spectroscopy

(FT-IR) analysis. The eggshell quality was evaluated by morphological observation

of the mammillary cores by scanning electron microscope and FT-IR analysis. The

high Ca concentration feed group showed a low bone density post-molting and a

high carbonate/phosphate ratio pre-molting. In high mineral concentration feed

group, the eggshell strength, the thickness, and the proportion of large mammillary

core areas were signicantly higher (p < 0.05) than control group post-molting.

These results suggest that the eggshell strength increases as the proportion of mam-

millary core areas increases. Furthermore, the high carbonate/phosphate ratio pro-

moted a decrease in bone density. Therefore, the concentration of dietary mineral is

strongly related with the maintenance of eggshells and bone quality post-molting.

Keyword Hens · Fourier transform infrared spectroscopy (FT-IR) · Scanning

electron microscope (SEM) · Carbonate/phosphate ratio (C/P ratio) · Bone density

· Mammillary cores · Calcium(Ca)

N. Ishikawa · C. Nishii · K.-e. Yamauchi · Y. Matsumoto (*)

Faculty of Agriculture, Kagawa University, Miki, Japan

e-mail: myoshiki@ag.kagawa-u.ac.jp

H. Mishima

Department of Dental Engineering, Tsurumi University School of Dental Medicine,

Yokohama, Japan

7.1 Introduction

A high egg laying ratio of laying hens is maintained by the metabolic mechanism of

calcium (Ca) which is the main ingredient of eggshell. Approximately 70% of Ca

derived from feed is accumulated in the marrow bones, a characteristic feature of

birds, and approximately 68% of Ca in the bones is used for eggshell formation

(Itoh 1971). Therefore, Ca storage in bones has a direct relationship with eggshell

production. Consequently, the decline in Ca metabolism of the aging hens which

leads to an increase in the number of broken eggs is a key issue in the poultry indus-

try. In Japan, forced molting by feeding restriction of hens is applied in the latter

stages of the laying period to improve the hen-day egg production and the eggshell

quality and to extend the laying period (Roland and Bushong 1978). However a

decrease in bone quality and bone density is caused by forced molting (Mazzuco

and Hester 2005). Therefore, it is necessary to address the decline of bone quality in

post-molting. Several studies have reported that dietary minerals and the particle

size of Ca can lead to an improvement in the bone and eggshell quality (Calvo etal.

1982; Guinotte etal. 1991; Mekada etal. 1976). However, the effect on bone quality

in post-molting has not yet been claried. This study evaluated the effects of the

particle size of Ca and the mineral components contained in feed on the eggshell

and bone quality in fasting hens during pre- and post-molting periods.

7.2 Material andMethod

7.2.1 Breeding Test

Sixteen egg-laying hens (Julia) were equally divided into four feeding groups, a

control group (Group C) and three experimental groups (Group D, E, and F): Ca

concentration (CaCO3) was 3.8% CaCO3 for Group C and 4.6% CaCO3for each

experimental group by weight. CaCO3 particle size was 40% CaCO3 of 1–1.4mm

diameter for Groups C, D, and F.Group E was fed with powdered CaCO3 less than

0.3mm diameter. All groups, except Group F, received the same concentration of

mineral additives, a mineral premix containing manganese sulfate, iron (III) sulfate,

zinc sulfate, copper (II) sulfate, calcium iodate, and cobalt (II) sulfate. Group F

received a richer mineral concentration.1 The hens were fed from the age of

160daysold. Feed was given every day and there was no restriction on water intake

by the hens. Forced moltingby feeding restriction was applied for 8days from the

age of 503days oldto 510daysold. After forced molting, hens were refeed according

to their group’s respective feeding specication. All experiments were conducted in

1 The amount of minerals added to feed is proprietary information and subject to a condentiality

agreement.

N. Ishikawa et al.

accordance with the regulations of the Kagawa University Animal Care and Use

Committee.

7.2.2 Sample Collection

When the sample hens were 458days old, two hens from each groups (a total of

eight hens) were slaughtered, and femur samples were collected. The samples were

regarded as pre-molting samples (PRE). When the hens were 512days old, two hens

from each groups (a total of eight hens) were slaughtered after 24h of refeeding,

and their femur samples were collected. These samples were regarded as post-

molting samples (POST). The femur heads were xed in 4% paraformaldehyde

phosphate buffer solution (pH 7.4) and demineralized with 0.5M EDTA (pH 8.0)

for 3weeks. Femur heads were embedded in parafn and sectioned to obtain 10μm

sections.

7.2.3 Bone Density

The sections were stained by the Azan-Mallory method. The trabecular and bone

marrow areas in femoral cancellous bone were observed at 10× magnication under

a uorescence microscope (Keyence Corp., Japan), and their central parts, 5500μm

away from periosteum, were analyzed using the software WinROOF version 7.4

(Mitani Corp., Japan). The area ratio of the trabecular to total area was determined

as bone density.

7.2.4 Fourier Transform Infrared Analysis oftheFemoral

Cortex

The qualitative characteristics of the femoral cortex were determined using Fourier

transform infrared spectroscopy (FT-IR) (Jasco Corp., Japan) analysis. The femoral

cortex was powdered and mixed with potassium bromide (KBr). Spectroscopic

analysis was based on two parameters: the calcication and the carbonate/phos-

phate ratio (C/P ratio) of the samples, both in PRE and POST.The calcication was

calculated as the ratio of the area of the phosphate bands (900–1200cm−1) to the

area of the amide I bands (1585–1720cm−1). The C/P ratio was calculated as the

ratio of the area of the carbonate bands (850–890cm−1) to that of the phosphate

bands (900–1200cm−1) (Boskey and Camacho 2007).

7 Relationship Between Bone Morphology andBone Quality inFemale Femurs…

7.2.5 Eggshell Quality Test

The eggshell quality tests were performed twice in both PRE and POST.Eggshell

thickness was determined three times on the eggshell equator for eight eggs per

group. Eggshell strength was determined by an eggshell strength meter (Fujihira

Industry Co., Ltd., Japan). The eggshells of 501 and 680daysold hens were used

as morphological samples. The eggshells were cut into about 5mm squares, and

the eggshell membrane was dissolved with a mixture of6% sodium hypochlorite,

4.12% sodium chloride, and 0.15% sodium hydroxide (Radwan etal. 2010). The

mammillary cores were observed at 200× magnication under a scanning electron

microscope (SEM) (JCM-6000: JEOL Ltd., Japan). The areas of approximately

100 mammillary cores were analyzed per piece, and 4 pieces per group were

examined. Thereafter, the histogram of the areas of the mammillary cores was

produced.

In the FT-IR analysis, the eggshells, including their membranes, were powdered

and mixed with KBr. Spectroscopic analysis was calculated using the ratio of the

peak at873cm−1 to the peak at 713 cm−1, a parameter to evaluate the carbonate

purity of the eggshells (Rodriguez-Navarro etal. 2015).

7.2.6 Statistics

Statistical processing was done using the IBM SPSS version 19 software (IBM

Corp., USA). The signicant differences among the groups were analyzed by the

Tukey’s HSD and Games-Howell post hoc tests. The level of signicance was set at

5%, and the trend level was set at 10%.

7.3 Results

7.3.1 Bone Density

The trabecular areas in POST from all groups were signicantly lower than those in

PRE (p<0.05). The area ratio of the trabecular to total area in PRE did not differ

between any groups. However, the area ratio of the trabecular to total area in POST

was signicantly lower in Groups D and E than those in Groups C and F (p<0.05)

(Fig.7.1a, b).

N. Ishikawa et al.

7.3.2 Fourier Transform Infrared Analysis ofFemoral Cortex

The calcication of the femoral cortex in Group F signicantly decreased in POST

(p<0.05), whereas that in the other groups showed a decreasing trend (p< 0.1)

(Fig.7.2a). The C/P ratio of Groups D and E were signicantly higher than Group

F in PRE (p<0.05) (Fig.7.2b).

7.3.3 Egg Quality Test

Eggshell strength in Group F was signicantly higher than those in Group C in POST

(p< 0.05) (Fig. 7.3a). Eggshell thickness in Group F was signicantly higher than

those in Groups C and E in POST (p<0.05) (Fig.7.3b). The mammillary cores were

observed as shown in Fig.7.4a, andthe proportions of the area of the eggshell mam-

millary cores did not differ in any group in PRE(Fig. 7.4b). There was a signicant

difference between Group C and Group F in POST with respect to their mammillary

core areas larger than 6000μm2 (p < 0.05)(Fig. 7.4b). The ratio of the peak at 873cm−1

to the peak at713cm−1 did not differ in any groups in PRE and POST (Fig.7.5).

Fig. 7.1 (a) The trabecular and bone marrow areas in PRE and POST (black area, trabecular area;

gray area, bone marrow area; white area, background). (b) The area ratio of the trabecular to total

area in PRE and POST (a, b bars with different superscripts within a group are different at p<0.05,

n=4)

7 Relationship Between Bone Morphology andBone Quality inFemale Femurs…

Fig. 7.3 (a) Eggshell strength in PRE and POST (a, b bars with different superscripts within a

group are different at p<0.05, n=16). (b) Eggshell thickness in PRE and POST (a, b bars with

different superscripts within a group are different at p<0.05, n=16)

Fig. 7.2 (a) The calcication of the femoral cortex (a, b bars with different superscripts within a

group are different at p<0.05, n=6). (b) The C/P ratio of the femoral cortex (a, b bars with dif-

ferent superscripts within a group are different at p<0.05, n=6)

N. Ishikawa et al.

7.4 Discussion

In this study, we focused on the effects of themineral components and the particle

size of CaCO3 contained in feed andwe investigated the morphology and quality of

bone and eggshell in PRE and POST.The area ratio of the trabecular to total area is

an index for evaluating bone density (Hagino, 2005). It was thought that a decrease

in bone density of the femoral cancellous bone was due to the inuence of forced

molting because of the area ratio of the trabecular to total area diminished in all

groups in POST (Fig.7.1b). Thus, three experimental groups’ feedsdid not sup-

press the decline of the bone density.

Calcication is an index for evaluating bone strength, and high calcication indi-

cates that the storage function of mineral in bone is high (Boskey and Camacho

2007). The decline of bone strength of the femoral cortex was also caused by the

impact of forced molting because the calcication decreased in all groups in POST

Fig. 7.4 (a) SEM microphotographs of eggshell mammillary core in PRE and POST. (b)

Histogram of the areas of mammillary cores in PRE and POST (n=4)

Fig. 7.5 The ratio of the

peak at873cm−1to the

peak at713cm−1 (a

parameter to evaluate the

carbonate purity of the

eggshell) in PRE and

POST (n=6)

7 Relationship Between Bone Morphology andBone Quality inFemale Femurs…

(Fig.7.2a). Furthermore, when the hardness of the part of the cortex of the femoral

diaphysis was determined by Vickers hardness tester, the result of bone hardness

and bone calcication were linked (data not shown), and further analysis is neces-

sary about bone strength.

The C/P ratio is used as an index of the purity of bone minerals, and a low C/P

ratio indicates a high degree of crystal purity (Malluche etal. 2012). The nding of

this study suggested that crystal purity was low in PRE in Groups D and E (Fig.7.2b).

The main constituent of bone minerals are present in the bone as calcium phosphate

and undergo a phase change to convert to stable apatite due to maturation. Apatite

in bones replaces ions, such as carbonate ions(CO32−) and phosphateions, in the

crystal lattice. Apatite containing a lot of CO32− is reported to have high solubility

and increase bone dissolution (Suetsugu 1996; Gourion-Arsiquaud et al. 2012).

This suggested that the solubility of apatite was high in Groups D and E in PRE

because a ratio of carbonate in Groups D and E were higher than Group F.Therefore,

we think that the reduction of bone density was promoted further in Groups D and

E in POST.These differences were thought to be due to the difference in the ratio of

Ca and other mineral concentration in feed.

Eggshell strength and thickness in Group F were signicantly higher than those

in Group C in POST (Fig.7.3a, b). Since zinc and manganese contained in mineral

additives in feed have a relationship in eggshell formation, it was conclusive that the

high mineral concentration feed improves eggshell quality and supports the previ-

ous research which mineral addition in feed improve eggshell quality (Mekada etal.

1976). Eggshell is formed by mammillary core formation on the eggshell membrane

and deposition of Ca as a starting point from the mammillary cores. It was reported

that as the number of large mammillary cores where several mammillary cores

fused increases, the eggshell strength increases (Solomon 2010;Stefanello et al.

2014). As a result of the large number of mammillary core areas larger than 6000μm2

in POST, it was thought that the eggshell strength was linked the size of mammillary

cores (Fig.7.4b). Therefore, in addition to evaluating conventional eggshell strength,

the morphological observation of the mammillary cores which is the microstructure

of eggshell suggested that it is a new means to evaluate eggshell quality. The mecha-

nism of action which mineral components in feed change the form of eggshell struc-

ture requires further investigation.

In the FT-IR analysis of eggshells, it was reported that the purity of carbonate

changes during eggshell formation (Rodriguez-Navarro etal. 2015).We examined

if the eggshell crystal quality could be evaluated after laying, however, theinuence

of feed on eggshell crystal quality was not detected.

The present result suggested that high mineral concentration feed improved

eggshell quality, and high Ca concentration feed promoted the reduction of bone

density in POST.In conclusion, the present study has demonstrated that the ratio

of theminerals except Ca and theCa concentration in the feed is important for

maintenance of eggshell quality and bone quality in pre- and post-molting. It is

necessary to analyze the bone metabolism in order to investigate the effect of feed

in the future.

N. Ishikawa et al.

Acknowledgments This study was supported by Ise Foods, Inc., Japan, and Nichiwa Sangyo Co.,

Ltd., Japan, by providing hens and feeds.

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Rodriguez-Navarro AB, Marie P, Nys Y, Hincke MT, Gautron JL (2015) Amorphous calcium car-

bonate controls avian eggshell mineralization: a new paradigm for understanding rapid egg-

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

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7 Relationship Between Bone Morphology andBone Quality inFemale Femurs…

73© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_8

Chapter 8

Spectroscopic Investigation ofShell

Pigments fromtheFamily Neritidae

(Mollusca: Gastropoda)

ToshiyukiKomura, HiroyukiKagi, MakikoIshikawa, ManaYasui,

andTakenoriSasaki

Abstract Molluscan shells display a wide variety of pigmentation patterns. The

diversity in molluscan shell color reects the variety of different chemical species

in the shell surface. Chemical characteristics of molluscan shell pigments have been

extensively investigated, and compounds including porphyrins, polyenes, and mela-

nins were identied as shell pigments. Here, we investigated shell pigments in 24

species in the family Neritidae using Raman spectroscopy. An excitation wave-

length of 514.5nm revealed two types of Raman spectra. One was characterized by

two peaks ranging in wavenumber from 1100–1200 to 1500–1600cm−1, which indi-

cate the presence of polyenes. Another type remained unassigned, implying the

presence of other pigments such as porphyrins or melanins. The Raman spectra

indicated a different distribution of the two types of pigments in shells. The patterns

of the Raman spectra had no obvious relationship with taxonomical classication

lower than the genus level and typical habitats. Measurement of the Raman spec-

trum at an excitation wavelength of 442nm suggested that the wavelength can dis-

tinguish polyenes from other types of pigments.

T. Komura (*) · H. Kagi

Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

e-mail: komura@eqchem.s.u-tokyo.ac.jp; kagi@eqchem.s.u-tokyo.ac.jp

M. Ishikawa

Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Faculty of Animal Health Technology, Yamazaki Gakuen University, Hachioji, Tokyo, Japan

e-mail: maki.ishikawa.gm@gmail.com

M. Yasui

Department of Resources and Environmental Engineering, School of Creative Science and

Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan

e-mail: mana@aoni.waseda.jp

T. Sasaki

The University Museum, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

e-mail: sasaki@um.u-tokyo.ac.jp

Keywords Mollusc · Neritidae · Shell pigment · Polyene · Porphyrin · Raman

spectroscopy

8.1 Introduction

The phylum Mollusca is the second largest taxon in the animal kingdom. It is very

diverse in size, shape, color, habitats, and other traits. The diverse pigmentation pat-

terns on shells are a key trait. Diversity in molluscan shell color reects the wide

variety of chemical species included in the shell. Investigating the origin of shell

pigmentation is very important to understand the evolutionary history of Mollusca

(Williams 2017). Furthermore, chemical speciation of shell pigment molecules

could provide insight into the interaction of pigment molecules with other shell

components (Hedegaard etal. 2006), which is important for the better understand-

ing of biomineralization during shell formation.

Shell pigments have been extensively investigated (Williams 2017; Ishikawa

et al. 2013 and references therein). Comfort (1949a, b) reported the presence of

porphyrin compounds in several groups of gastropods including the family Neritidae

(described later) using ultraviolet uorescence and absorption chromatography.

Subsequently, the presence of polyene compounds was reported using Raman spec-

troscopy. For example, Hedegaard etal. (2006) estimated the chain length of conju-

gated polyenes and discussed the chemical modication of polyenes contained in

the shell of the gastropod Cypraea moneta (Cypraeidae). Polyene pigments were

observed in various taxa. However, the target samples used in previous studies were

limited to common shells with strong colors. Here, we comprehensively studied the

family Neritidae and conducted a preliminary study to detect porphyrin pigments

using Raman spectroscopy. The family Neritidae comprises small- or medium-sized

snails that inhabit a wide variety of shallow-water environments such as brackish

areas and intertidal rocky zones. The family is characterized by clear and vivid color

bands (Fig.8.1), which make spectroscopic analysis easy to perform. Their various

habitats are expected to have an effect on pigmentation because shell colors are

affected by environmental factors (e.g., Sokolova and Berger 2000).

8.2 Materials andMethods

We investigated 24 species from 5 genera of the family Neritidae by using Raman

spectroscopy (Table8.1). Raman spectra were obtained from each differently col-

ored area using an exposure time of 10s for each measurement. Ten spectra were

obtained for each area. Estimated energy resolution ranged from 1 to 2cm−1. Two

Raman spectrometers with different excitation wavelengths (514.5 and 442 nm)

were used (Table8.2). The excitation wavelength of 514.5nm has been previously

demonstrated to detect polyenes. The excitation wavelength of 442nm is near the

Soret maxima of porphyrins and thus is expected to excite porphyrins that are

T. Komura et al.

Fig. 8.1 Neritina waigiensis in the family Neritidae. A section of the shell denoted by the white

square was used for Raman analysis (see Fig.8.3)

Table 8.1 Collection numbers, species, locality, and typical habitats of the investigated samples

Coll.

number Species Locality Typical habitat

32906

Clithon oualaniense (Lesson,

1831)

Bohol Island,

Philippines

Brackish, on sandy or

muddy substrate

32907

Clithon (Pictoneritina)

chlorostoma (Broderip, 1833)

Ishigaki Island,

Japan

Under rocks, in estuaries

32908

Clithon cryptum (Eichhorst, 2016) Amami Island,

Japan

Under rocks, in estuaries

32909

Nerita (Amphinerita) insculpta

(Récluz, 1841)

Okinawa Island,

Japan

Intertidal, on rocks

32910

Nerita (Cymostyla) tristis (Pilsbry,

1901)

Amami Oshima

Island, Japan

Intertidal, on rocks

32911

Nerita (Cymostyla) striata

(Burrow, 1815)

Okinawa Island,

Japan

Intertidal, on rocks

32912

Nerita (Ritena) plicata (Linnaeus,

1758)

Ikema Island, Japan Intertidal, on rocks

32913

Nerita (Ritena) costata (Gmelin,

1791)

Amami Oshima

Island, Japan

Intertidal, on rocks

32914

Nerita (Theliostyla) picea (Récluz,

1841)

Australia Uncertain

32915

Nerita (Theliostyla) exuvia

(Linnaeus, 1758)

Cebu Island,

Philippines

Intertidal, on rocks

32916

Nerita (Theliostyls) albicala

(Linnaeus, 1758)

Amami Island,

Japan

Intertidal, on rocks

32917

Nerita (Argonerita) signata

(Lamarck, 1822)

Bohol Island,

Philippines

Intertidal, on rocks

32918

Nerita (Argonerita) chammaeleon

(Linnaeus, 1758)

Ishigaki Island,

Japan

Intertidal, on rocks

32919

Nerita (Linnerita) incerta (von

dem Busch, 1844)

Zamami Island,

Japan

Intertidal, on rocks

(continued)

8 Spectroscopic Investigation ofShell Pigments fromtheFamily Neritidae (Mollusca…

present in the mollusc shell (Comfort 1949a). We performed intact shell surface

analysis without chemical treatments. The samples examined were registered in The

University Museum, The University of Tokyo.

Table 8.1 (continued)

Coll.

number Species Locality Typical habitat

32920

Neritina (Linnerita) rumphi

(Récluz, 1841)

Malakal Island,

Palau

Intertidal, on rocks

32921

Neritina (Linnerita) polita

(Linnaeus, 1758)

Okinawa Island,

Japan

Intertidal, on rocks near

sand

32922

Neritina (Neritina) pulligera

(Linnaeus, 1767)

Okinawa Island,

Japan

On rocks in rivers

32923

Neritina (Vittina) paralela

(Röding, 1798)

Cebu Island,

Philippines

Brackish area

32924

Neritina (Vittina) waigiensis

(Lesson, 1831)

Cebu Island,

Philippines

Mangrove swamp

32925

Neritina (Vittina) turritta (Gmelin,

1791)

Cebu Island,

Philippines

On mud in mangrove

swamp

32926

Neripteron (Dostia) cornucopia

(Benson, 1836)

Cebu Island,

Philippines

Brackish area and

mangrove swamp

32927

Smaragdia rangiana (Récluz,

1842)

Balicasag Island,

Philippines

On seagrasses

32928

Neritodryas dubia (Gmelin, 1791) Cebu Island,

Philippines

Intertidal, brackish area

32929

Neritodryas sp.Cebu Island,

Philippines

Intertidal, brackish area

Scientic names and typical habitats follow the designation schemes of Tsuchiya and Kano (2017)

and Eichhorst (2016)

Table 8.2 Excitation wavelength, laser source, laser power, calibration standard, and objective

lens magnication data of the two Raman spectrometers

Excitation wavelength

(nm)

Laser

source

Laser power

(mW)

Calibration

standard

Objective lens

magnication

514.5 Ar+ laser 30 Naphthalene ×20

442 He-Cd

laser

120 Silicon ×20

Laser power could be attenuated through the optical paths for both instruments

T. Komura et al.

8.3 Results andDiscussion

8.3.1

Raman Spectra at 514.5nm

Figure 8.2 shows representative Raman spectra obtained from yellow, red, and black

color bands of N. waigiensis. The Raman spectra of the black and red parts were

similar, while the spectrum of the yellow part was markedly different from both.

The spectrum obtained from the yellow part was characterized by two peaks with

wavenumbers ranging from 1100–1200cm−1 (ν5) to 1500–1600 cm−1 (ν1). These

two peaks are assigned to polyene pigments contained in the shell (e.g., Bergamonti

etal. 2013). These polyene-type spectra were obtained from the species listed in

Table8.3. While Raman shifts of ν5 and ν1 varied in all species, the observed values

were within a certain range.

The spectra obtained from red and black parts of N. waigiensis had no obvious

peaks that could be assigned to polyenes. This type of spectrum was also obtained

from the species listed in Table 8.4. Similar spectra from the gastropod Clunculus

pharaonius (Trochidae) were reported by Merlin and Delé-Dubois (1986) and

Williams etal. (2016). The red and black pigments of C. pharaonius were identied

as uroporphyrin and eumelanin, respectively, using high-performance liquid chro-

matography (Williams etal. 2016). However, the authors argued that the Raman

spectra did not convey any denitive information concerning shell pigments, and it

Fig. 8.2 Raman spectra obtained from each color band of N. waigiensis using 514.5nm excita-

tion. White circles in the gure indicate measurement points for Raman analysis

8 Spectroscopic Investigation ofShell Pigments fromtheFamily Neritidae (Mollusca…

was quite unclear whether the obtained spectra were assignable to uroporphyrin or

eumelanin included in the shell. Presently, we also could not conclusively identify

shell pigment from the Raman spectra. However, the difference in chemical origins

of the black and red pigmentations of N. waigiensis from that of yellow pigmenta-

tion is a novel nding. It is worth noting that the Raman peaks obtained from red

and black regions of N. waigiensis appeared at a similar frequency to those in the

spectrum of eumelanin reported by Mbonyiryivuze etal. (2015). It is conceivable

that the peaks we observed were due to background uorescence.

Shell pigmentation may be relevant to taxonomical classication (Comfort

1949a, Williams 2017). However, the patterns and peak wavenumber of the Raman

spectra (Tables 8.1, 8.2, and 8.3) had no obvious relationship with the taxonomical

classication at the genus or lower level or with typical habitats. Different types of

spectra could be observed even in the same individuals.

We fabricated the shell section perpendicular to the apertural margin of N.

waigiensis (Fig.8.3). The thickness of red- and black-colored layers was approxi-

mately 30μm. The Raman spectra obtained from ve points are shown in Fig.8.4.

No obvious peaks assignable to polyenes were observed from points 1 to 2. Two

weak peaks assignable to polyenes were detected at point 3. The obtained spectra

Table 8.3 Colors and wavenumbers of the ν5 and ν1 peaks using the 514.5nm excitation from

species containing polyenes

Species Color

ν5

(cm−1)

ν1

(cm−1)

Clithonoualaniense (Lesson, 1831) Pale yellow 1139 1532

Nerita (Amphinerita) insculpta (Récluz, 1841) Black 1138 1528

Nerita (Linnerita) incerta (von dem Busch,

1844)

Green 1138 1526

Nerita (Cymostyla) tristis (Pilsbry, 1901) Deep green 1138 1530

Nerita (Cymostyla) striata (Burrow, 1815) Black 1135 1525

Nerita (Cymostyla) striata (Burrow, 1815) Light green 1136 1527

Nerita (Ritena) plicata (Linnaeus, 1758) Black 1135 1524

Nerita (Argonerita) signata (Lamarck, 1822) Red 1133 1523

Nerita (Argonerita) chammaeleon (Linnaeus,

1758)

Black 1138 1524

Nerita (Theliostyls) albicilla (Linnaeus, 1758) Deep green 1137 1527

Neritina (Linnerita) rumphii (Récluz, 1841) Black 1136 1526

Neritina (Linnerita) rumphii (Récluz, 1841) Green 1137 1525

Neritina (Linnerita) polita (Linnaeus, 1758) Deep green 1135 1524

Neritina (Linnerita) polita (Linnaeus, 1758) Green 1135 1524

Neritina (Neritina) pulligera (Linnaeus, 1767) Deep green 1138 1528

Neritina (Neritina) pulligera (Linnaeus, 1767) Orange 1139 1530

Neritina (Vittina) waigiensis (Lesson, 1831) Yellow 1142 1527

Smaragdia rangiana (Récluz, 1842) Green 1139 1534

Nerita (Ritena) costata (Gmelin, 1791) Black (between spiral

ribs)

1134 1524

T. Komura et al.

raise the possibility that the concentration of polyenes increases continuously from

the interior of the shell to the exterior surface. In contrast, black and red pigmenta-

tion appeared only in the narrow range on the surface, indicating that their distribu-

tions are quite different from the distribution of polyenes.

Table 8.4 Colors and Raman shifts of representative peaks obtained at 514.5 nm excitation

wavelength from species displaying other spectra

Species Color

Raman shifts of representative

peak (cm−1)

Clithon oualaniense (Lesson, 1831) Black 1562

Clithon (Pictoneritina) chlorostoma

(Broderip, 1833)

Black 1562

Clithon cryptum (Eichhorst, 2016) Black 1566

Nerita (Linnerita) incerta (von dem

Busch, 1844)

Deep green 1562

Nerita (Theliostyla) picea (Récluz, 1841) Black 1560

Nerita (Theliostyla) exuvia (Linnaeus,

1758)

Black 1548

Neripteron (Dostia) cornucopia (Benson,

1836)

Deep green 1559

Neritina (Vittina) waigiensis (Lesson,

1831)

Red 1562

Neritina (Vittina) waigiensis (Lesson,

1831)

Black 1557

Neritina (Vittina) turritta (Gmelin, 1791) Black 1566

Neritina (Vittina) parallella (Röding,

1798)

Black 1558

Neritodryas dubia (Gmelin, 1791) Deep purple 1560

Neritodryas sp.Red 1560

Neritodryas sp.Black 1565

Nerita (Ritena) costata (Gmelin, 1791) Black (on spiral

ribs)

1554

Fig. 8.3 (a) A section perpendicular to the apertural margin of N. waigiensis. The black and yel-

low patterns on the shell exterior are visible on the upper side of the gure. The numbers 1–5

indicate measured points. (b) Scanning electron microscopy image of the section of N. waigiensis.

The multilayered structure of the shell is evident. The outermost region is a prismatic layer (P) that

corresponds to the colored layer in (a). The inner layer has a crossed-lamellar structure (CL).

These two gures were obtained from different individuals of N. waigiensis

8 Spectroscopic Investigation ofShell Pigments fromtheFamily Neritidae (Mollusca…

8.3.2 Raman Spectra at 442nm Excitation

Figure 8.5 shows Raman spectra obtained from N. waigiensis using the 442 nm

excitation wavelength. Clear peaks assignable to polyenes were observed from the

yellow and red parts of the shell surface, but not from the black part. Polyene-type

Fig. 8.5 Raman spectrum obtained from each color band of N. waigiensis using the 442nm exci-

tation wavelength. The white circles in the gure indicate the measurement points for Raman

analysis

Fig. 8.4 Raman spectrum obtained from each measured point in Fig.8.3

T. Komura et al.

spectrum from the red part may be attributed to the polyenes of the yellow part

existing just under the red color band on the surface. Williams etal. (2016) argued

that it is difcult to distinguish polyenes from porphyrins only by the Raman spec-

tra. However, our results suggest that polyenes can be distinguished from other

pigments because the background uorescence was relatively low compared to that

for the peak heights at ν5 and ν1.

8.4 Conclusions

Analysis using the 514.5nm excitation wavelength demonstrated that the pigmenta-

tion in the family Neritidae has at least two types of origins: polyenes and other

unknown pigments such as porphyrins or melanins. The data from a shell section of

N. waigiensis suggest that these two types of pigments are distributed in a different

manner in the section. The patterns of the Raman spectra did not display obvious

relationships with taxonomical classication and habitats. The Raman spectra

obtained using the 442nm excitation wavelength suggest that polyenes can be dis-

tinguished from other pigments by the presence of strong peaks of ν5 and ν1.

However, denitive information on shell pigments was difcult using only Raman

spectroscopy. Other analytical techniques such as high-performance liquid chroma-

tography could be needed and will be studied.

Acknowledgment We thank Dr. Natsuhiko Sugimura from the Research Support Center at

Waseda University for providing support for the measurement of the Raman spectra.

References

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in molluscan shells. Biochem J44:111–117

Comfort A (1949b) Acid-soluble pigments of shells. 4. Identication of shell porphyrins with par-

ticular reference to conchoporphyrin. Biochem J45:208–210

Eichhorst TE (2016) Neritidae in the world. ConchBooks, Harxheim

Hedegaard C, Bardeau J-F, Chateigner D (2006) Molluscan shell pigments: an in situ resonance

Raman study. JMolluscan Stud 72:157–162

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ments. Earth Mon 35(12):712–719 (In Japanese)

Mbonyiryivuze A, Omollo I, Ngom BD, Mwakikunga B, Dhlamini SM, Park E, Maaza M (2015)

Natural dye sensitizer for Grӓtzel cells: Sepia Melanin. Phys Mater Chem 3(1):1–6

Merlin JC, Delé-Dubois ML (1986) Resonance Raman characterization of polyacetylenic pig-

ments in the calcareous skeleton. Comp Biochem Physiol 84B(1):97–103

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Sokolova IM, Berger VJ (2000) Physiological variation related to shell colour polymorphism in

White Sea Littorina saxatilis. JExp Mar Biol Ecol 245:1–23

Tsuchiya K, Kano Y (2017) Family Neritidae. In: Okutani T (ed) Marine Mollusks in Japan, 2nd

edn. Tokai University Press, Tokyo

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Williams ST, Ito S, Wakamatsu K, Goral T, Edwards NP, Wogelius RA etal (2016) Identication of

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Gastropoda). PLoSONE 11(7):e0156664

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

T. Komura et al.

83© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_9

Chapter 9

3D Visualization ofCalcied

andNon-calcied Molluscan Tissues

Using Computed Tomography

TakenoriSasaki, YuMaekawa, YusukeTakeda, MakiAtsushiba,

ChongChen, KojiNoshita, KentaroUesugi, andMasatoHoshino

Abstract Three-dimensional (3D) reconstruction is an essential approach in mor-

phological studies in biology and paleontology. Seeking an optimized protocol for

nondestructive observations, we attempted 3D visualization of various molluscan

shells and animals with X-ray micro-computed tomography (micro-CT). Calcied

parts of molluscs were easily visualized except for cases with marked differences in

thickness heterogeneity. 3D imaging of shell microstructure was difcult.

Visualization of soft tissue requires staining to enhance the image contrast.

Especially for soft tissues, synchrotron X-ray microtomography is the most

advanced method to generate clear 3D images. 3D data facilitates morphological

quantication, enabling calculations of length and volume even for very complex

forms. X-ray micro-CT is extremely useful in the morphologic examination of

T. Sasaki (*) · Y. Maekawa · M. Atsushiba

The University Museum, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

e-mail: atsushiba-7122@g.ecc.u-tokyo.ac.jp; yu.maekawa@um.u-tokyo.ac.jp;

sasaki@um.u-tokyo.ac.jp

Y. Takeda

The University Museum, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Department of Earth and Planetary Science, Hokkaido University,

Kita-ku, Sapporo, Hokkaido, Japan

e-mail: ytakeda@sci.hokudai.ac.jp

C. Chen

Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa, Japan

e-mail: cchen@jamstec.go.jp

K. Noshita

Graduate School of Agricultural and Life Sciences, The University of Tokyo,

Bunkyo-ku, Tokyo, Japan

e-mail: noshita@morphometrics.jp

K. Uesugi · M. Hoshino

Japan Synchrotron Radiation Research Institute, Sayo-gun, Hyogo, Japan

e-mail: hoshino@spring8.or.jp; ueken@spring8.or.jp

mineralized and soft tissues, although microstructural and histological details

should be supplemented by other microscopic techniques.

Keywords Micro-CT · Synchrotron CT · Reconstruction · Anatomy

9.1 Introduction

The importance of computed tomography (CT) has been established in biology and

paleontology. CT has also been used in examinations of vertebrate morphology

(Gignac etal. 2016; du Plessis etal. 2017), and micro-CT has been used in inverte-

brate biology, mainly in entomology studies (Friedrich etal. 2014; Wiper et al.

2016). The approach has been infrequently used for other animal groups. In mala-

cology, the use of micro-CT is restricted to a small number of case studies involving

anatomy (Golding and Jones 2007; Golding et al. 2009), ontogeny (Kerbl et al.

2013), paleontology (Takeda et al. 2016), and shell morphology (Monnet et al.

2009; Liew and Schilthuizen 2016; Noshita etal. 2016).

The application of CT is still in the testing and development stage. Having an

optimized protocol for the nondestructive micro-CT analysis of various morpho-

logical characteristics would help expand the scope of CT applications and was the

focus of the present study.

9.2 Material andMethods

The molluscan specimens used in this study are all registered and deposited in The

University Museum, The University of Tokyo (UMUT). The abbreviations RM and

CM in the registration numbers indicate recent and Cenozoic molluscs, respectively.

Animals were xed in 5% formaldehyde solution for several days, washed in tap

water, and then preserved in 70% ethanol. Intact animals were extracted from shells

by soaking the specimens in approximately 10% HCl to completely dissolve the

shells. Most specimens were rst digitally imaged (Fig.9.1a–e) using a ScanXmate

B100TSS110 industrial micro-CT device (Comscantecno Co., Ltd.) at UMUT.Scan

parameters were adjusted according to samples and included source voltage (70–

100kV), source current (approximately 40–150μA), and exposure time per frame

(0.4–1.0s). The total number of frames was 1200, the number of pixels of the detec-

tor was 1024×1012, and the highest resolution was approximately 2μm. Samples

larger than 3 cm were scanned in the laboratory of Comscantecno Co. Ltd. For

scanning, embedding the sample in a heat-generated hole in Styrofoam on a rotating

stage was the most effective method of sample xation. For soft portions of sam-

ples, high-resolution X-ray CT was also performed (Fig.9.1f–h) at the hutch 3 of

the beamline BL20B2in SPring-8 (Hyogo, Japan) with a resolution of 13.16μm per

pixel and an X-ray energy of 25keV. Three-dimensional (3D) visualization was

conducted using Molcer Plus (White Rabbit Corporation), OsiriX (OsiriX

Foundation), and Amira 3.5.5 (Visage Imaging, Inc.) software.

T. Sasaki et al.

9.3 Results

9.3.1 Shells

Most samples of calcied shells were scanned without difculty using conventional

industrial micro-CT (Fig.9.2). The internal structure of shells was also perfectly

reconstructed from scan data. Exceptions included shells of specic groups of gas-

tropods, with shells from different regions having extremely disparate thicknesses.

For example, members of the families Ellobiidae (Figs. 9.2c, d), Olividae, and

Conidae have shells in which the internal whorls are much thinner than the outer

wall owing to the secondary resorption that occurs during growth. In such cases, the

thinner inner whorls disappear or became patchy in distribution when the settings

were optimized for the outer shell surface. Conversely, if contrast were enhanced to

reveal the inner structures, outer surfaces were rendered unusable by the increased

noise levels (the overexposure effect). So far, no viable solution to this problem has

been identied in our current system.

Digitization using micro-CT is an excellent approach to display the overall 3D

morphology and the internal structure of molluscan shells. Examples of specimens

of small Cenozoic fossil gastropods are presented in Fig.9.2e–j. Virtual slicing of

type specimens along an arbitrary plane is possible only using micro-CT.Prominent

columellar folds (Fig.9.2f, h) or denticles in the outer lip (Fig.9.2h) were important

features to morphologically diagnose species. Construction of images from apical

(Fig.9.2i) and basal (Fig. 9.2j) views for fragile and minute specimens is easily

acquired. However, imaging of the same specimens by photography with a digital

camera and binocular microscope carries the risk of specimen loss or damage.

Micro-CT allows the detailed nondestructive observation of the delicate structures

of shells.

Fig. 9.1 CT facilities used in this study. (a–e) Industrial CT (UMUT). (b) X-ray tube. (c) Sample

stage. (d) X-ray detector. (e) Sample holder. (f–h) Synchrotron CT (SPring-8 BL20B2). (g)

Magnied view of sample stage. Arrowhead indicates sample. (h) X-ray detector

9 3D Visualization ofCalcied andNon-calcied Molluscan Tissues Using Computed…

However, the use of micro-CT for shells is not entirely versatile. Most impor-

tantly, growth lines, shell layers, and shell microstructure cannot be observed. We

were unable to detect any structure on sliced shell planes in the CT-derived 3D data.

The observation of these ne structures still necessitates mechanical destruction or

cutting of the actual shell samples.

Another issue is that high-resolution scanning of a specic part of a large shell is

not possible. To achieve high-resolution scan data, it is necessary to place a sample

Fig. 9.2 Example of 3D reconstruction of calcied shells from industrial micro-CT data. (a, b)

Volutoconus bednalli (Brazier, 1878) (Volutidae). UMUT RM32762. (c, d) Ellobium aurismidae

(Linnaeus, 1758) (Ellobiidae). UMUT RM32763. Arrowhead indicates missing inner wall as arti-

fact. (e, f) Cerithiella trisulcata (Yokoyama, 1922) (Newtoniellidae). UMUT CM20781. Holotype.

(g–i) Tiberia pseudopulchella (Yokoyama, 1920) (Pyramidellidae). UMUT CM20242. Holotype.

(i) Apical view. (j) Adapical view. Shell height: a, b=90.6mm; c, d 88.8mm; e, f=5.2mm; g,

h=7.0mm. Software: Molcer Plus

T. Sasaki et al.

as close to the X-ray source as possible (for any given detector image size, the real-

ized resolution depends on the eld of view and, thus, the size of the specimen). In

conventional micro-CT, it is necessary to rotate the specimen. Thus, if the sample is

large, it must be placed further away from the X-ray source, which will decrease the

resolution. This dilemma cannot be overcome at the present time.

9.3.2 Soft Tissues

Simultaneous visualization of both the shell and the soft regions from a single speci-

men could not be achieved with satisfactory results. If various parameters are opti-

mized for shells, the settings are far from optimal for the soft regions, and vice

versa. The only way to achieve high-quality scans for both features from a single

individual is to obtain the shell scan rst and to decalcify it to scan the soft regions.

This destroys the shell. Therefore, development of a new algorithm or methodology

is desired to enable the nondestructive scans of precious specimens (such as intact

types) to reveal the anatomy of soft portions.

Applying CT to soft parts is more challenging than applying it to calcied parts.

Animal tissues usually do not display any contrast in CT images without the aid of

contrast-enhancing substances. Figure9.3 shows an example of Patellogastropoda.

In this organism, only iron-mineralized radular teeth are visible if the specimen is

not treated with a contrast agent (e.g., iodine).

Installation of samples on the rotating stage is also an extremely sensitive part of

the scanning process. If a wet sample is scanned while it is exposed to air, it quickly

becomes dehydrated and deformed, which will result in a very blurred image that is

Fig. 9.3 Soft part of Lottia

dorsuosa (Lottiidae).

UMUT RM32764.

Comparison of scanning

results between iodine-free

(left) and iodine-stained

(right) samples with

industrial micro-CT. R

radular teeth, M metal ball

for position correction.

Body length=22mm

9 3D Visualization ofCalcied andNon-calcied Molluscan Tissues Using Computed…

unusable. Therefore, samples should be contained in a liquid medium such as etha-

nol, water, or physiological saline water in a (relatively) X-ray transparent con-

tainer. Wrapping a sample with X-ray transparent lm in a Styrofoam container can

be an effective solution. Expelling air bubbles from the medium is essential because

the bubbles can cause sample movement when they expand as they are subjected to

heat generated during prolonged scanning.

In our experience, despite repeated experiments, scanning of soft parts with a

conventional industrial micro-CT frequently resulted in insufcient contrast and

clarity (Fig.9.4). To explore other solutions, we attempted to use synchrotron CT

instead of industrial CT (Fig.9.1f–h). The results were clearly superior to the indus-

trial CT data, as exemplied in an experiment in which the same specimen was used

for both methods (Fig.9.5). The synchrotron CT data were characterized by the

clear edge of the outline and sharp boundaries of the internal organs. The congura-

tions of internal organs were more reliably identied using this method.

Fig. 9.4 Soft part of Conus ebraeus Linnaeus, 1758 (Conidae). UMUT RM32765. (a) Formalin-

xed animal with shell decalcied. (b–d) 3D reconstruction with software Molcer Plus from scan

data of industrial CT.Body length=19mm

Fig. 9.5 Soft part of Lottia dorsuosa (Gould, 1859) (Lottiidae). UMUT RM32764. Comparison

of scanning results between conventional industrial micro-CT (a) and synchrotron CT (b SPring-8

BL20B2). dg digestive gland, es esophagus, f foot, i intestine, mm mantle margin, pb pedal blood

sinus, pc pallial cavity, rds radular sac, st stomach. Body length=22mm

T. Sasaki et al.

A widely utilized method that was also used by us is to immerse samples in 1%

iodine solution as the contrast agent for both laboratory and synchrotron CT scan-

ning. This staining method is very handy and inexpensive. In our experiments,

iodine rapidly penetrated into the samples, and immersion in iodine for 1 to several

days was sufcient for samples smaller than 2cm. However, slight shrinkage of the

samples was unavoidable with iodine staining. This artifact was especially evident

with the thin and membranous mantle tissue, which displayed numerous minute

wrinkles after staining with iodine. In this study, we could not sufciently test dif-

ferent contrast agents such as phosphomolybdic acid and osmium tetroxide. This

will be the subject of a future study.

We tested Molcer Plus, OsiriX, and Amira software. Each has its own inherent

advantages. Molcer Plus was the best software for visualizing calcied shells.

OsiriX was able to create vivid 3D images of soft portions (Fig.9.6b–d) with the

most straightforward operation. Amira was superior in terms of results and opera-

tion for internal anatomy (Fig.9.6e, f). By adjusting the threshold of brightness, the

surface outline of a specimen could be automatically extracted (Figs.9.6e and 9.7a–

c). Different parts of internal organs could be illustrated in various colors after the

segmentation procedure (Fig.9.6f). Figure9.7 is an example of the 3D visualization

from three different angles, showing the animal’s surface outline, digestive tracts

with a translucent outline, and isolated digestive tracts only. This method allowed us

to quantify the size and volume of various parts of the animal body.

Fig. 9.6 Examples of 3D visualization of soft parts from synchrotron CT data. (a–d) Spirostoma

japonicum (A. Adams, 1867) (Cyclophoridae). UMUT RM32766. Body length = 9.7 mm. (a)

Transmission image. (b) Ventral view, (c) dorsal view, (d) frontal view. (e, f) Granata lyrata

(Pilsbry, 1890) (Chilodontidae). UMUT RM32767. Body length=9.9mm. (e) Body surface. (f)

Digestive tracts with translucent body surface. Software: (a–d) OsiriX. (e, f) Amira

9 3D Visualization ofCalcied andNon-calcied Molluscan Tissues Using Computed…

9.4 Discussion

Through this experimental study, we conrmed that micro-CT is a very useful

method for 3D presentation of various hard and soft features of molluscs. CT scan-

ning has three important advantages. First, observations of internal shell structures

are possible without having to cut the shells. Second, acquisition of a complete

series of serially sectioned images without damaging soft portions is feasible. Third,

the use of synchrotron CT allows 3D reconstruction of soft parts in much less time.

Scanning calcium carbonate shells is relatively easily performed and should be

applied widely to other biominerals of invertebrates such as corals, barnacles, and

Fig. 9.7 Examples of 3D visualization of soft parts from synchrotron CT data (SPring-8 BL20B2).

Phenacolepas unguiformis (Gould, 1859) (Phenacolepadidae). UMUT RM32768. (a–c) Exterior.

(d–f) Digestive tracts with translucent outline. (g–i) Extracted digestive tracts. (a, d, g) Dorsal

view. (b, e, h) Left lateral view. (c, f, i) Ventral view. Esophagus(blue), stomach(red), and intes-

tine(yellow) are indicated in different colors. Body length=6.0mm. Software: Amira

T. Sasaki et al.

echinoderms (Okanishi etal. 2017). The most comprehensive method for under-

standing shell morphology is achieved by the combination of micro-CT and scan-

ning electron microscopy (SEM). Micro-CT has the advantages of 3D digitization

and nondestructive internal observation. For example, it is possible to reveal inter-

nal morphological characteristics of specimens without cutting through them

(Fig.9.2) and can be achieved from any direction and for specimens of any size. On

the other hand, SEM has a much higher resolution, which enables the observation

of microscopic sculptures at higher magnication. Therefore, SEM is still necessary

for characterizing micromolluscs (Sasaki 2008). In addition, shell microstructure

and shell layer structure are most reliably observed with SEM (Nishida etal. 2012;

Sato and Sasaki 2015).

We conrmed that micro-CT is also extremely effective for the study of soft

parts. In particular, this method has a tremendous benet for samples that are dif-

cult to section with a microtome. For example, the digestive tracts of deposit feeders

such as infaunal bivalves are often lled with many sediment grains, which easily

damage knives during histological sectioning. In patellogastropod limpets (Fig.9.3)

and chitons, the radular teeth are heavily mineralized and also cause serious knife

damage. Using micro-CT avoids these problems.

Before the introduction of micro-CT, serial histological sectioning was the only

method available for 3D reconstruction (Chen etal. 2015; see Ruthensteiner 2008

for detailed methodology). However, with micro-CT, we are liberated from the dif-

cult and time-consuming demand of making a complete series of serial sections of

whole animals. For 3D reconstruction from serial sections, considerable time is

spent aligning the stacks. This issue does not exist in micro-CT because the image

stack generated is completely aligned without skew. In addition, complete anatomi-

cal reconstruction of large specimens becomes possible (the realistic upper size

limit of resin-embedded serial sectioning is approximately 5mm wide, and parafn

sections are too skewed to be useful for 3D visualization). Therefore, CT presents a

high-throughput, highly efcient approach compared to sectioning, as has previ-

ously been pointed out by Golding and Jones (2007) and Kerbl etal. (2013).

Generating 3D data from a 2D image stack is still a time-consuming process with

regard to internal anatomy. At present, we do not have a practical algorithm for

automatic segmentation of the various internal organs. This continues to hinder

advances in 3D morphological analysis.

The resolution of CT data is still much lower than that of histological sectioning.

In addition, we cannot distinguish different organs using distinctive staining, as is

used for parafn-embedded sectioning or immunohistochemistry. For example,

muscle and connective tissue can be vividly distinguished by trichrome staining

(Katsuno and Sasaki 2008), but such a distinction is not possible with CT data.

Therefore, it is advisable to reconstruct various 3D morphologies from CT data and

supplement the details from histological sections. In this approach, much less his-

tology data are needed for an overall understanding of the specimen. In this way, we

can greatly improve the efciency of morphological studies with CT.

3D data with segmentation may be exploited for various morphological analyses,

especially quantitative analysis. For example, we can calculate the length and

9 3D Visualization ofCalcied andNon-calcied Molluscan Tissues Using Computed…

volume of various structures that are difcult to measure in actual specimens.

Quantication of shape and its applications are topics for the future.

In conclusion, X-ray micro-CT is extremely useful in the morphological charac-

terization of both mineralized and non-mineralized tissues of molluscs. Although

this method has the potential to greatly improve the efciency of morphological

studies, it does not entirely replace conventional approaches and should be supple-

mented by data from other destructive microscopic methods.

Acknowledgments Dr. Osamu Sasaki (The Tohoku University Museum) and Dr. Akiteru Maeno

(National Institute of Genetics) kindly provided technical advice when we introduced the

Comscantechno CT scanner to The University Museum, The University of Tokyo. This study was

funded by a JSPS Kakenhi Grant, number 15K14589 and JP16J06269. The synchrotron radiation

experiments were performed at the BL20B2 of SPring-8 with the approval of the Japan Synchrotron

Radiation Research Institute (JASRI) (Proposal Nos. 2015B1833, 2016A1706, 2017A1720,

2017B1767).

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based contrast-enhanced computed tomography (diceCT): an emerging tool for rapid, high-

resolution, 3-D imaging of metazoan soft tissues. JAnat 228:889–909

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

9 3D Visualization ofCalcied andNon-calcied Molluscan Tissues Using Computed…

Part II

Molecular and Cellular Regulation of

Biomineralization

97© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_10

Chapter 10

Calcium Ion andMineral Pathways

inBiomineralization: APerspective

GalMorKhalifa, KerenKahil, LiaAddadi, andSteveWeiner

Abstract Calcium transport from the environment to the nal site of mineral depo-

sition involves uptake from the water or the food into cells. Within the cells calcium

ions are translocated to various organelles and vesicles where they accumulate, in

such a way as to not raise the very low calcium concentrations in the cytosol. In

various biomineralizing systems, the calcium is stored in vesicles as a highly disor-

dered hence relatively soluble solid phase. The concentrated calcium phase is then

translocated out of the cell to the site of mineralization. Additional pathways may

involve transport through the vasculature as ions and possibly mineral from distant

sites. Understanding calcium pathways is the foundation for not only better under-

standing biomineralization processes but also for better understanding calcium and

its fundamental role in cell signaling.

Keywords Calcium uptake and transport · Seawater vacuoles · Calcium signaling

· Mineral-containing vesicles

10.1 Introduction

One common attribute of all biologically mineralizing processes is that huge

amounts of ions must be acquired from the environment, andtransported to the site

of mineralization, where they are deposited. In many mineralizing processes, the

ions are temporarily stored in the form of highly unstable and disordered membrane-

bound mineral phases inside the cells (Weiner and Addadi 2011). Furthermore, in

many cases the mineral rst deposited at the site of mineralized tissue formation is

also an unstable disordered mineral (“precursor phase”) that subsequently crystal-

lizes into the mature phase (Beniash etal. 1997; Crane etal. 2006; Mahamid etal.

2010; Weiss etal. 2002).

G. M. Khalifa · K. Kahil · L. Addadi · S. Weiner (*)

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel

e-mail: Gal.Mor@weizmann.ac.il; keren.kahil@weizmann.ac.il; lia.addadi@weizmann.ac.il;

steve.weiner@weizmann.ac.il

Ion uptake and transport into cells and its temporary storage in cells are by no

means conned to mineralization processes. In fact all cells need a suite of ions in

order to function. Probably the best-studied ion in this respect is calcium. Calcium

is used by all cells for signaling (Altin and Bygrave 1988; Carafoli 2005). For this

to work, the cell needs to maintain a very low concentration of calcium in its cyto-

sol– about 100–200nM (Carafoli 2005), which is at least 10,000 times less concen-

trated than calcium in seawater (about 10 mmol) (Pietrobon et al. 1990). This

signaling role of calcium requires the cell to take up calcium and store it inlocations

that do not contaminate the cytosol. This requirement must be even more stringent

for cells that are involved in the calcium mineralization process, as they need to

transport large amounts of calcium but also must have a functioning calcium signal-

ing system.

Although a lot is known about the functions of calcium in cell metabolism, the

actual distributions and concentrations of calcium ions in cells (not only in the cyto-

sol) are not well documented. The main reasons for this are probably technical, as

such mapping and measurements cannot be carried out on xed and dehydrated

specimens but require either invivo measurements or a suitable xation method. In

vivo options for identifying ion concentrations are uorescent probes which are

sensitive to nanomolar concentrations (Rudolf et al. 2003) but not to the much

higher concentrations that are relevant for the different stages in biomineralization.

Identication of minerals invivo is at present limited to micro-Raman spectroscopy,

although the laser could conceivably induce amorphous minerals to crystallize. A

most suitable xation method for preserving ion concentrations and stabilizing

unstable mineral phases is cryo-xation. Cryo-xation is the very rapid freezing of

water such that ice crystals do not form, but the water is essentially frozen into a

glassy or vitried state. This method was developed by Dubochet (Dubochet etal.

1988). Mapping and measuring calcium concentrations invivo or after cryo-xation

are, however, still technically very challenging. One promising option is cryo-soft

X-ray tomography (Pereiro and Chichόn 2014; Sviben etal. 2016).

10.2 Calcium Uptake andTransport

The ultimate source of ions is from the environment in which the organism lives.

The ions can be extracted directly from the aqueous medium (seawater or freshwa-

ter) and/or from the food. Active ion-by-ion uptake takes place through ion-specic

channels and pumps located in the cell membranes. Many proteins that bind one or

several ions are known, and many are thought to be involved in ion transport

(Pietrobon etal. 1990). These ion uptake and transport processes also occur in cells

that are responsible for depositing mineral. Even if sufcient ions can be trans-

ported in these ways during rapid mineralization, or even during normal mineraliza-

tion, are these the only pathways used? Much still remains to be learned about ion

and mineral pathways in biology.

G. M. Khalifa et al.

One approach is to use uorescent molecules that are not able to pass through

membranes: such as calcein and large uorescently labeled polymers such as dex-

tran (e.g., Butko etal. 1996). Organisms are grown in a medium in which one or

both of these molecules are dissolved. If the uorescent label enters the cell, then

the marker molecules must have entered without passing through the membrane.

One such process is endocytosis. As calcein is also a calcium-binding molecule, this

is of particular relevance to tracking calcium in biomineralization. Bentov etal.

(2009) monitored both these molecules in rapidly re-mineralizing benthic foramin-

ifera whose shells had been previously dissolved so that the uorescent signal could

be imaged. These re-mineralizing foraminifera do indeed take up the uorescent

molecules, and these uorescent molecules end up in the shell itself (Bentov etal.

2009). In this way Bentov etal. (2009) demonstrated that these organisms must be

incorporating seawater droplets and at least some of the components of these drop-

lets could be incorporated in the shell. Seawater droplet uptake was subsequently

conrmed for intact foraminifera by imaging cryo-xed fracture surfaces onto

which uorescent maps of the same cryo-fracture surface were superimposed

(Khalifa etal. 2016). The calcein distribution in the foraminifer cytoplasm, investi-

gated using this technique, shows that the foraminifer cell incorporates seawater

droplets in a variety of sizes, starting from small micron-sized vesicles up to vacu-

oles tens of microns in size (Fig.10.1). Seawater uptake was subsequently shown to

occur in sea urchin larvae also using uorescent dextran and calcein (Vidavsky etal.

2016). Surprisingly, the uorescent markers labeled many sea urchin larval cells, in

addition to the primary mesenchymal cells (PMCs) that are responsible for calcite

spicule formation. Here the uorescent calcein, but not the dextran, ends up in the

calcitic spicule (Vidavsky etal. 2016). As seawater endocytosis is now known from

Fig. 10.1 Cryo-uorescence images superimposed on a micrograph of high-pressure frozen and

freeze-fractured Amphistegina lessonii. (a) A cryo-uorescent image of a whole A. lessonii speci-

men superimposed on a freeze-fractured surface showing symbiont autouorescence (red) and

calcein labeling (green). The white arrow shows the location of one large seawater vacuole. Scale

bar = 100μm. (b) Magnied correlative cryo-SEM- uorescence image of the seawater vacuole

identied in image a. Scale bar = 20μm

10 Calcium Ion andMineral Pathways inBiomineralization: APerspective

100

two very different organisms, we suspect that the seawater occlusion uptake path-

way may be widespread. This in turn opens up a fascinating question of how seawa-

ter can be manipulated chemically and/or biologically to enable specic ions, such

as calcium, to be extracted without extracting all the other ions such as magnesium

or strontium that are also present in seawater, in some cases in greater abundance

than calcium.

In sea urchin larvae, seawater is incorporated into the body cavity (blastocoel).

Many lopodia traverse the blastocoel, and it has been observed that the cells con-

centrate the calcein label into intracellular vesicles, and these vesicles move along

the lopodia. They are transporting calcium in the form of a mineral, as shown by

Raman spectroscopy, to the epithelial and mesenchymal cells (Vidavsky etal. 2015).

Another possible mode of seawater uptake from the blastocoel was observed using

cryo-FIB SEM to reconstruct the 3D organization of the constituents of the primary

mesenchyme cells (PMCs) of sea urchin larvae– the cells responsible for calcite

spicule formation. These cells contain vesicles, some of which have openings to the

seawater-containing blastocoel (Fig.10.2). In one case it was shown that such a

vesicle is connected to other vesicles and/or vacuoles to form a large membrane-

bound intracellular network (Vidavsky etal. 2016). It is conceivable, but not proven,

that such a conduit might allow seawater with its calcium ions to move through the

cell without contaminating the cytosol. Another option is to transport ions and/or

minerals outside cells– either between cells as has been observed in coral epithelia

responsible for skeletal mineralization (Gattuso etal. 1999) or as mineral- containing

vesicles that have been observed in blood vessels of vertebrates (Kerschnitzki etal.

2016) and mollusks (Mount etal. 2004) and in the vertebrate extracellular matrix

(so-called matrix vesicles) (Anderson 1995).

Fig. 10.2 Cryo-FIB-SEM micrograph of a single section of a high-pressure frozen sea urchin

larva at the prism stage. (a) A primary mesenchyme cell attached to the spicule (S). A vesicle con-

taining a uniform content similar in texture and gray levels to the cytoplasm and extracellular uids

is marked by an asterisk. This vesicle is close to the plasma membrane and has an opening to the

extracellular uids. (b) Higher magnication of the vesicle in a

G. M. Khalifa et al.

101

10.3 Temporary Calcium Storage inCells

Intracellular calcium is known to be stored in the sarcoplasmic reticulum, the endo-

plasmic reticulum, mitochondria, and various vesicles including the acidosomes

(Docampo etal. 2005; García etal. 2006; Pezzati etal. 1997). Apparently what is

not known is how the calcium is transported to these organelles or vesicles without

contaminating the cytosol. It is also not known in which forms the calcium is stored

at these locations. It is however known that in some sea urchin larval cells, calcium

is stored in vesicles in the form of a highly disordered mineral phase, amorphous

calcium carbonate (Beniash etal. 1999; Weiner and Addadi 2011). A particularly

interesting mineral storage vesicle is present in Coccolithophoridae. This vesicle

stores a calcium polyphosphate mineral (Sviben etal. 2016). This is surprising as

Coccolithophoridae produce calcitic bodies (coccoliths). Intracellular mineral stor-

age vesicles are also known in cells that are involved in bone formation (Akiva etal.

2015; Mahamid etal. 2011).

10.4 Many Open Questions andChallenges Remain

Many mineralization processes take place either in the extracellular environment or

in relatively large vacuoles within the cell. The rst formed mineral phase, however,

is often found in membrane-bound vesicles within the cell (Weiner and Addadi

2011). Almost nothing is known about whether the temporarily stored calcium in

the membrane-bound vesicles is transferred to the extracellular or intracellular sites

of mineralized skeletal formation, and if so how is the calcium transferred out of the

cell. In one case mineral-containing vesicles were observed to be exocytosed into

the extracellular environment during bone formation (Boonrungsiman etal. 2012).

Foraminifera and sea urchin larvae are now known to take up ions by introducing

seawater into the intracellular environment (Bentov et al. 2009; Vidavsky et al.

2016). This in turn raises the question of how this seawater is manipulated chemi-

cally and/or biologically to extract the calcium ions. One interesting observation is

the presence of mineral bodies rich in magnesium in the seawater vesicles of fora-

minifera (Khalifa etal. 2016). How the Mg-rich mineral forms and for what purpose

are as yet unanswered questions.

It was reported that the vasculature in developing chick bones contains membrane-

bound mineral particles (Kerschnitzki etal. 2016). These vesicles must have formed

at some other location and were introduced into the blood system. It is not known

where these mineral particles form, but the implication is that bone formation

involves many different cells, some of which may be located at considerable dis-

tances from the site of bone mineralization.

A 3D map of the distributions of ions in a cell would contribute signicantly to

our understanding of how all cells translocate and store large concentrations of ions

without contaminating the cytosol. We still however do not have appropriate tech-

niques to map the full range of ion concentrations in cells.

10 Calcium Ion andMineral Pathways inBiomineralization: APerspective

102

10.5 Concluding Comment

Biologically mineralizing processes involve the uptake, transport, and deposition of

large amounts of ions, often within a relatively short time. These systems are there-

fore advantageous for elucidating ion and mineral pathways in biology. Some of

these pathways may be conned to mineralizing systems, but other pathways may

also operate in all cells, as all cells require calcium and many other ions for per-

forming basic functions. Thus elucidating these ion and mineral pathways in

biomineralization may well contribute to our understanding of a fundamental bio-

logical process.

Acknowledgments LA and SW are the incumbents of the Dorothy and Patrick Gorman

Professorial Chair of Biological Ultrastructure and the Dr. Trude Burchardt Professorial Chair of

Structural Biology, respectively.

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

10 Calcium Ion andMineral Pathways inBiomineralization: APerspective

105© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_11

Chapter 11

Identication ofBarnacle Shell Proteins

byTranscriptome andProteomic

Approaches

YueHimWong, NoriakiOzaki, Wei-PangZhang, JinSun, ErinaYoshimura,

MiekoOguro-Okano, YasuyukiNogata, Hsiu-ChinLin, BennyK.K.Chan,

Pei-YuanQian, andKeijuOkano

Abstract In barnacle shell, the calcied shell layer is laid on top of the epicuticle.

Here, we report our strategy and some preliminary results on the identication of

potential shell proteins of the barnacle Megabalanus rosa. At rst, M. rosa proteins

from acid-soluble and acid-insoluble shell extracts were subjected to proteomic

analysis and searched against M. rosa complete transcriptome. Then using the infor-

mation that the calcied shell is formed just after the larval-adult molt, juvenile

Y. H. Wong · N. Ozaki · K. Okano (*)

Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University,

Akita, Japan

e-mail: timwong@akita-pu.ac.jp; ozanor@akita-pu.ac.jp; keijuo@akita-pu.ac.jp

W.-P. Zhang · J. Sun · P.-Y. Qian

Division of Life Science, School of Science, The Hong Kong University of Science and

Technology, Hong Kong, China

e-mail: wzhangae@connect.ust.hk; boqianpy@ust.hk

E. Yoshimura · Y. Nogata

Environmental Science Research Laboratory, Central Research Institute of Electric Power

Industry, Chiba, Japan

e-mail: yoshimura@ceresco.jp; noga@criepi.denken.or.jp

M. Oguro-Okano

Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University,

Akita, Japan

Department of Animal, Health Technology, Yamazaki Gakuen University, Tokyo, Japan

e-mail: m-oguro@yamazaki.ac.jp

H.-C. Lin

Department of Marine Biotechnology and Resources, National Sun Yat-sen University,

Kaohsiung, Taiwan

e-mail: hsiuchinlin@mail.nsysu.edu.tw

B. K. K. Chan

Biodiversity Research Center, Academia Sinica, Taipei, Taiwan

e-mail: chankk@gate.sinica.edu.tw

106

differentially expressed genes against larval stages were screened. Sixty secretory

protein sequences were identied as primary candidates of M. rosa shell proteins,

among which 37 are novel proteins.

Keywords Barnacle · Shell formation · Transcriptome · Proteomics

11.1 Introduction

Thoracican barnacles produce a heavily calcied shell that, unlike their crustacean

relatives, does not shed away (Walley 1969). The calcied shell offers physical

protection against predators and wave actions (Astachov etal. 2011). The shell also

enables barnacles to prevent desiccation in the intertidal region.

The calcied shell plates produced by the subtidal thoracican barnacle

Megabalanus rosa are composed of the operculum plates (OPs), the lateral wall

plates (WPs), and the basal shell plate (BP) (Fig.11.1). OPs consist of two pairs of

shell plates, namely, terga and scuta. These are movable plates that serve as the shut-

ter of the soft body. M. rosa produces four pairs of WPs, which are the major shell

plates that protect the interior soft body from mechanical impact. BP is the calcied

shell plate at the bottom of the barnacle, separating the barnacle soft body from the

rock substratum surface. Hence, each of these plates has different functions. Details

of barnacle shell anatomy can be found in Chan etal. (2009).

Obviously, it is important to understand what molecules are involved in the for-

mation of these shell structures. Matsubara etal. (2008) isolated two C-type lectin

proteins BRA-2 and BRA-3 from the regenerating M. rosa shell. Zhang etal. (2015)

reported a list of acid-soluble shell proteins, including carbonic anhydrase and an

aspartic acid-rich partial protein sequence from the intertidal barnacle Amphibalanus

amphitrite. The aspartic acid-rich partial protein, however, has shown no sequence

homology to the molluscan Asprich shell protein family (Gotliv et al. 2005),

Fig. 11.1 The thoracican barnacle Megabalanus rosa and a schemetic drawing of the adult shell

structure. (a) 2-month M. rosa juvenile, with cirri reaching out from its calcied shell for feeding

on planktons. (b) A schemetic drawing of M. rosa shell structure. M. rosa shell composes of the

operculum plates (OPs), the wall plates (WPs), and a basal plate (BP)

Y. H. Wong et al.

107

suggesting that aspartic acid-rich proteins in barnacles and mollusks have indepen-

dently evolved.

In this study, we used M. rosa as the study model for barnacle shell formation.

We present an integrated transcriptomic and proteomic approach for the identica-

tion of barnacle shell protein candidates.

11.2 Material andMethods

11.2.1 M. rosa andIts Larval Culture Construction

Adult individuals of M. rosa were obtained in theOga Peninsula, Akita, Japan,

from the bouydeployed by local shermen. Upon arriving the lab, the M. rosa

individuals were maintained in a 20°C aquarium until being sampled. Larval sam-

ples were prepared at Central Research Institute of Electric Power Industry, accord-

ing to Yoshimura etal. (2006a, b).

11.2.2 Transcriptome Data Analysis

The details of the preparation of M. rosa Illumina transcriptome will be reported

elsewhere. The M. rosa transcriptome assembly covered various larval stages,

attached larvae, metamorphosing larvae, 1-day and 3-day-old juveniles, and adult

tissues. The transcriptome data were mainly assembled using Trinity v2.3.2

(Grabherr etal. 2011). But we soon discovered that Trinity failed to resolve highly

repetitive tandem repeat structures often found among potential shell protein tran-

scripts, owing to the use of xed short kmer length (kmer=25bp). Therefore, we

switched to a multiple kmer strategy, in which the Illumina read data were assem-

bled using Transabyss v1.6.3 (Robertson etal. 2010) with kmer size ranging from

21 to 161bp, for every 10bp. These assemblies were then merged using the script

Transabyss-merge. Transcript abundance estimation was performed using the soft-

ware RSEM (Li and Dewey 2011). The levels of transcript expression were normal-

ized according to coverage and then converted to the relative expression levels in

FPKM values. Differential expression analysis was performed using the software

edgeR (Robinson etal. 2010).

11.2.3 Preparation ofShell-Soluble andShell-Insoluble

Fractions

To extract shell proteins, the mineralized shell plates, namely, OPs, WPs, and BP as

described above, were subjected to acid decalcication. After removing the pro-

mosa (all soft body parts) from the shell, OPs, WPs, and BP were then cleaned by

11 Identication ofBarnacle Shell Proteins byTranscriptome andProteomic Approaches

108

10% bleach solution (household bleach) for 3 days, with daily exchange of the

bleach solution. After bleaching, the shell plates were washed with distilled water

extensively and then dried in freeze drier for 1day. The dried shell plates were then

crushed into ne powder smaller than 110μm (lteredby a mesh lter). Altogether,

20 M. rosa specimens (integument diameter ranging from 2 to 5cm) were being

sacriced. Then, around 12g of OP powder, 20g of WP powder, and 20g of BP

powder were independently decalcied by 10% acetic acid overnight in 4°C.By

4000 × g centrifugation, the supernatants, which represent the acid-soluble fractions

(ASF), and the pellets, which represent the acid-insoluble fractions (AIF) of OP,

WP, and BP, were separated. Acetic acid in the ASF of OP, WP, and BP was

exchanged with Milli-Q water by dialysis (6–8kDa dialysis membrane). Proteins in

the ASFs were then concentrated by 3 kDa cutoff centrifugal ltering device

(Amicon Ultra, Millipore). After removal of lipids by methanol-chloroform precipi-

tation, puried ASF protein samples were redissolved in 40μl 2 M thiourea. To

extract proteins from AIF, Milli-Q water-washed insoluble pellets from OP, WP, and

BP were treated with 1ml of 8M urea for 1h at room temperature. The retrieved

solubilized AIFs were then subjected to methanol-chloroform precipitation. The

puried AIF proteins were redissolved in 40μl 2M thiourea. ASF and AIF protein

samples were then loaded onto a 4–20% SDS-PAGE gradient gel. The gel was sub-

sequently stained with Coomassie Brilliant Blue G-250.

11.2.4 In-Gel Digestion, LC-MS/MS Analysis, andProtein

Identication

The AIF and ASF samples from OPs, WPs, and BP were divided into nine size frac-

tions and independently retrieved for in-gel digestion. Destaining, reduction, alkyla-

tion, in-gel digestion, and the nal peptide digest retrieval were performed as in Sun

etal. (2012), except that 1μg of trypsin (sequencing grade) dissolved in 100μl of

10mM of triethylammonium bicarbonate was applied to thegel piecesfrom each

sample; standard shotgun proteomics techniques were used to analyze the samples

on a Thermo Scientic LTQ VelosTM platform (Thermo Fisher Scientic, Bremen,

Germany). Operation details of the LC-MS/MS can refer to Zhang etal. (2014).

Identication of protein was performed using MASCOT search engine (v2.3.02),

with the parameter setting the same as in Zhang etal. (2014). The transcriptome

data generated from M. rosa (method and sample details mentioned above) were in

silico translated to a protein database using Transdecoder v.3.0.3 (Haas and

Papanicolaou 2016), with minimum length of the open reading frame set at 100

amino acid. Decoy sequences consisting of shufed protein sequences for each of

the proteins were concatenated to the original protein database to generate a target-

decoy database for protein identication.

Y. H. Wong et al.

109

11.2.5 Analysis ofM. rosa Shell Protein Candidates

The identied protein sequences were analyzed for the presence of N-terminal sig-

nal sequence using SignalP server 4.1 (Nielsen 2017), followed by manual correc-

tion. The identied proteins with N-terminal signal peptide were then selected and

categorized based on their annotation status.

11.3 Results andDiscussion

11.3.1 Development ofShell Formation inM. rosa Juvenile

Figure 11.2a shows the life cycle of thoracican barnacles including M. rosa. M. rosa

larvae are in general encased in chitinous cuticles. They do not produce any calci-

ed shell structure at any larval stage. Formation of calcium carbonate shell occurs

only after post-settlement metamorphosis, that is, the transition of a cypris larva into

a juvenile barnacle. This means that the genes responsible for barnacle shell forma-

tion start to be expressed during metamorphosis and in the early juvenile stage. As

shown in Fig.11.2b, in M. rosa, the progression of calcication was the most obvi-

ous at the rst 5days of juvenile development. At 5days after metamorphosis, all

shell plates are completely nontransparent, indicating these shell plates are all

mineralized.

11.3.2 Identication ofProteins inM. rosa Shell Extract

In total, 431 translated protein sequences (Fig.11.3a) from all the 6samples, that is,

the AIF and ASF of BP/OPs/WPs, were identied using the translated protein data-

base generated from the multiple kmer Transabyss pipeline (contains 35,998 trans-

lated nonredundant protein sequences).

11.3.3 Filtering Shell Proteome Data withTranscriptome

Analysis

We noticed that, among the 431 identied translated protein sequences identi-

edfrom all the shell samples, ovary proteins such as vitellogenin were detected

(Fig. 11.3a), indicating that the shell plate samples were contaminated by ovary

tissue, and bleaching obviously was not effective in removing all of these ovary

11 Identication ofBarnacle Shell Proteins byTranscriptome andProteomic Approaches

110

proteins stuck to the shell during sampling. From our preliminary SEM and EDX

analyses, we have observed that at the juvenile stage, shell material is beingactively

synthesized. We therefore selected the identied proteins whose transcripts were

four-folds (16 times) more highly expressed in the juvenile stages than in the larval

stages (FDR <0.001). This criterion ltered out 349 proteins, leaving 82 relevant

shell proteins, among which 60 contain a signal peptide (Fig. 11.3b). With the

juvenile- specic expression pattern and the presence of signal peptide as the crite-

ria, the list of identied shell protein candidates became much smaller yet specic.

Among the 60 shell protein candidates, some of them were implicated in shell for-

mation in other systems, including carbonic anhydrase, C-type lectin domain pro-

tein, and chitin-binding domain proteins (Miyamoto etal. 1996; Suzuki etal. 2007;

Joubert etal. 2010). In addition, 37 unknown/novel proteins were also discovered,

and these proteins are now under our major investigation. Our results clearly

Fig. 11.2 Life cycle of M. rosa and the development of young juvenile. (a) The barnacle spawned

as nauplius larva, which undergoes four successive molts and then transforms into a cypris larva.

The cypris larva actively searches for an appropriate substratum and will then fully commit to

settlement, which involves attachment of the larval body onto the substratum by cement secretion,

and metamorphosis, which involves ecdysis of the cyprid body molt. The soft newly metamor-

phosed juvenile soon begins to feed on planktons using the cirri and develops into an adult barna-

cle, protected by calcied shells. (b) Development of young juvenile. The 0day juvenile, that is,

the stage when ecdysis was just completed. Five days after completion of metamorphosis, the

juvenile has developed mineralized shell plates

Y. H. Wong et al.

111

indicate that our transcriptome-based ltering method, that is, to lter the shell pro-

teome data with the corresponding transcript expression patterns, is an excellent

pipeline for the purpose of relevant shell protein identication.

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

Y. H. Wong et al.

113© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_12

Chapter 12

The Optical Characteristics ofCultured

Akoya Pearl Are Inuenced by Both

Donor andRecipient Oysters

ToshiharuIwai, MasaharuTakahashi, ChiemiMiura, andTakeshiMiura

Abstract The characteristics of a cultured pearl are inuenced by two kinds of

pearl oysters. One is the donor pearl oyster, which provides a small piece of mantle

to be transplanted, and the other is the recipient pearl oyster, in which the pearl

nucleus and a small piece of mantle are transplanted. Generally, the brightness,

luster, and color of pearls are affected by the donor oyster, while the thickness of

nacre is affected by the recipient oyster. Previously, we have indicated that the sex

of recipient pearl oyster directly affects the quality of pearl, and the optical charac-

teristics measured by FT-IR (Fourier transform infrared spectroscopy) of pearl pro-

duced from male and female pearl oysters signicantly differ (Iwai etal. Aquaculture

437:333–338, 2015). Moreover, using the various strains of Akoya pearl oyster as

recipient and the same donor oyster, the produced Akoya pearl had different spectra

for each strain. Also, besides the culture of the Akoya pearl oyster, the transplanta-

tion also produced different optically characterized pearls by breeding them in vari-

ous environments. These results suggested that the optical characteristics underlying

pearl quality are not only the inuence by donor oyster but also the sex, the strain,

and the breeding conditions of recipient oyster.

Keywords Cultured pearl · Optic characteristic · FT-IR

T. Iwai · T. Miura (*)

Graduate School of Agriculture, University of Ehime, Matsuyama, Ehime, Japan

e-mail: t-iwai@agr.ehime-u.ac.jp; miutake@agr.ehime-u.ac.jp

M. Takahashi

Pearl Oyster Research Laboratory, Shimonada Fisheries Cooperative, Uwajima, Japan

e-mail: simonada-jf-t-i@md.pikara.ne.jp

C. Miura

Graduate School of Agriculture, University of Ehime, Matsuyama, Ehime, Japan

Department of Global Environment Studies, Faculty of Environmental Studies,

Hiroshima Institute of Technology, Hiroshima, Japan

e-mail: chiemi8@agr.ehime-u.ac.jp

114

12.1 Introduction

The technique of producing pearls in the body of Akoya pearl oyster by transplanta-

tion is a biotechnology developed in Japan that skillfully utilized the biomineraliza-

tion ability of oysters (Southgate and Lucas 2008; Wada 1999). The characteristics

of a cultured pearl are inuenced by two kinds of pearl oysters. One is the donor

pearl oyster, which provides a small piece of mantle to be transplanted; the other is

the recipient pearl oyster, in which the pearl nucleus and a small piece of mantle are

transplanted to produce a pearl. Generally, the brightness, luster, and color of pearls

are affected by the donor oyster, while the thickness of nacre is affected by the

recipient oyster (Wada and Komaru 1996). Only the recipient oyster is required to

have good growth rate and disease-resistance trait; hence, the effect of recipient

oysters on the quality of produced pearls has been underestimated.

In transplantation, the pearl nucleus and a small piece of mantle were trans-

planted into the gonads of recipient pearl oyster. The sex and state of gonads affect

the characteristics and quality of cultured pearl. In our previous study, we have

clearly indicated that the sex of recipient pearl oyster directly affects the quality of

pearl, and the optical characteristics measured by FT-IR of pearl produced from

male and female pearl oysters signicantly differed (Iwai etal. 2015). These results

showed that the optical characteristics and quality of pearls have strong inuence

not only by donor oysters but also by recipient oysters. Moreover, it is also known

that the characteristics and quality of pearls are affected by the difference of pearl

cultivation area in general. In this study, we measured the FT-IR spectra of the

pearls produced with various recipient and donor strain combinations and pearls

cultured in various culture area and, then based on these characteristics, clustered

and investigated what kind of factors inuence the quality of pearls.

12.2 Materials andMethods

12.2.1 Akoya Pearl Oysters

Akoya pearl oysters, Pinctada fucata, were obtained from K, S, U, and M pearl

farmers. The strains “U-H” and “U-T” were two types of Akoya pearl oyster strains

produced by U pearl farmer. The strain “M-H” was one of several strains produced

by M pearl farmer.

12.2.2 Pearl Culture

Various conditions, such as transplantation date and harvest date, in pearl culture

used in each test are described in each gure legend. The method of pearl culture

used for this study was generally in accordance with the method that the pearl

farmer carried out.

T. Iwai et al.

115

12.2.3 Fourier Transform Infrared Spectroscopy andData

Analysis

The methods in this study were described previously (Iwai etal. 2015). Briey, a

Fourier transform infrared (FT-IR) ALPHA Platinum ATR spectrometer (Bruker

Optics, Germany) was used to acquire spectral data of the pearl surface. A spectral

resolution of 4cm−1 was applied, and 64 scans were co-added and averaged for each

spectrum. Transmission/absorption FT-IR spectra were collected, and data from 400

to 4000 wavenumbers were stored on a computer while purging the instrument con-

tinuously with dry air to reduce water vapor absorption. Ward’s algorithm was used

for hierarchical clustering as described previously (Helm etal. 1991). The hierarchi-

cal clustering was performed with the cluster analysis module of OPUS 7.2 soft-

ware (Bruker Optics, Germany). Hierarchical cluster analysis was used to objectively

assess clustering of the FT-IR vector-normalized spectra obtained from the different

culture site or recipient and donor combination.

12.3 Results

12.3.1 The Cultured Pearls fromVarious Culture Sites

In order to evaluate the characteristics of cultured pearls, ten culture areas mainly

carrying pearl culture in Uwa Sea, Ehime Prefecture, Japan, were selected

(Fig.12.1a). Nine months after transplantation at various culture areas, the pearls

were collected and analyzed by FT-IR spectrometry (Fig.12.1b, c). However, visual

evaluation of the quality of these pearls was difcult. Accordingly, we examined the

differences between the pearls from various culture area based on the optical prop-

erties of the pearl surface using the FT-IR spectrometer (Fig.12.1c). By performing

hierarchical cluster analysis with FT-IR spectrometry, these pearls were classied

into two clusters. When comparing this result with the actual culture area, the cul-

ture pearls were separated into a north area and a south area. These results indicated

that the optical characteristics of pearls from various culture areas were signi-

cantly different. Furthermore, the optical characteristics of each pearl were deter-

mined according to the culture area of Akoya pearl oyster.

12.3.2 The Cultured Pearls fromCombination ofDonor

andRecipient Pearl Oysters

In order to investigate how the characteristics of pearls change by combination of

donor and recipient oysters, pearls were produced using three kinds of Akoya pearl

oyster strains as donors and two kinds as recipients (Fig.12.2a). Unlike pearls pro-

duced by various culture area, pearls differed greatly in appearance. Pearls using

12 The Optical Character istics ofCultured Akoya Pearl Are Inuenced by Both…

116

A: Uwajima-KushimaB: Uwajima-Torikubi C: Miura-OkiD: Miura-Oku

E: Shimonada

-Tanohama

H: Shimonada

-Tsuboi

F: Shimonada

-Suge

J: Mishouwan

-Takahata

I: Mishouwan

-Shinnkan

G:Shimonada

-Hirai

Uwajima

Ainan

©OpenStreetMapcontributors

A: Uwajima-Kushima

B: Uwajima-Torikubi

C: Miura-Oki

D: Miura-Oku

E: Shimonada-Tanohama

H: Shimonada-Tsuboi

F: Shimonada-Suge

J: Mishouwan-Takahata

I: Mishouwan-Shinnkan

G:Shimonada-Hirai

North-Area

South-Area

Heredity

North-Area South-Area

5 km

Fig. 12.1 The cultured pearls from various culture sites. The pearls were cultured by the following

conditions: transplantation date, July 20, 2014; harvest date, Feb 23, 2015–Mar 6, 2015; pearl

nucleus size, 6.67mm at single transplantation; donor oyster, Akoya pearl oyster strain-K; recipi-

ent oyster, Akoya pearl oyster strain-S; transplantation was operated by single pearl farmer. (a) A

map showing the point where pearls were cultivated. (b) A picture of the cultured pearls. (c)

Dendrogram of a hierarchical cluster analysis showing objective spectral diversity in pearlsfrom

vrious culture sites. Cluster analysis was performed with vector-normalized spectra. The spectral

distances were calculated with Pearson’s correlation coefcient, and Ward’s algorithm was used

for hierarchical clustering. Hierarchical clustering is a statistical data analysis procedure for the

classication of similar objects into North-Area and South-Area groups. The name of each sample

is as follows: taking “A-7T,” for example, “A” indicates the cultured area shown in (a, b), “7”

means a serial number of the obtained pearl, and “T” is the initial of Tounen in Japanese and means

that the cultivated period is within 1year

T. Iwai et al.

117

Donor

Recipient

Akoya Pearl Oyster

AkoyaPearl Oyster

Heredity

Donor:

Recipient:

: Akoya -

Fig. 12.2 The cultured pearls from combination of donor and recipient pearl oyster. The pearls

were cultured by the following conditions: transplantation date, July 2–3, 2015; harvest date, Jan

19, 2016; pearl nucleus size, 6.67 mm at single transplantation were operated by single pearl

farmer. (a) A picture of the cultured pearl. (b) Dendrogram of a hierarchical cluster analysis show-

ing objective spectral diversity in pearls from combination of donor and recipient pearl oyster.

Cluster analysis was performed with vector-normalized spectra. The spectral distances were calcu-

lated with Pearson’s correlation coefcient, and Ward’s algorithm was used for hierarchical clus-

tering. Hierarchical clustering is a statistical data analysis procedure for the classication of similar

objects into strain “U-T” and “U-H” as recipient pearl oyster groups

12 The Optical Characteristics ofCultured Akoya Pearl Are Inuenced by Both…

118

Akoya pearl oyster strain “M-H” as a donor clearly produced white pearls compared

to the other four pearls, irrespective of using the Akoya pearl oyster strain “U-H” or

“U-T” as recipient. Accordingly, we examined the differences between the pearls

from various combinations of donor and recipient oysters based on the optical prop-

erties of the pearl surface using the FT-IR spectrometer (Fig.12.2b). By performing

hierarchical cluster analysis with FT-IR spectrometry, these pearls were classied

into two clusters. These two clusters were highly dependent on the recipient strain.

These results suggested that the effect on the optical characteristics of pearls was

stronger in recipients than in donor.

12.4 Discussion

What is the factor that affects the quality of pearls which has been the great attention

of pearl culture farmers and researchers since the beginning of pearl culture? From

various experiments and experience of pearl farmers, it was known that the donor’s

characteristics have a great inuence on the quality of pearls, and research on donors

has been actively conducted in recent research (Fujimura and Komaru 2017;

Odawara etal. 2017). However, the recipient oysters that actually produce the pearl

were considered to affect only the size of the pearl, such as the thickness of the

nacre, and attention has not been paid to recipient oysters. Our study revealed that

the sex of the recipient oysters affects not only the thickness of the pearl nacre but

also the optical characteristics and quality of the pearl (Iwai etal. 2015). Therefore,

in this study, we investigated the inuence of donor and recipient on pearls, which

is a factor inuencing the optical characteristics of pearls. As a result, it was revealed

that the inuence of the recipient on the optical characteristics of the pearl surface

by the FT-IR is larger than that of the donor which has been conventionally men-

tioned. It was suggested that in order to further improve quality in the future pearl

culture, it was necessary to carry out seedling production and selective breeding of

pearl oyster as a recipient with consideration of the inuence on the quality of

pearls. Moreover, the quality of pearls was also known to be strongly inuenced by

the pearl culture area. However, there were not many cases comparing pearls with

different pearl culture environments. In this study, comparison of produced pearls

was possible according to Akoya pearl oysters, which was transplanted under the

same condition and was bred in each culture area, and the optical characteristics of

pearls using FT-IR could be evaluated objectively. As a result of the comparison, the

pearls produced in each culture area of Uwa Sea had two different optical character-

istics, and it was able to distinguish from the north area and the south area clearly.

These results are consistent with the actual impression of the pearl culture farmer,

and it became clear that the method of distinguishing pearls by FT-IR is useful. It is

necessary to investigate in detail what kind of environmental factors in breeding

culture areas are affecting the optical characteristics of pearls. We investigate vari-

ous factors on the quality and optical characteristics of culture pearls, and we could

contribute to the improvement of pearl quality by investigating the inuence.

T. Iwai et al.

119

Acknowledgments This study was supported by a grant-in-aid from the Science and Technology

Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (26019A).

This research was supported by grants from the Project of the NARO Bio-oriented Technology

Research Advancement Institution (The Special Scheme to Create Dynamism in Agriculture,

Forestry, and Fisheries Through Deploying Highly Advanced Technology). This research was sup-

ported by grants from the Project of the NARO Bio-oriented Technology Research Advancement

Institution (The Special Scheme Project on Regional Developing Strategy).

References

Fujimura T, Komaru A (2017) The inuence of nacreous crystal thickness of donor oysters on

interference color appearance and crystal thickness of pearls in Pinctada fucata (Japanese pearl

oyster). Nippon Suisan Gakkaishi 83:971–980 (in Japanese with English abstract)

Helm D, Labischinski H, Schallehn G, Naumann D (1991) Classication and identication of

bacteria by Fourier-transform infrared spectroscopy. JGen Microbiol 137:69–79

Iwai T, Takahashi M, Ido A, Miura C, Miura T (2015) Effect of gender on Akoya pearl quality.

Aquaculture 437:333–338

Odawara K, Ozaki R, Takagi M (2017) Inuence of the thickness of the nacreous elemental lam-

ina of the pearl oyster Pinctada fucata used as donor oysters on the pearls. Nippon Suisan

Gakkaishi 83:981–995 (in Japanese with English abstract)

Southgate PC, Lucas JS (2008) The pearl oyster. Elsevier, Oxford

Wada KT (1999) Science of the pearl oyster. Shinju Shinbunsha, Tokyo (in Japanese)

Wada KT, Komaru A (1996) Color and weight of pearls produced by grafting the mantle tissue

from a selected population for white shell color of the Japanese pearl oyster Pinctada fucata.

Aquaculture 142:25–32

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

12 The Optical Character istics ofCultured Akoya Pearl Are Inuenced by Both…

121© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_13

Chapter 13

Inuence ofB Vitamins onProliferation

andDifferentiation ofOsteoblastic Bovine

Cell Cultures: AnInVitro Study

KentUrban, JuliaAuer, SebastianBürklein, andUlrichPlate

Abstract Minerals and vitamins affect bone formation, genetics, nutrition, and

hormones. Studies mainly focus on the elucidation of the metabolic pathways dur-

ing biomineralization to get an idea of how to promote the process of biomineraliza-

tion invivo and invitro. One qualied approach to reach this is to investigate the

inuence of different substances on the proliferation and differentiation of osteo-

blastic cell cultures invitro. The aim of this study was to investigate the effects of

different types of single B vitamins (B6, B9, and B12) and a vitamin B complex (B1,

B2, B3, B5, B6, B9, and B12) on proliferation and differentiation of primary bovine

osteoblastic cells invitro. The proliferation of osteoblastic cells during the experi-

ments was evaluated by cell number analysis while cultivating. The expression of

marking proteins of the osteogenic differentiation was evaluated by immunohisto-

chemistry. Previous experiments with seven different B vitamins in different con-

centrations revealed a positive effect on cell proliferation with increasing

concentration caused by three B vitamins: pyridoxal (B6), folic acid (B9), and cobal-

amine (B12). The use of vitamin B6, B9, and B12 in different concentrations resulted

in a signicant increase of cell proliferation (p<0.05). But neither the B vitamins

nor the B vitamin complexes stimulated the expression of the typical bone cell

proteins.

Keywords Vitamins · Vitamin B · Bone formation · Bone regeneration · Bone

metabolism · In vitro biomineralization

K. Urban

Department of Periodontology and Operative Dentistry in the School of Dentistry,

University of Münster, Münster, Germany

e-mail: Kent.Urban@ukmuenster.de

J. Auer · U. Plate (*)

Department of Maxillofacial Surgery, VABOS, University of Münster, Münster, Germany

e-mail: plateu@uni-muenster.de

S. Bürklein

Central Interdisciplinary Ambulance in the School of Dentistry, University of Münster,

Münster, Germany

e-mail: Sebastian.Buerklein@ukmuenster.de

122

13.1 Introduction

Focussing on the rapid development of implant- and bone-substitute materials as

well as their integration in autologous tissue and optimized wound healing pro-

cesses, it gets more and more important to give well-known therapies new perspec-

tives. For a better adaption of implants in surrounding tissue, not only material’s

biocompatibility but also bone regeneration as a part of wound healing becomes

subject of scientic research. Many existing therapies on bone regeneration dealing

with vitamin D and calcium supplementation are well established (Avenell etal.

2014; Javed etal. 2016; Vandenbroucke etal. 2017). The effect of some other single

vitamins or vitamin complexes is already investigated (Masse et al. 2010; Elste

etal. 2017). Nevertheless, the effects in direct supplementation with vitamins in

bone defects on bone regeneration are not completely understood yet (Owen etal.

1990; Herrmann etal. 2013).

13.2 Materials andMethods

For the experiments, two different cell culture mediums were used:

(a) Medium MP (High Growth Enhancement Medium without L-glutamine, MP

Biomedicals GmbH, Eschwege, Germany) and

(b) Medium PAN (High Growth Enhancement Medium without L-glutamine and

without B vitamins; PAN Bio-Tek, Bad Frierdrichshall, Germany).

Both mediums were supplemented with 4% FCS (fetal calf serum) (Biochrom,

Berlin, Germany), 10.000IU/ml penicillin, 10.000μg/ml streptomycin, 250μg/ml

amphotericin B, and 200 mM L-glutamine (Biochrom KG Seromed, Berlin,

Germany). Primary bovine osteoblast-like cells were used in this study. The cells

were derived from the periosteum of calf metacarpus according to the instructions

of Jones and Boyde (1977). Tissue explants were cultured for 4weeks in medium

MP supplemented with 10% FCS, 10.000IU/ml penicillin, 10.000μg/ml strepto-

mycin, 250 μg/ml amphotericin B, 10 mM ß-glycerophosphate, and

200mML-glutamine (Biochrom KG Seromed, Berlin, Germany), at 37°C and 5%

CO2 in humidied air. The medium was replaced once a week. When cells reached

conuence, they were harvested (20min incubation at 37°C with 0.4g collagenase,

98.8mg nutrient mixture (HAM’s F– 10) in 10ml HEPES (2-[4-(2-hydroxyethyl)-

1-piperazinyl] ethane sulfonic acid), repeatedly washed with phosphate-buffered

saline (PBS), subsequently incubated for 15 min, and centrifuged. Pellets were

resuspended in PBS and the cell number was determined in a cell counter (CASYWI

Modell TT, Schärfe System, Reutlingen, Germany). Osteoblasts (10.000/cm2) were

seeded on 24-well plate plastic petri dishes (Nunc TFS, Roskilde, Denmark) with

different mediums and B vitamin concentrations. Cell proliferation was determined

after 1, 3, and 5days, respectively. Cell morphology evaluation was performed by

K. Urban et al.

123

means of light microscopy. To determine the cell number, digital photos were taken

under standardized conditions and cells were counted using the software program

ImageJ with the plug-in Cell Counter. The experiments were repeated six times and

all data were analyzed using one-way analysis of variance and post hoc Scheffé

Test. As basic level of vitamin complex, the standard concentration of included B

vitamins in medium MP was used (4mg/l thiamine, 0, 4mg/l riboavin, 4mg/l

niacin, 4mg/l pantothenic acid, 4mg/l pyridoxal, and 4mg/l folic acid). This con-

centration was called medium MP0 and served as negative control for each group.

Vitamin concentration was increased to achieve different concentrations (from

basic level up to fourfold concentration) and called MP1–MP3 (Table13.1). Vitamin

B12 was not included and only solely added to medium MP.Based on medium PAN

without B vitamins, single B vitamins were also added in different concentrations

(from 4 to 12 μg/ml) (Table 13.1). Medium PAN without any B vitamins was

called PAN0 and was used as negative control for all experiments with medium

PAN.An increase in cell number after 3 and 5days was observed in all test groups

(Figs.13.1 and 13.2). For cell differentiation, collagen I, osteonectin, and osteo-

calcin were assessed by immunohistochemistry (Dako EnVision System, Dako,

Hamburg, Germany) under standardized conditions with uorescence microscopy

Table 13.1 Different B-vitamin concentrations in μg/ml for (a) medium MP, MP0=standard B

vitamin concentration (negative control), MP1 = double concentration of vitamin, MP2 = triple

concentration of vitamin, MP3 = fourfold concentration of vitamin and (b) medium PAN, PAN0 =

without vitamin (negative control), PAN1 = 4μg/ml, PAN2 = 8μg/ml, and PAN3 = 12μg/ml vitamin

Medium/vitamin B1 B2 B3 B5 B6 B9 B12

MP04 0,4 4 4 4 4 0

PAN000 00 000

MP1 B6 4 0,4 4 4 8 4 0

MP2 B6 4 0,4 4 4 12 4 0

MP3 B6 4 0,4 4 4 16 4 0

MP1 B9 4 0,4 4 4 4 8 0

MP2 B9 4 0,4 4 4 4 12 0

MP3 B9 4 0,4 4 4 4 16 0

MP1 B12 4 0,4 4 4 4 4 8

MP2 B12 4 0,4 4 4 4 4 12

MP3 B12 4 0,4 4 4 4 4 16

PAN1 B6 0 0 0 0 4 0 0

PAN2 B6 0 0 0 0 8 0 0

PAN3 B6 0 0 0 0 12 0 0

PAN1 B9 0 0 0 0 0 4 0

PAN2 B9 0 0 0 0 0 8 0

PAN3 B9 0 0 0 0 0 12 0

PAN1 B12 0 0 0 0 0 0 4

PAN2 B12 0 0 0 0 0 0 8

PAN3 B12 0 0 0 0 0 0 12

13 Inuence ofB Vitamins onProliferation andDifferentiation ofOsteoblastic Bovine…

124

(Axioplan 2, Carl Zeiss, Germany) and processed using AxioVision 3.1 software

(Carl Zeiss, Germany).

Sixty thousand osteoblasts/cm2 were seeded in 100 × 20mm plastic petri dishes

(TPP, Trasadingen, Schweiz). After cultivation for 14days at 37 °C in an atmo-

sphere of 5% CO2 in the different media, osteoblastic cells were xed with methanol

and primary antibodies were used (diluted 1:100 with Blocking Solution): anti-

collagen I (Biotrend, Cologne, Germany), anti-osteocalcin (TaKaRa Bio, MoBiTec,

Goettingen, Germany), and anti-osteonectin (TaKaRa Bio, MoBiTec, Goettingen,

Germany). Digital images were taken under standardized conditions by uores-

cence microscopy (Axioplan 2 Carl Zeiss, Germany) and processed using the soft-

ware AxioVision 3.1 (Carl Zeiss, Germany).

13.3 Results andDiscussion

Proliferation of osteoblastic cells during the experiments was evaluated by cell

number analysis during culture. Expression of marking proteins of osteogenic

differentiation was assessed by immunohistochemistry. Proliferation and

Fig. 13.1 Cell proliferation with B vitamin complex and single B vitamins in medium MP.Medium

MP with B vitamin complex was used with different single vitamin B concentrations over 5days.

All groups started with nearly the same cell number at day 1 p<0.05 (data not shown). An increase

in cell number after 3 and 5days was observed in all groups

K. Urban et al.

125

differentiation of osteoblasts enable the production of extracellular matrix (ECM)

and is therefore the initial step in the formation of calcied tissue, especially bone.

This study mainly focuses on the elucidation of the metabolic pathways during

biomineralization to get an idea of these processes invivo and invitro. Previous

experiments with seven different B vitamins in different concentrations revealed a

positive effect on cell proliferation with increasing concentrations caused by three

B vitamins pyridoxal (B6), folic acid (B9), and cobalamine (B12) (Dhonukshe-Rutten

etal. 2003; Swart etal. 2016).

Characteristics of the B vitamins are:

• Essential nutrients that must be added to the body for normal cell formation,

growth, and development

• Catalyzing and regulatory functions as cofactors and enzymes

• Being water-soluble, without danger of hypervitaminosis when overdosed

Under the conditions of the present study, the use of vitamin B6, B9, and B12

in different concentrations resulted in a signicant increase of cell proliferation

(p < 0.05). The negative control groups MP0 and PAN0 differed signicantly

from all other groupsMP1,2,3 and PAN1,2,3(p < 0,05)(Table 13.1, Figs.13.1, 13.2,

and 13.3).

Fig. 13.2 Cell proliferations with single B vitamins in medium PAN.Medium PAN was used only

with different single vitamin B concentrations over 3 and 5days. All groups started with nearly the

same cell number at day 1 p<0.05 (data not shown). An increase in cell number after 3 and 5days

was observed in all groups

13 Inuence ofB Vitamins onProliferation andDifferentiation ofOsteoblastic Bovine…

126

Finally, supplementation of certain vitamins in an appropriate concentration sig-

nicantly increased proliferation and improved growth of osteoblast-like cells.

Probably, this increased cell growth leads to a superior wound healing and bone

regeneration.

Using vitamin culture media to enhance proliferation and collagen formation

(Herrmann etal. 2007) of osteoblast-like cells during culturing seems to be quite

reasonable.

During different stages of differentiation, several proteins are synthesized by the

osteoblasts (Roach 1994; Kim etal. 1996):

(a) Collagen type I, the main component of the ECM

(b) Non-collagenous proteins like alkaline phosphatase

(d) Later in the differentiation progress osteocalcin

While collagen type I as well as the protein marker osteonectin could be detected by

immunohistochemistry at the end of the experiments (Fig.13.4), none of the cell

cultures showed any signs of osteocalcin expression. Neither the B vitamins nor the

B vitamin complexes signicantly stimulated the expression of the typical bone cell

proteins.

Supplementation of other vitamins, e.g., ascorbic acid, supports the synthesis of

collagen, and the ECM (extracellular matrix) (Urban etal. 2012) seemed to have a

positive effect compared to vitamin-free cultures (Najeeb etal. 2016; Fratoni and

Brandi 2015; Zhaoli Dai and Koh 2015). Bioactive vitamins placed on implant sur-

Fig. 13.3 Osteoblastic cells (a) after 3days of incubation medium PAN0; (b) after 3days of incu-

bation medium PAN3 (with vitamin B9); (c) after 5days of incubation medium PAN0, (d) after

5days of incubation medium PAN3 (with vitamin B9)

K. Urban et al.

127

face may positively affect wound healing due to direct transmission into surround-

ing tissue. Mixing those vitamins into bone-substitute materials could be another

benecial aspect in bone regeneration by diffusion into the wound (Bartold etal.

2016). Further investigations should follow with the aim to increase supportive

effects of vitamins on biological processes such as wound healing, bone regenera-

tion, and revised healing of bone implants.

Acknowledgment Financial support of the Arbeitsgemeinschaft für Elektronenoptik e.V.,

Germany, is gratefully acknowledged.

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JP, Herrmann W (2007) The effect of B-vitamins on biochemical bone turnover Osteocalcin

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blind placebo controlled trial. Clin Chem Lab Med 45(12):1785–1792

Fig. 13.4 Immunohistochemical analyses of collagen (primary antibody “anti-collagen I

(Biotrend, Cologne, Germany)”) in medium PAN with single vitamins B6, B9, and B12

13 Inuence ofB Vitamins onProliferation andDifferentiation ofOsteoblastic Bovine…

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

K. Urban et al.

129© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_14

Chapter 14

Rice Plant Biomineralization: Electron

Microscopic Study onPlant Opals

andExploration ofOrganic Matrices

Involved inBiosilica Formation

NoriakiOzaki, TakuyaIshida, AkiyoshiOsawa, YumiSasaki, HiromiSato,

MichioSuzuki, KeijuOkano, andYukoYoshizawa

Abstract Biologically formed amorphous silica (biosilica) is widely found in dia-

toms, marine sponges, terrestrial plants, and bacteria, some of which have been well

characterized. Although rice plants produce large amounts of biosilica (plant opal)

in their leaf blades and rice husks, the molecular mechanism of biomineralization is

still poorly understood. In the present study, we investigated the fundamental prop-

erties of plant opal in leaf blades of the rice plants (Oryza sativa) by scanning elec-

tron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy. The

number of fan-shaped plant opal increases in the motor cells (bubble-shaped epider-

mal cells) during heading time. High-resolution SEM analysis revealed that the

plant opals are composed of nanoparticles, as is the case with diatom silica and

siliceous spicule of sponge. Organic matrices in biominerals have been considered

to control mineralization. Biosilicas in diatom and marine sponge are formed under

ambient conditions using organic matrices, unique proteins, and long-chain poly-

amines. In this study, we report the establishment of purication method of plant

opals from rice leaf blades. Finally, we succeeded in extracting organic matrices

from the puried plant opal.

Keywords Biomineralization · Biosilica · Organic matrix · Plant opal · Rice plant

N. Ozaki (*) · T. Ishida · A. Osawa · Y. Sasaki · H. Sato · K. Okano · Y. Yoshizawa

Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University,

Akita, Japan

e-mail: ozanor@akita-pu.ac.jp; m14g005@akita-pu.ac.jp; keijuo@akita-pu.ac.jp;

yyoshizawak@akita-pu.ac.jp

M. Suzuki

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life

Sciences, The University of Tokyo, Tokyo, Japan

e-mail: amichiwo@mail.ecc.u-tokyo.ac.jp

130

14.1 Introduction

Biomineralization is widespread phenomenon by which organisms produce miner-

als by using organic matrices under ambient conditions (Lowenstam and Weiner

1989). The resulting minerals, termed biominerals, have a specic morphology and

demonstrate excellent physical properties. Biogenic amorphous silica (biosilica) is

known as one of the representative biominerals. Biosilica is widely observed in

skeleton of diatoms, spicules of marine sponges, spore coats of bacteria, and epicu-

ticles of certain higher plants. As in the case of other biominerals, organic matrices

in biosilicas are thought to be associated with silica formation. Until now, in dia-

toms, glass sponges, and certain facultative bacteria, several organic matrices

involved in silica formation have previously been identied, such as unique proteins

(silafn, glassin, and CotB1) and long-chain polyamines (LCPAs) (Sumper and

Kröger 2004; Shimizu etal. 2015; Matsunaga etal. 2007; Motomura etal. 2016).

These matrices are highly charged and have been shown to promote silica formation

from monosilicic acid solution near a neutral pH.On the other hand, there are few

studies on silica formation of higher plants. The best-known example of silicon

accumulating plants, rice plants (Oryza sativa), produces a large amount of biosili-

cas (plant opal) in their leaf blades and rice husks. Silicon uptake mechanism from

soil is transporter mediated and energy dependent (Ma etal. 2006). Plant opal depo-

sition has been shown to improve disease resistance, light interception, and mechan-

ical properties (Ma and Takahashi 2002). Despite the importance of plant opal in

rice plants, information on the molecular mechanisms involved in plant opal forma-

tion is very limited. To date, there are no published reports on organic matrices from

plant opal of rice, as far as we know. In the present work, we have investigated the

fundamental properties of plant opals by microscopic analyses and extracted an

organic matrix from fan-shaped plant opals.

14.2 Materials andMethods

14.2.1 Plant Materials andMicroscopy

The leaf blades of rice plants (Oryza sativa cv. Akita-sake-komachi) were collected

from paddy eld in Akita Prefectural University. Optical microscope (BX51,

Olympus) and eld emission SEM (SU-8010, Hitachi) were used to analyze the

microstructure of leaf blades and morphology of plant opals. The chemical compo-

sition of silica was conrmed with an energy-dispersive X-ray spectroscope (EDX;

EMAX x-act, HORIBA). Prior to counting the number of fan-shaped opal, the leaf

blade was incinerated at 550 °C. The ashed sample was carefully placed on

Superfrost micro slides (Matsunami) and observed with the optical microscope.

N. Ozaki et al.

131

14.2.2 Extraction ofOrganic Matrices fromPlant Opals

Plant opals were separated from mature leaves according to the method of Setoguchi

etal. (1990) with slight modications. After washing with distilled water, rice leaf

blades were cut into small pieces and ground with a mixer mill. The homogenate

was passed through a nylon mesh lter of 258μm pore size (NB60, Aton). The

ltrate containing plant opals is put on a watch glass, and heavier fan-shaped plant

opals were separated from lighter small leaf fragment by a series of decantation.

Cell walls bound to fan-shaped plant opals were removed by sulfuric acid and cel-

lulase (Onozuka R-10, Wako, Osaka) treatments. The resulting fan-shaped opals

were suspended in 4M hydrogen uoride (HF) solution and left for 2h at room

temperature. After centrifugation for 10min at 2000 g, the supernatant was sub-

jected to dialysis (Float-A-Lyzer, Spectra-Por) against distilled water. The dialysate

(HF-soluble fraction) was lyophilized and the resulting organic matrices were sub-

jected to tricine-SDS-PAGE.Organic matrices were detected by Coomassie Brilliant

blue (CBB; EzStain AQua, ATTO) and silver (SilverXpress, Thermo Fisher) stain-

ing. The HF-soluble fraction passed through the 30kDa molecular weight cutoff

lter (Amicon Ultra 15, Millipore) was used to raise antibody in rabbits through a

commercial source. Another set of sample was subjected to SDS-PAGE and subse-

quent blotting to a PVDF membrane. The membrane was blocked with 5% skim

milk diluted in TBS (Tris-buffered saline; 50mM Tris, 0.85% NaCl, pH 7.2) with

Tween 20 (0.05%) for 1 h and then incubated in the primary antibody solution

(1:100) for 1 h at room temperature. After washing, it was incubated in 1:3000

diluted solution of AP-conjugated goat anti-rabbit IgG secondary antibody (Bio-

Rad) for 1h at room temperature. The target protein was visualized using a BCIP/

NBT substrate system (Bio-Rad).

14.3 Results andDiscussion

14.3.1 Morphology andFunction ofPlant Opals

Representative SEM micrographs of several types of plant opal from rice leaf blades

are shown in Fig.14.1. SEM-EDX analysis proved that all types of plant opals were

composed of silicon, oxygen, and carbon (data not shown). The surface of leaf

blades consists of silicied cells, called long cells making plate-shaped opals

(Fig. 14.1a), prickle hair (Fig. 14.1b), and short cells making dumbbell-shaped

opals (Fig.14.1c). Our preliminary study revealed that plant opals on leaf surface

are formed and silicied within 2weeks after germination (data not shown). The

possible function of these opals on the leaf surface is considered to protect against

biotic and abiotic stress, such as pathogenic bacteria and dryness. Only the

14 Rice Plant Biomineralization: Electron Microscopic Study onPlant Opals…

132

fan- shaped plant opal (Fig. 14.1d) is formed inside the leaf blade. Our research

group conrmed that fan-shaped opal contains highest concentration of silica (Sato

etal. 2017). Based on higher magnication image of fan-shaped opals, we found

that the opal comprised ne particles with several tens of nanometer in diameter

(Fig.14.1e). Similar silica nanoparticles are also found in the spicules of marine

sponges (Aizenberg etal. 2005)

In order to investigate the formation period of the fan-shaped opal, we collected

rice leaves from July to October. To count the number of opals, each collected leaf

blades were treated by calcination at 550°C.The optical micrograph of surface of

leaf blade after calcination is shown in Fig.14.2a. Fan-shaped plant opals (arrow-

heads) were observed in the motor cells between the veins of the leaf (white dotted

line) and were aligned like a backbone. As shown in Fig.14.2b, the number of

fan- shaped opals sharply increased around the heading time. The heading time

(dotted line) in Akita prefecture was early August with strong sunlight. Although

the function of the fan-shaped opal had not been claried (Kaufman etal. 1979;

Fig. 14.1 Representative scanning electron microscope (SEM) micrographs of plant opals in rice

leaf blades. (a) Plate-shaped opal. (b) Silicied prickle hair. (c) Dumbbell-shaped opals. (d)

Puried fan-shaped opal. (e) Silica nanoparticles constituting fan-shaped opal

N. Ozaki et al.

133

Agarie etal. 1996), we suggested the possibility that fan-shaped opals have a role

in guiding light to chloroplast by optical simulation and actual optical experiment

(Sato etal. 2016). Another possibility is that the fan-shaped opals formed inside

the leaf play a role like a bone and improve the posture for light interception (Ma

and Takahashi 2002). In either case, the fan-shaped opal may promote photosyn-

thesis necessary for panicle formation.

14.3.2 Organic Matrices fromSeparated Plant Opals

Although organic matrices from biosilica are considered to play important roles in

silica formation, there are few reports about organic compounds in plant opal (phy-

tolith) of higher plants (Elbaum etal. 2009). It was difcult to extract organic matri-

ces from opals, due to complex structure consisting of cuticle and silica layer. So,

we developed a novel silica purication method and succeeded in extracting organic

matrices from the separated plant opal. Cell walls bound to the silica were decom-

posed by successive treatments with sulfuric acid and cellulase. The HF-soluble

fraction from opals was subjected to tricine-SDS-PAGE.At least two main bands

(arrowheads) were detected using CBB staining (Fig.14.3a). On the other hand,

only one band was stained with silver staining (Fig.14.3b, arrowhead). From these

results, the band with an apparent molecular mass of 10kDa is referred to as the

HF-soluble protein. Only the 10 kDa protein strongly reacted with the antibody

raised against the fraction containing HF-soluble protein (Fig.14.3c, arrowhead),

indicating that the antibody was highly specic to this protein. The amino acid

sequence analysis by LC-MS/MS and immunohistochemical analysis of this protein

are in progress. Detailed results will be reported elsewhere.

Fig. 14.2 Fan-shaped plant opal formation in the ag leaf of rice. (a) Optical micrograph of sur-

face of ashed leaf blade. White dotted lines indicate vascular bundle. When looking at fan-shaped

opals from another angle, it looks like rectangle structures (arrowheads). (b) Time course of forma-

tion of fan-shaped plant opals. Dotted line indicates the heading time (ear-forming period). The

number of fan-shaped opal was measured by counting rectangle structures (arrowhead in a)

14 Rice Plant Biomineralization: Electron Microscopic Study onPlant Opals…

134

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abc

kDa

MSSS

Fig. 14.3 Tricine-SDS-PAGE (12.5%) and Western blot analyses of the HF-soluble organic matri-

ces extracted from fan-shaped plant opals. (a) Two major bands (arrowheads) were detected by

CBB staining. (b) Only one band (arrowhead) was positive for silver staining. (c) Western blotting

with an anti-HF-soluble proteins antiserum. Only one immunoreactive band was detected at about

10kDa. M molecular mass standards. S HF-soluble fraction

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Setoguchi H, Okazaki M, Suga S (1990) Calcication in higher plants with special reference to

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14 Rice Plant Biomineralization: Electron Microscopic Study onPlant Opals…

137© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_15

Chapter 15

DMP1 Binds Specically toType I

Collagen andRegulates Mineral

Nucleation andGrowth

AnneGeorge, ElizabethGuirado, andYinghuaChen

Abstract Extracellular matrix of bone and dentin is highly complex and involves a

dynamic process of deposition and removal. Cells are the main architect that build

this designer matrix that is highly specialized to calcied tissues. Osteoblasts or

odontoblasts secrete both collagen and noncollagenous proteins in a temporal and

spatial manner. Type I collagen self-assembles and forms a fabric-like template onto

which noncollagenous proteins and mineral bind in a well-regulated manner. Dentin

matrix protein 1 (DMP1) is one such noncollagenous protein that contains several

acidic groups that can bind calcium ions which in turn binds phosphate and initiates

the calcication process. In this study, we demonstrate that DMP1 is localized at

specic sites on the self-assembled collagen matrix of dentin. In vitro nucleation

studies on demineralized and deproteinized dentin slice adsorbed with DMP1 show

bundles of well-ordered needle-shaped nanohydroxyapatite deposited on the dentin

matrix. The nucleated mineral structures had uniform length and width and their long

axis was oriented parallel to the collagen bril axis. Overall, the physiologically self-

assembled collagen and DMP1 mediated ordered deposition of nanocrystalline HAP.

Keywords Dentin matrix protein 1 · Collagen · Hydroxyapatite · Mineralization ·

Extracellular matrix

15.1 Introduction

Biological composites such as bone, dentin, and cementum consist of a crystalline

inorganic phase mainly carbonated hydroxyapatite embedded within a biopoly-

meric organic matrix (Veis 1993; Veis and Dorvee 2013). The cells that produce

these mineralized matrices exert a regulatory and exquisite control over the minerals

they deposit, creating materials of varied shapes, sizes, and high tensile strength.

A. George (*) · E. Guirado · Y. Chen

Brodie Tooth Development Genetics & Regenerative Medicine Research Laboratory,

Department of Oral Biology, University of Illinois at Chicago, Chicago, IL, USA

e-mail: anneg@uic.edu; eguira@uic.edu; yinghua@uic.edu

138

The stereospecic interaction of macromolecules with biominerals represents a

unique phenomenon in nature (Addadi etal. 2001; Grzesiak etal. 2017).

Mineralized tissue formation results from the coordinated activity of highly

differentiated cells. During the differentiation process, these cells secrete an

extracellular matrix which performs various functions (Grzesiak etal. 2017; Hirata

etal. 2005; Kang etal. 2015). A distinct feature of the ECM in calcied tissues is

that it contains both crystal growth promoters and inhibitors (Goldberg and Smith

2004). Components in the ECM are involved in directing the deposition of specic

calcium phosphate polymorphs during the formation of the calcied matrix.

The deposition of extracellular matrix and subsequent mineralization are a

temporal and spatial event. They are assembled from a collagenous brillar network

containing small amounts of tissue-specic regulatory proteins and other widely

circulatory proteins. Phosphoproteins are one of the major component groups

among the noncollagenous proteins found in all calcied tissues (Ravindran and

George 2014). They have been postulated to play an important role in the initiation

of calcication and possibly in the regulation of crystal growth. These regulatory

proteins are responsible for controlled crystal growth within the collagenous matrix.

Dentin matrix protein (DMP1) is an example of a regulatory protein localized in

the ECM of bone and dentin (George etal. 1993, 1994, 1995; He and George 2004).

Understanding the players and the mechanism by which the extracellular matrix cal-

cies is important to understand the function of mineralized biological tissues. In the

current study, we demonstrate that DMP1 is localized to specic sites on the dense

physiologically arranged collagen brils of demineralized dentin matrix. Using a

demineralized and deproteinized dentin slice, we demonstrate that adsorbed recom-

binant DMP1 can promote the deposition of organized nanocrystalline hydroxyapa-

tite ribbons. Modeling of DMP1 shows that the protein could have a exible carboxyl

terminal domain, which might help in binding collagen and Ca2+ in the ECM.

15.2 Methods

15.2.1 Expression ofRecombinant DMP1 Protein

Full-length rat recombinant DMP1 was produced as previously published (Bedran-

Russo etal. 2013; Srinivasan etal. 1999). BSA (bovine serum albumin) was used as

a negative control.

15.2.2 Preparation ofDemineralized Dentin Wafers

forImmunogold Labeling

Third molars without caries were selected and kept frozen following approval of the

Institutional Review Board at the University of Illinois at Chicago (protocol 2009-

0198). Coronal dentin cross-sections 1.5mm thick were cut from each tooth and

A. George et al.

139

further sectioned to produce 250-μm-thick slices using a hydrated diamond blade

saw (IsoMet 1000, Buehler, Lake Bluff, IL, USA). These slices represent the sam-

ples hereinafter referred to as dentin wafers.

Wafers measuring 1.5 × 3 × 0.250mm thick were placed together in 14% EDTA

at 4 °C for 10 days. Demineralization was veried by X-ray microradiography

(MX-20 Faxitron, LLC, Lincolnshire, IL). These dentin wafers were dehydrated in

a series of ethanol from 30% to 100%, trimmed, and embedded in epoxy resin, and

ultrathin sections 70nm were placed on 300 mesh formvar-coated nickel grids.

Grid-mounted tissue sections were processed for colloidal gold

immunohistochemistry by incubating the sections with rabbit primary antibody

against DMP1 (1:250) as published before. After which the sections were incubated

with anti- rabbit IgG colloidal gold particles (10nm gold particles) and washed. For

controls, the sections were incubated with 20 nm gold-conjugated anti-rabbit

IgG.All the sections were imaged either unstained or stained with uranyl acetate

and imaged using JEOL JEM 1220 Electron Microscope and digital images obtained

using Erlangshen ESW 1000w 785 camera.

15.2.3 Preparation ofDemineralized andDeproteinized Dentin

Wafers forNucleation Experiments

Demineralized dentin wafers were subjected to treatment with 1M NaCl for 1h at

room temperature to disrupt loosely bound noncollagenous proteins. Samples were

then incubated in 0.25% trypsin-EDTA twice for 4h at 37°C to remove strongly

bound, endogenous noncollagenous proteins. These wafers were cryosectioned to

5μm, stained with Stains-All® (Sigma-Aldrich), and imaged to conrm the com-

pleteness of removal of noncollagenous proteins (Padovano etal. 2015).

In vitro nucleation was performed as reported earlier for 14days (He etal. 2003).

At the end of the time point, samples were washed and dehydrated by passing

through a series of graded ethanol solutions, 30%, 50%, 90%, and 100% for 10min

each. The samples were nally dehydrated by immersing them in a solution of hexa-

methyldisilazane embedded in epoxy resin, and ultrathin sections of 70nm were

placed on copper TEM grid and examined in a JEOL JEM 1220 TEM (JEOL Ltd.,

Tokyo, JAPAN) Gatan accelerating voltage of 60kV.Images were recorded using a

CCD camera (Gatan Inc., Pleasanton, CA).

15.2.4 Modeling ofDMP1

The I-TASSER (Iterative Threading ASSEmbly Renement) server was used for

DMP1 3D structure prediction (Roy etal. 2010; Yang etal. 2015; Yang and Zhang

2015a, b; Zhang 2009). The polypeptide strand was built by using human DMP1

sequence data from NCBI protein database (NP_004398.1). The program uses ab

15 DMP1 Binds Specically toType ICollagen andRegulates Mineral Nucleation…

140

initio modeling as well as the LOMETS multiple threading program to retrieve pro-

teins with similar folds from the PDB library. Replica-exchange Monte Carlo simu-

lations generated a pool of protein structures from which low free-energy states

were selected. Iterative template fragment assembly simulations identied the top

ve near-native models. C scores between −5 and 2 are assigned to these models,

with more positive scores indicating greater condence.

15.3 Results

15.3.1 DMP1 Binds toDensely Packed Collagen Fibrils

oftheDentin Matrix

Immunogold labeling experiment showed that gold particles were distributed

specically over the dense collagen bril of the demineralized dentin matrix

(Fig.15.1a). Demineralization of dentin led to the dissolution of the mineral only

and did not alter the antigenic properties of DMP1in the matrix. Staining the dentin

slice with uranyl acetate and lead citrate showed that DMP1 was localized on the

collagen matrix. Image presented in Fig.15.1b shows that the labeling was mostly

localized at the edge of the 67nm periodic band of type I collagen. This region

would correspond to the gap region of the self-assembled collagen matrix.

Fig. 15.1 Localization of DMP1 on the dentin wafer. (a) Unstained transmission electron

micrograph of the demineralized dentin slice on which immunogold labeling with anti-DMP1 was

performed prior to TEM.Scale bar represents 200nm. (b) Same experiment wherein the dentin

slice was stained with uranyl acetate and lead citrate. Scale bar represents 0.2μm

A. George et al.

141

15.3.2 Structural Characterization oftheMineral Deposited

ontheCollagen Matrix ofDentin

In vitro nucleation studies showed that demineralized and deproteinized dentin

slices adsorbed with DMP1 initiated calcium phosphate deposition. At the end of

14days, TEM analysis showed the presence of nanostructured mineral structures

which were aligned to the dentin collagen brils (Fig.15.2). At low magnication

(Fig.15.2a), it was apparent that the dentin slice was coated with electron-dense

calcium phosphate mineral, particularly concentrated around the dentinal tubule.

The high magnication image (Fig. 15.2b) shows that the dark electron-dense

deposits are bundles of mineral lamellae which are about 3–5nm apart. The needle-

like mineral lamellae were all oriented with their long axis nearly parallel to the

collagen brils. Selected area electron diffraction analysis (SAED) of the acicular

deposits (Fig.15.3a) showed the presence of nanocrystalline hydroxyapatite as they

displayed the 002 reection pattern (Fig.15.3b).

15.3.3 Computation andAb Initio Models ofDMP1

Computer modeling was used to gain insight into the molecular shape of DMP1.

Two predicted models are shown in Fig.15.4a, b. Of these, (b) represents the high-

est scoring model C-score of (−1.59), while, model a had a C-score of (−2.54).

Fig. 15.2 Structural morphology of DMP1 mediated mineral deposition: (a) transmission electron

microscopy (TEM) image of DMP1 mediated HA nanocrystal on demineralized and deproteinized

dentin slice. Scale bar represents 0.5μm. (b) Higher magnication image showing bands of closely

aligned mineral bers aligned parallel to the collagen brils. Scale bar represents 50nm

15 DMP1 Binds Specically toType ICollagen andRegulates Mineral Nucleation…

142

Model a shows a “globular” N-terminus with an “extended arm” at the C-terminus.

However, Model b with a lower free-energy shows that the C-terminus had the pro-

pensity to fold into beta sheets, while the N-terminus remained globular.

15.4 Discussion

The constituents of the extracellular matrix of bone and dentin are responsible for

modulating nucleation of apatite nanoparticles and their growth into micrometer-

sized crystals. The organic matrix mainly consists of type I collagen which forms

the template that directs ordered deposition of mineral crystals. The rest of the

Fig. 15.3 Characterization of the Ca-P deposits: (a) TEM image shows nanocrystalline Ca-P

deposits with acicular morphology. (b) Selected area electron diffraction pattern indexed to nano-

crystalline HAP.Note that the 002 reections are in the process of forming arcs indicating pre-

ferred orientation of the c-axis of the HAP parallel to that of the collagen brils

Fig. 15.4 Energy-minimized models of full-length DMP1: the I-TASSER (Iterative Threading

ASSEmbly Renement) server was used for DMP1 3D structure prediction (NP_004398.1). The

program uses ab initio modeling as well as the LOMETS multiple threading program to retrieve

proteins with similar folds from the PDB library

A. George et al.

143

components of the organic matrix consist of noncollagenous proteins, lipids, and

proteoglycans. Each of these classes of macromolecules play decisive roles in the

controlled growth of HAP on the collagen matrix.

In this study, we used the demineralized and deproteinized dentin slice as a

template to demonstrate the role of extracellular dentin matrix protein 1, a member

of the noncollagenous class of proteins in calcied tissues. The processed dentin

slice contains brillar cross-linked collagen arranged in a three-dimensional

supramolecular architecture.

We rst showed the spatial localization of DMP1 on the demineralized collagen

matrix. Demineralization was able to expose the epitope site for the antibody to bind

to DMP1. Gold particles were specically localized on the collagen bril. As DMP1

is a calcium-binding protein, there is a good possibility that some might be removed

from the dentin matrix during the process of demineralization. Specic localization

of DMP1 on the collagen template suggests that initiation of mineral deposition

would be site specic.

In vitro nucleation experiments conducted on the dentin wafer adsorbed with

DMP1 show abundant Ca-P deposits around the dentinal tubule. Higher magnica-

tion of the deposits shows that nucleation is not a random event, but the process is

initiated in spatially distinct nucleation sites. Closer inspection shows that the nano-

crystals are deposited as bundles of mineral structures in a highly organized manner

with their c-axis oriented nearly parallel to the collagen brils. The nanocrystalline

deposits had nearly similar length and widths suggesting that the self-assembled

collagen brils could dictate the morphology and growth of the mineral deposits.

The mineral structures were characterized as nanocrystalline HAP. McNally etal.

also reported the presence of similar rigid hydroxyapatite struts encased in the col-

lagen matrix of the human femoral bone (McNally etal. 2012). In a new model for

the ultrastructure of bone based on TEM analysis, Schwarcz reports the presence of

stacks of mineral lamellae packed around the brils and that the lamellae are spaced

less than 1nm apart (Schwarcz 2015). Similar ultrastructure of the mineral phase

was observed in this study.

With the use of molecular modeling, we have demonstrated that DMP1 contains

a exible loop between the C- and N-termini where the proteolytic cleavage site

resides. The stringent model (Fig.15.4b) showed that C-DMP1 had the propensity

to form beta sheets. This could be envisaged as DMP1in the presence of calcium

adopts a beta sheet structure (He etal. 2003). We have shown that the C-terminal

portion of DMP1 is highly acidic due to abundant glutamic acid residues along

with serines which could be phosphorylated, thereby increasing the negative charge

(He etal. 2005). We have also demonstrated that intermolecular assembly of the

peptides at the C-terminus in the presence of calcium ions was important to form a

stereospecic template for hydroxyapatite nucleation. It is possible that the nano-

crystalline hydroxyapatite brillar deposits observed in this study could be depen-

dent on the beta sheet conformation of C-DMP1 and the conned gap regions

present in the three-dimensional supramolecular architecture of self-assembled

type I collagen.

15 DMP1 Binds Specically toType ICollagen andRegulates Mineral Nucleation…

144

Overall, this study demonstrates that biomineralization is a complex cell-

mediated process and the DMP1 has an intimate relationship with the collagen

matrix. Such an interaction dictates the site-specic nucleation of Ca-P nanocrystal-

line hydroxyapatite. Subsequently, other proteins in the ECM might be responsible

for crystal fusion and controlled growth of the initially nucleated crystals.

Acknowledgments This work was supported by National Institutes of Health Grant DE 11657

and the Brodie Endowment Fund (AG). “This work made use of instruments in the Electron

Microscopy Service (Research Resources Center, UIC.”

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basis for repair and tissue engineering. Crit Rev Oral Biol Med 15:13–27

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

15 DMP1 Binds Specically toType ICollagen andRegulates Mineral Nucleation…

147© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_16

Chapter 16

Exploration ofGenes Associated with

Sponge Silicon Biomineralization inthe

Whole Genome Sequence oftheHexactinellid

Euplectella curvistellata

KatsuhikoShimizu, HirokiKobayashi, MichikaNishi, MasatoshiTsukahara,

TomohiroBito, andJiroArima

Abstract Silicatein is the rst protein isolated from the silicon biominerals and

characterized as constituent of the axial lament in the silica spicules of the demo-

sponge Tethya aurantia, by signicant sequence similarity with cathepsin L, an ani-

mal lysosomal protease, and as a catalyst of silica polycondensation at neutral pH

and room temperature. This protein was then identied in a wide range of the class

Demospongiae and in some species of the class Hexactinellida. Our attempt to iso-

late silicatein from the silica skeleton of Euplectella was unsuccessful, but instead

we discovered glassin, a protein directing acceleration of silica polycondensation

and sharing no signicant relationship with any proteins including silicatein. The

present study aims to verify the existence of silicatein by exploring the whole genome

DNA sequence database of E. curvistellata with the sequence similarity search.

Although we identied the sequences of glassin, cathepsin L and chitin synthetase,

an enzyme synthesizing chitin, which has already been found in the silicon biominer-

K. Shimizu (*)

Platform for Community-Based Research and Education, Tottori University, Tottori, Japan

e-mail: kshimizu@tottori-u.ac.jp

H. Kobayashi

The United Graduate School of Agricultural Sciences, Tottori University, Tottori, Japan

M. Nishi

Faculty of Agriculture, Tottori University, Tottori, Japan

e-mail: B14A3142U@edu.tottori-u.ac.jp

M. Tsukahara

BioJet Ltd, Uruma, Okinawa, Japan

e-mail: tsuka@biojet.jp

T. Bito · J. Arima

Faculty of Agriculture, Tottori University, Tottori, Japan

e-mail: bito@muses.tottori-u.ac.jp; arima@muses.tottori-u.ac.jp

148

als in E. aspergillum, silicatein failed to be identied. Our result indicates that silica-

tein is not essential for poriferan silicon biomineralization in the presence of glassin.

Keywords biosilicication · Silica · Silicatein · Glassin · Chitin

16.1 Introduction

Silicon biominerals are produced by the living organisms through physiological

activities in contrast to silicon-based manmade products, often manufactured

through processes with high energy consumption and harsh impacts on the

environment. Understanding of the mechanisms on silicon biomineralization is

expected to offer the prospect of developing environmentally benign routes to

synthesize silicon-based materials. Silicon biominerals generally contain a small

amount of organic substances, which may help production of silicon biominerals at

physiological conditions.

Phylum Porifera (sponges) consists of four classes, Hexactinellida (glass

sponges), Demospongiae (demosponges), Calcarea (calcareous sponges), and

Homoscleromorpha, among which Hexactinellida, Demospongiae, and

Homoscleromorpha produce silica biominerals while calcium carbonate biominerals

occur in Calcarea.

The demosponge Tethya aurantia produces a large quantity of silicon biomineral

in a form of silica as needlelike structures or spicules, allowing us to isolate and

analyze the organic molecules occluded in the biomineral. Silicatein, the rst protein

isolated from silicon biomineral and characterized, constitutes the axial lament in

the spicules; shares signicant sequence similarity with cathepsin L, an animal lyso-

somal protease; and catalyzes silica polycondensation at neutral pH and room tem-

perature (Shimizu etal. 1998; Cha etal. 1999). This protein and its gene were then

identied in a wide range of the class Demospongiae (Krasko etal. 2000; Pozzolini

etal. 2004; Funayama etal. 2005; Müller etal. 2007). In addition, PCR products

encoding a partial silicatein sequences were amplied in the class Hexactinellida

including Crateromorpha meyeri (Müller etal. 2008a), Monorhaphis chuni (Müller

et al. 2008b), and Euplectella aspergillum (unpublished data. The sequence was

deposited to GenBank database by Müller et al. in 2011. The accession number

FR748156). Our attempt to isolate silicatein from the silica skeleton of the E. asper-

gillum and E. curvistellata was unsuccessful, but instead we discovered glassin as a

protein directing acceleration of silica polycondensation (Shimizu et al. 2015).

Sequences encoding silicatein have not identied from the transcriptome analysis of

Aphrocallistes vastus by Riesgo etal. (2015). Veremeichik etal. (2011) tried to iso-

late silicatein genes from Pheronema raphanus, Aulosaccus schulzei, and Bathydorus

levis, resulting in only identication of Aulosaccus sp. silicatein-like sequence with

cysteine at the catalytic residue instead of serine as seen in silicateins of other spe-

cies. Collectively, the existence of silicateins has been unsettled in Hexactinellida.

The present study aims to verify the existence of genetic information on silica-

tein by exploring the whole genome DNA sequence database of E. curvistellata

K. Shimizu et al.

149

with the sequence similarity search and discusses on relationship of silicatein and

glassin in silicon biomineralization of Porifera.

16.2 Materials andMethods

Live specimens of E. curvistellata were collected at a depth of 236m at 32°30 N,

129°10 E in the East China Sea on March 4, 2012, as described previously (Shimizu

etal. 2015) and then stored in ethanol at −20°C.Genomic DNA was extracted

from the specimen stored in ethanol with DNeasy Blood & Tissue Kit (QIAGEN,

Hilden, Germany). The genomic DNA library was prepared from mechanically

fragmented genomic DNA with TruSeq DNA prep kit (Illumina, San Diego, CA,

USA). Then, the library was sequenced with MiSeq (Illumina) three times. The

raw reads were trimmed and assembled using Genomics Workbench (CLC Bio

Inc., Aarhus, Denmark). Sequence similarities were analyzed with NCBI BLAST

program.

16.3 Results andDiscussion

16.3.1 Construction ofWhole Genome DNA Library ofE.

curvistellata

The library of E. curvistellata whole genome DNA was constructed with the

next- generation DNA sequencer. Total three runs of sequencing gave rise to

27,939,250 reads, being assembled to 442,583 contigs with the average length of

427 and the median length of 420 (Table16.1). The longest contig covers 145,960

while the shortest is 18 nucleotides. Total number of the nucleotides reached to

190,209,345. This number can be roughly considered as a genome size of the spe-

cies, with fairly matching to that of Amphimedon queenslandica, being 167 Mbp

(Srivastava etal. 2010).

To evaluate the quality of the library, we run the blast program with Aphrocallistes

vastus Cox3 gene (GenBank accession no. EU000309.1) (Rosengarten etal. 2008)

as a query. As a result, we obtained the single contig_1075 containing not only

Cox3 gene but also the whole mitochondrion DNA sequence, 19,700bp. This

result indicates that the library is qualitatively sound and can be useful for gene

searching.

Table 16.1 Summary of whole genome sequencing and assembly

Reads

Number of

contigs

Maximum

contig (base)

Minimum

contig (base)

N50

(base)

Average

length

(base)

Total length

(base)

27,939,250 442,583 145,960 18 420 427 190,209,345

16 Exploration ofGenes Associated withSponge Silicon Biomineralization…

150

16.3.2 Search forSilicatein Gene

The axial lament was obtained in the intact form by dissolving the silica spicules

of T. aurantia (Shimizu etal. 1998). Although the axial lament was observed in the

cross section of Euplectella silica spicules under the scanning electron microscope

(Weaver et al. 2007), the axial laments or any lamentous materials were not

obtained in our attempt. The extract contained proteins, but these proteins had no

silicatein sequences as long as we examined. On the other hand, a partial silicatein

cDNA from E. aspergillum was archived in DNA sequence database (FR748156)

(Table16.2). In addition, silicateins or silicatein-like sequences have been reported

from the hexactinellid sponges Aulosaccus sp. (Veremeichik etal. 2011), C. meyeri

(Müller etal. 2008a), and M. chuni (Müller etal. 2008b).

To verify the silicatein sequence in E. curvistellata genome, the local blast pro-

gram was executed with these hexactinellid silicatein sequences as well as T. auran-

tia silicateins as queries and E. curvistellata genomic DNA library as a database.

For the partial silicatein cDNA from E. aspergillum, no contig with E values less

than 10 was hit. Similarly, no hit was obtained when T. aurantia silicateins α

(AF032117) and β (AF098670), Aulosaccus sp. silicatein-like (ACU86976),

C. meyeri silicatein (CAP49202), and M. chuni silicatein (CAZ04880) were used as

queries.

The amino acid sequence KNSWG was widely conserved in silicateins and

cathepsin L; 296–300 of T. aurantia silicatein α (Shimizu etal. 1998, AF032117),

296–300 of Suberites domuncula silicatein (Krasko etal. 2000, AJ272013), 292–

Table 16.2 Identication of the genes related to hexactinellid biosilica

Protein Previous description E. curvistellata genome search

Silicatein C. meyeri (Müller etal. 2008a;

CAP49202)

No hit with queries as follows:

E. aspergillum (Müller etal. 2011;

FR748156)

T. aurantia silicateins α and β (Shimizu

etal. 1998; AF032117, and AF098670,

respectively)

Aulosaccus sp. silicatein-like

(Veremeichik etal. 2011; ACU86976)

C. meyeri (Müller etal. 2008a;

CAP49202)

M. chuni (Müller etal. 2008b;

CAZ04880)

M. chuni (Müller etal. 2008b;

CAZ04880)

E. aspergillum (Müller etal. 2011;

FR748156)

No silicatein gene in A. vastus (Riesgo

etal. 2015)

Cathepsin

The gene identied in A. vastus

transcriptome data (Riesgo etal. 2015)

1343bp (324 AAs) composed of four

exons in contig_50860 (306 bp) and

contig_7,117 (8869bp)

Glassin A protein occluded in spicules of

Euplectella (Shimizu etal. 2015)

1638bp (546 AAs) in contig_14,569

(2400bp) and contig_22,997 (1331bp)

Chitin Fluorescent dye staining, X-ray

diffraction, chemical analysis (Ehrlich

and Worch 2007)

Chitin synthase gene 4335bp ORF (1445

AAs) in contig_18,557 (12,682bp)

K. Shimizu et al.

151

296 of S. domuncula cathepsin L (Müller et al. 2003, AJ784224), and 299–303

human cathepsin L (Gal and Gottesman 1988, X12451). In the case of this sequence

used as a query, contig_7,117 (8869bp) was hit. The contig contains the stop codon,

but not the rst Met. The 5′ region, contig_50860, was obtained by running blast

program with the contig_7,117 as a query. Total length of the coding region is

1343bp composed of 4 exons coding 324 amino acid residues and 3 introns. The

predicted protein sequence is more similar to those of sponge and human cathepsin

L than silicateins. In addition, cysteine at the position corresponding to the catalytic

residue and the surrounding sequences in cathepsin L are conserved in the contig,

indicating that the gene encodes cathepsin L but not silicatein. The boundaries of all

the four exons in the cathepsin L of E. curvistellata are identical to those of exon 2/

exon 3, exon 3/exon 4, and exon 4/exon 5in human cathepsin L gene consists of

eight exons and seven introns (Chauhan etal. 1993), suggesting that the exon-intron

structure of cathepsin L in E. curvistellata is conserved in the human gene.

The result of our blast search for silicatein in E. curvistellata genome is consis-

tent with the fact that the silicatein or silicatein-like proteins were not obtained in

dissolution of silica spicules. Previous transcriptome analysis concluded that any

silicatein gene was identied in the hexactinellid A. vastus (Riesgo etal. 2015). It is

unlikely that silicatein exists in all species of the class Hexactinellida. However,

further research should be performed using the hexactinellid species which have

been reported to have the evidence for the existence of silicateins before the

conclusion is drawn.

16.3.3 Search ofGenes Associated withSilicon

Biomineralization

Genes for glassin were assigned by conducting the similarity research with glassin

cDNA as a query. The two contigs 14,569 and 22,997 cover 5′ and 3′ regions of

glassin gene, respectively, while overlapping each other. Some mismatches were

observed in the overlapped and 3′ regions, indicating the assembly in complicated

sequences including the repetitive sequences is incomplete. Therefore, further

renement on the library may be required.

Ehrlich and Worch (2007) reported chitin in E. aspergillum as an organic compo-

nent of their silicious skeletal systems. A gene encoding chitin synthase was

searched using A. queenslandica chitin synthase2 and 3-like protein sequences

(XP_011402997 and XP_003389565, respectively) as queries, and contig_18,557

(12,682 bp) containing 4335 bp open reading ame encoding 1445 amino acid

residues was obtained. Our result suggests that Euplectella is capable of chitin

synthesis and thus is consistent with previous observation on occurrence of chitin in

Euplectella.

16 Exploration ofGenes Associated withSponge Silicon Biomineralization…

152

16.4 Conclusion

The present study aims to verify the existence of genetic information on silicatein

by exploring the whole genome DNA sequence database of E. curvistellata with the

sequence similarity search. We identied the sequences of glassin, cathepsin L and

chitin synthetase, an enzyme synthesizing chitin, which has already been found in

the silicon biominerals in E. aspergillum (Ehrlich and Worch 2007). However,

silicatein failed to be identied in the genome data consistent with the previous

extraction experiment (Shimizu et al. 2015). Although PCR products encoding

partial silicatein sequences have been amplied in some hexactinellid sponges

(Müller etal. 2008a, b), silicatein was not identied in the transcriptome analysis of

A. vastus (Riesgo etal. 2015). Collectively, the existence of silicatein is not evident

in Hexactinellid. At least, silicatein is not essential and glassin is responsible for

silicon biomineralization in E. curvistellata. Therefore, the evidences imply that

there are at least two ways for silicon biomineralization in Porifera in terms of usage

of the protein for silica polymerization, silicatein or glassin, and that the selection

of either protein depends on the species but not on the taxonomic classes.

Acknowledgment This work is supported by JSPS Kakenhi grant number 15K06581.

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

16 Exploration ofGenes Associated withSponge Silicon Biomineralization…

Part III

Genome-Based Analysis of

Biomineralization

157© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_17

Chapter 17

The Origin andEarly Evolution ofSCPP

Genes andTissue Mineralization

inVertebrates

KazuhikoKawasaki

Abstract Various secretory calcium-binding phosphoprotein (SCPP) genes are

involved in the formation of the bone, dentin, enamel, and enameloid in bony verte-

brates. By contrast, no SCPP gene is found in cartilaginous vertebrates. In order to

explain this difference, I investigated the origin and early evolution of SCPP genes.

First, I examined the phylogeny of SPARC-family genes that include evolutionary

precursors of SCPP genes. Then, I analyzed the genomic arrangement of the SCPP

genes and three SPARC-family genes, SPARCL1, SPARCL1L1, and SPARCR1. The

results are consistent with our previous hypothesis that an SCPP gene-like structure

arose in the 5′ half of SPARCL1L1 in a common ancestor of jawed vertebrates, at

about the same time as the origin of mineralized skeleton. It is possible that carti-

laginous vertebrates secondarily lost early SCPP genes, while bony vertebrates

gained various new SCPP genes. Some of these new SCPP genes appear to have

specically involved in scale formation; however, these scale genes were lost in

tetrapods.

Keywords SCPP genes · SPARC gene family · Mineralized skeleton · Bony

vertebrates · Cartilaginous vertebrates · Jawed vertebrates · Scale formation · Gene

duplication · Genome duplication · Vertebrate evolution

17.1 Introduction

Among cardinal traits evolved in vertebrates is mineralized skeleton, which arose in

a common ancestor of jawed vertebrates after the divergence of the lineage leading

to modern jawless vertebrates (Fig.17.1) (Donoghue and Sansom 2002). The bone,

dentin, enamel, and enameloid are principal mineralized skeletal tissues (Donoghue

and Sansom 2002). Among these tissues, the bone was secondarily lost in cartilagi-

nous vertebrates (Eames etal. 2007), and enamel is thought to have originated in

bony vertebrates (Schultze 2016).

K. Kawasaki (*)

Department of Anthropology, Pennsylvania State University, University Park, PA, USA

e-mail: kuk2@psu.edu

158

CSN1S1

CSN2

STATH

HTN3

HTN1

PRR27

ODAM

FDCSP

CSN3

PROL5

PROL3

PROL1

MUC7

AMTN

AMBN

ENAM

Human

RCHY1

G3BP2

USO1

SCPPPQ1

SPARCL1

DSPP

DMP1

IBSP

MEPE

SPP1

AMEL

Coelacanth

amel

dmp1

dsppl1

ibsp

mepe

spp1

scppa3

enam

ambn

amtn

uso1

g3bp2

sparcl1l1

scpppq3

scpppq2

odam

scpppq5

scpppq4

sparcl1

rchy1

scpppq8

sparcr1

Gar

lpq20

g3bp2

uso1

sparcl1l1

lpq8

lpq7

enam

scpp5

scpp7

lpq6

ambn

lpq5

lpq4

scpp3dl

scpp3cl

scpp3bl

scpp3al

scpp9

lpq2

lpq1

sparcl1

dmp1

dsppl1

ibsp

mepe1

mepe2

rchy1

JH128762

JH1294864

sparcr1

Zebrafish

g3bp2

uso1

scpp1

sparcl1

spp1

scpp8

scpp11b

scpp11a

scpp14

scpp13

rchy1

gsp37

scpp12

lpq17

lpq16b

lpq16a

lpq15

lpq14

lpq13

lpq12

lpq19

lpq11

lpq18

lpq10

lpq9

enam

scpp5

scpp7

ambn

scpp3b

scpp3a

odam

scpp9

Elephant

shark

KI635875 KE994958

rchy1

g3bp2

uso1

sparcl1l1

Lamprey

g3bp2

rchy1

uso1

sparcb

spock3

odam

lpq3

JH128571

JH126997 22

JH128070

JH128571

JH129474

JH128739

X/Y

mineralized skeleton jawlessjawed

vertebrates

Fig. 17.1 The arrangement of SCPP genes, SPARC family genes, USO1, G3BP2, and RCHY1 in

the human, coelacanth, gar, zebrash, elephant shark, and lamprey genomes. The phylogeny of

these vertebrates and the origin of mineralized skeleton are shown on the top. Vertical bars repre-

sent chromosomes or contigs (names on the top). Regions separated by >200 kilobases are shown

by double slashes. Horizontal bars represent P/Q-rich SCPP genes (red), acidic SCPP genes (blue),

SPARC-family genes (yellow, circled), and other genes (green, circled). Genes with different tran-

scriptional directions are shown on different sides (right, plus strand; left, minus strand). Orthologs

are connected with a dashed line (a question mark represents unconrmed orthologs). See

Kawasaki etal. (2017) for more details, including lamprey spock3

K. Kawasaki

159

In bony vertebrates, formation of mineralized tissues involves various secretory

calcium-binding phosphoprotein (SCPP) genes, which arose by gene duplication

and form gene clusters (Fig.17.1) (Kawasaki and Weiss 2003). Two types of SCPP

genes are known; one encodes acidic SCPPs and the other Pro and Glu (P/Q)-rich

SCPPs. Most acidic SCPP genes are employed in the formation of the bone and/or

dentin, whereas many P/Q-rich SCPP genes are expressed during the enamel and/or

enameloid formation (Kawasaki 2011). In contrast to bony vertebrates, no SCPP

gene is found in the genomes of cartilaginous vertebrates (Venkatesh etal. 2014). In

order to explain this difference, I investigated the origin and early evolution of

SCPP genes.

SCPP genes evolve rapidly, but all SCPP genes retain a characteristic exon-intron

structure, which allows us to identify SCPP genes without relying on sequence sim-

ilarities (Kawasaki and Weiss 2003). This characteristic exon-intron structure is also

found in the 5′ half of the SPARC-like 1 (SPARCL1) and SPARCL1-like 1

(SPARCL1L1), both located adjacent to SCPP genes in the genomes of coelacanth

and gar (Fig.17.1) (Kawasaki etal. 2004, 2017). Moreover, the 5′ half of elephant

shark sparcl1l1 is highly similar to SPP1 and other acidic SCPP genes, encoding an

extremely acidic sequence, a cluster of Ser-Xaa-Glu (Xaa represents any amino

acids, and the Ser residue is thought to be phosphorylated) near the N-terminus, and

one or more Arg-Gly-Asp (RGD) integrin-binding sequences (Fig.17.2). Based on

these ndings, we proposed that SCPP genes arose from the 5′ half of SPARCL1L1

(Kawasaki etal. 2017).

The 3′ half of SPARCL1 and SPARCL1L1 encodes evolutionarily conserved

amino acid sequences, known as the follistatin-like (FS) domain and the extracel-

lular calcium-binding EF-hand (EC) motif (Bradshaw 2012). Genes encoding the

SPP1

SPARCL1L

SPARCL1

SPARC

jawless - jawed

vertebrates

chartilaginous - bony

vertebrates

VGD1

VGD2

tandem

rayfin - lobefin

vertebrates

divergenc

tandem

SPARCR1

protostome -

deuterostome

small

acidic

N-terminus

large

acidic

N-terminus

pSer

RGD

SPARCR2

mineralization

Fig. 17.2 Duplication

history of SPARC-family

genes and SPP1.

Correlations of gene

duplications (tandem or

VGD1/VGD2), the

divergence of animal

clades (arrowhead), and

the origin of skeletal

mineralization are shown

on the top (not in scale).

Newly evolved

characteristics are shown

under the stem

17 The Origin andEarly Evolution ofSCPP Genes andTissue Mineralization…

160

FS domain and the EC motif constitute the SPARC gene family, which includes

SPARC, SPARCL1, SPARCL1L1, and two SPARC-related genes, SPARCR1 and

SPARCR2 (Kawasaki etal. 2017). Previous studies suggested the duplication his-

tory of these genes (Fig. 17.2) (Bertrand et al. 2013; Torres-Núñez et al. 2015;

Kawasaki etal. 2017). A tandem duplication split the SPARC and SPARCR lineages

before the divergence of protostomes and deuterostomes. In the SPARC lineage,

SPARC and the common ancestor of SPARCL1 and SPARCL1L1 arose in two verte-

brate genome duplications (VGD1/VGD2) (Ohno 1970), thought to have occurred

in the common ancestor of jawless vertebrates and jawed vertebrates (Kuraku etal.

2009). In the SPARCR lineage, SPARCR1 and SPARCR2 originated also in VGD1/

VGD2. SPARCL1 and SPARCL1L1 arose subsequently by tandem duplication in the

common ancestor of cartilaginous vertebrates and bony vertebrates (Fig.17.2).

Duplicated SPARC family genes appear to have differentiated asymmetrically;

while one duplicate maintained ancient characteristics, the other duplicate obtained

new characteristics. The new characteristics, encoded by the differentiated genes,

include a small N-terminal acidic domain arisen early in the SPARC lineage, a large

N-terminal acidic domain in the common ancestor of SPARCL1 and SPARCL1L1,

and a cluster of phospho-Ser (pSer) residues in the N-terminus and an RGD integrin-

binding sequence in SPARCL1L1 (Fig.17.2) (Kawasaki etal. 2017). We hypothe-

sized that the most derived characteristics arose in SPARCL1L1 evolved into SPP1

and other acidic SCPP genes (Fig.17.2). This hypothesis infers that an SPP1-like

structure originated in the common ancestor of jawed vertebrates, at about the same

time as the origin of skeletal mineralization (Fig.17.2) (Kawasaki etal. 2017). In

the present study, I reexamined this hypothesis and analyzed the genomic arrange-

ment of SCPP genes and their adjacent genes.

17.2 Materials andMethods

In the present study, four genes were added to our previous analysis (Kawasaki

et al. 2017). These genes are sparcl1 in the whale shark and Asian arowana

(GenBank XM_020510428.1 and XM_018735764.1, respectively) and sparc in

the Pacic hagsh and gray bichir (reconstructed from GenBank SRX2541845

and SRX796491, respectively). Amino acid sequences of the FS domain and the

EC motif, deduced from the nucleotide sequences, were used to construct a maxi-

mum likelihood (ML) tree and a Bayesian inference (BI) tree, as described previ-

ously (Kawasaki etal. 2017). I considered 95% or higher bootstrap values in the

ML tree and 95% or higher posterior probabilities in the BI tree as statistically

signicant.

K. Kawasaki

161

17.3 Results andDiscussion

17.3.1 Phylogenetic Analysis

Among the newly analyzed genes, the whale shark gene, annotated as “sparcl1,”

was clustered with elephant shark sparcl1l1 (100% in the BI tree; Fig. 17.3).

Elephant shark sparcl1l1 is characterized by a large exon, which encodes a highly

acidic sequence and is anked by a phase-0 intron at the 5′ border and a phase-1

intron at the 3′ border (Kawasaki etal. 2017). A similar large exon was also identi-

ed in this whale shark gene (649–1243 nucleotides of XM_020510428.1), but not

known from any other SPARC family genes. Based on these ndings, I consider this

whale shark gene as the SPARCL1L1 ortholog. Unfortunately, exons 1, 2, 3, and the

5′ end of exon 4 of whale shark sparcl1l1 were not identied, and details of these

exons remain to be elucidated.

Similar to our previous analysis (Kawasaki etal. 2017), SPARC genes in all

jawed vertebrates formed a single cluster (95% and 100% in the ML and BI trees,

respectively), whereas the phylogeny between SPARCL1L1 in cartilaginous verte-

brates, SPARCL1L1 in bony vertebrates, and SPARCL1 in bony vertebrates was not

resolved by signicant statistical supports (Fig.17.3). This result suggests a close

phylogenetic relationship of SPARCL1 and SPARCL1L1.

human

pig

quail

alligator

painted turtle

coelacanth

medaka

tilapia

zebrafish

gar

Asian arowana

coelacanth

gar

whale shark

elephant shark

human

pig

quail

alligator

painted turtle

tropical clawed frog

lungfish

medaka

tilapia

zebrafish

gar

grey bichir

coelacanth

nurse shark

little scate

elephant shark

sea lamprey (sparca)

sea lamprey (sparcb)

Pacific hagfish

Ciona

Branchiostoma

sea urchin

100

SPARC

SPARCL1L1

vertebrates

jawed

jawless

SPARCL1

ML BI

0.50

pig

alligator

0.20

zebrafish

coelacanth

sea lamprey (sparca)

sea urchin

pig

coelacanth

human

painted turtle

Ciona

medaka

alligator

quail

zebrafish

quail

gar

elephant shark

little scate

Asian arowana

tropical clawed frog

Branchiostoma

grey bichir

medaka

nurse shark

tilapia

whale shark

Pacific hagfish

human

sea lamprey (sparcb)

tilapia

coelacanth

painted turtle

lungfish

100

Fig. 17.3 Phylogenetic trees constructed by the ML and BI methods. Different colors show

SPARC, SPARCL1, SPARCL1L1, and sparcb. Non-vertebrate SPARC genes are the common ances-

tor of SPARC, SPARCL1, and SPARCL1L1. Bootstrap values of >70% in the ML tree, and posterior

probabilities of >80% in the BI tree are shown at the node. Vertical bars indicate genes in jawed

and jawless vertebrates. Horizontal bars represent scales

17 The Origin andEarly Evolution ofSCPP Genes andTissue Mineralization…

162

In the present study, I used three jawless vertebrate genes, sparca and sparcb in

lampreys and a hagsh gene. I tentatively call this hagsh gene sparc, because this

gene encodes a small N-terminal acidic domain (71 residues), similar to SPARC in

jawed vertebrates (Kawasaki etal. 2004). In the BI tree, SPARC, SPARCL1, and

SPARCL1L1 in all jawed vertebrates formed a cluster (100%), and this cluster was

most closely related to sparca (100%; Fig.17.3), which is thought to be orthologous

to SPARC (Kawasaki etal. 2017). However, the ML tree did not well support this

relationship and did not resolve the phylogeny of these genes, sparcb, and hagsh

sparc (80% or less; Fig.17.3).

17.3.2 Arrangements ofSCPP Genes andSPARC Family

Genes inVertebrate Genomes

In the genome of the elephant shark, gar, and coelacanth, g3bp2, uso1, and spar-

cl1l1 are clustered, and their order and directions are conserved (Fig.17.1). The

arrangement of these genes suggests that the last common ancestor of jawed verte-

brates had a similar cluster. In the lamprey genome, sparcb is located adjacent to

uso1, which reinforces our previous hypothesis that sparcb is co-orthologous to

SPARCL1 and SPARCL1L1 (Fig.17.1) (Kawasaki etal. 2017). Unlike SPARCL1 or

SPARCL1L1, sparcb encodes a small N-terminal acidic domain, similar to SPARC

(Kawasaki et al. 2017). This is presumably because the large N-terminal acidic

domain evolved in the common ancestor of SPARCL1 and SPARCL1L1 in the jawed

vertebrate lineage after the divergence of the lineage leading to modern jawless

vertebrates (Fig.17.2).

The phylogeny of sparcb as a co-ortholog of SPARCL1 and SPARCL1L1 is not

consistent with the topology of the phylogenetic trees; sparcb, SPARC (including

sparca), SPARCL1, and SPARCL1L1 are intermingled (Fig.17.3). The size of the

encoded N-terminal acidic domain is small in SPARC and sparcb but large in

SPARCL1 and SPARCL1L1, suggesting an asymmetrical functional divergence of

the common ancestor of SPARCL1 and SPARCL1L1. The functional divergence

probably led to differential sequence changes, which may partly explain the low

resolution of these genes in the phylogenetic analysis.

In the gar genome, sparcl1l1, 18 P/Q-rich SCPP genes, sparcl1, and six acidic

SCPP genes are clustered on chromosome 4, while spp1, rchy1, 12 P/Q-rich SCPP

genes, and sparcr1 form a cluster on chromosome 2 (Fig.17.1). Adjacent locations

of MEPE and SPP1 in the human and coelacanth genomes suggest that these two

large SCPP gene clusters in the gar genome were originally connected to each other

(double arrow in Fig.17.1). The original cluster was presumably composed of spar-

cl1l1, P/Q-rich SCPP genes, sparcl1, acidic SCPP genes, rchy1, P/Q-rich SCPP

genes, and sparcr1 in this order. The arrangement of the orthologs of these genes in

the coelacanth genome suggests that the last common ancestor of bony vertebrates

K. Kawasaki

163

had a similar gene cluster, containing at least ve acidic and three P/Q-rich SCPP

genes (Fig.17.1).

It was shown that two zebrash SCPP gene clusters, located on chromosomes 5

and 10, originated by the teleost genome duplication (Braasch et al. 2016). The

locations of spp1 and rchy1 in the gar and zebrash genomes suggest that these two

zebrash SCPP gene clusters are co-orthologons of the SCPP gene cluster on gar

chromosome 2 (Fig.17.1). Twelve P/Q-rich SCPP genes are found on gar chromo-

some 2 and seven P/Q-rich SCPP genes on zebrash chromosomes 5 and 10

(Fig.17.1). By contrast, only scpppq8 (XR_322354.2) was identied in the syntenic

region in the coelacanth genome and none in the tetrapod genomes (Fig. 17.1).

Among these P/Q-rich SCPP genes, the only gene investigated to date is gsp37,

which encodes a matrix protein of the surface layer of scales (Miyabe etal. 2012).

Moreover, expression of ten P/Q-rich SCPP genes on gar chromosome 2 was

detected in the skin that overlies scales but not in the teeth or bone (Kawasaki etal.

2017). Interestingly, both coelacanth scpppq8 and zebrash gsp37 encode similar

sequence elements, including a cluster of pSer residues, a Cys residue (rare in SCPP

genes), and an RGD integrin-binding sequence. These ndings suggest that one or

more P/Q-rich SCPP genes involved primarily in scale formation arose in the com-

mon ancestor of bony vertebrates and that these scale SCPP genes were secondarily

lost in tetrapods.

In summary, the present analysis is consistent with our previous hypothesis

(Kawasaki etal. 2017): an SPP1-like structure originated in the 5′ half of SPARCL1L1

in a common ancestor of jawed vertebrates, roughly contemporaneous with the ori-

gin of skeletal mineralization. It is possible that early SCPP genes were secondarily

lost in cartilaginous vertebrates, while common ancestors of bony vertebrates

gained various acidic and P/Q-rich SCPP genes. Some of these P/Q-rich SCPP

genes may have specically involved in scale formation, but these scale genes were

lost in tetrapods.

Acknowledgments I am grateful to Prof. Joan Richtsmeier at Penn State for encouragement and

Prof. Hiromichi Nagasawa at the University of Tokyo for inviting me to BiominXIV. This work

was made possible by the nancial support from the Department of Anthropology at Penn State

and from the National Institute of Health (P01HD078233).

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

K. Kawasaki

Part IV

Evolution in Biomineralization

167© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_18

Chapter 18

Immunolocalization ofEnamel Matrix

Protein-Like Proteins intheTooth

Enameloid ofActinopterygian Bony Fish

IchiroSasagawa, ShunyaOka, MasatoMikami, HiroyukiYokosuka,

andMikioIshiyama

Abstract Tooth enameloid in bony sh is a well-mineralized tissue resembling

enamel in mammals. It was assumed that the dental epithelial cells are deeply

involved in the formation of enameloid. However, unlike enamel matrix which fully

consists of several ectodermal enamel matrix proteins (EMPs), whether enameloid

matrix contains ectodermal EMPs has been debated for a long time. In the present

study, transmission electron microscopy-based immunohistochemical examina-

tions, using the protein A-gold method with antibodies and antiserum against mam-

malian amelogenin, were performed in order to search for EMP-like proteins in the

cap enameloid of basic actinopterygians, Polypterus and gar. Positive immunoreac-

tivity was detected in the cap enameloid matrix just before the appearance of many

crystallites along collagen brils, indicating that the cap enameloid contains EMP-

like proteins. Immunolabelling was usually found along the collagen brils but was

not seen on the electron-dense brous structures. Therefore, it is conceivable that

the ectodermal EMP-like proteins in cap enameloid are involved in crystallite for-

mation along collagen brils.

I. Sasagawa (*)

Advanced Research Center, The Nippon Dental University, Niigata, Japan

e-mail: ichsasgw@ngt.ndu.ac.jp

S. Oka

Department of Biology, The Nippon Dental University, Niigata, Japan

e-mail: okashun@ngt.ndu.ac.jp

M. Mikami

Department of Microbiology, The Nippon Dental University, Niigata, Japan

e-mail: mikami@ngt.ndu.ac.jp

H. Yokosuka · M. Ishiyama

Department of Histology, School of Life Dentistry at Niigata, The Nippon Dental University,

Niigata, Japan

e-mail: yokosuka@ngt.ndu.ac.jp; ishiyama@ngt.ndu.ac.jp

168

Keywords Bony sh · Enameloid · Enamel matrix protein ·

Immunohistochemistry · Tooth · Transmission electron microscopy

18.1 Introduction

Cap enameloid is a well-mineralized tissue that occupies the tooth tip of actinopter-

ygians (ray-nned bony sh) and corresponds to mammalian tooth enamel.

Enameloid is an attractive tissue for elucidating biomineralization in sh and evolu-

tion of dental hard tissues in early vertebrates because its process of formation is

different from that of mammalian enamel. Unlike amelogenesis in mammals,

enameloid is formed by both odontoblasts and dental epithelial cells. Most of the

organic matrix, including abundant collagen brils, is provided by odontoblasts dur-

ing the matrix formation stage of enameloid, and many odontoblast processes are

present in the matrix. Therefore, enameloid is homologous to the outermost layer of

dentin. Dental epithelial cells are mainly engaged in the degeneration and removal

of organic matrix and in the formation of large crystals during the maturation stage

of enameloid formation (Sasagawa and Ishiyama 2005a, b).

The pattern of mineralization in enameloid is accordingly different from that in

enamel. The mineralization process in cap enameloid is summarized as follows.

During the matrix formation stage, many matrix vesicles (MVs) and electron-dense

brous structures (EDFSs) that are probably derived from MVs (Sasagawa and

Ishiyama 2003) are observed in the collagen-rich enameloid matrix. MVs are often

the rst sites at which crystallites appear. Then, many slender crystallites are depos-

ited along the collagen brils in the enameloid, in a manner similar to the dentin and

bone, during the mineralization stage. During the next maturation stage, the dental

epithelial cells remove the degenerated organic matrix, including collagen brils,

from enameloid and supply inorganic ions to boost crystal growth, like the matura-

tion stage of amelogenesis in mammals. In matured enameloid, bundles are formed

from thick elongated crystals that become twisted with each other, which are thought

to keep the structure of degenerated collagen brils. However, the presence of ecto-

dermal EMPs in enameloid is an unsolved question, in spite of expected active

participation of the inner dental epithelial (IDE) cells.

Polypterus and gar possess both collar enamel in their teeth and ganoine in the

scales, other than cap enameloid (Fig.18.1a). EMP-like proteins were detected in

collar enamel and the ganoine layer by means of immunohistochemistry (Sasagawa

etal. 2012, 2014, 2016). It was assumed that the collar enamel and the ganoine are

an ectodermal element, and EMP-like proteins play an important role in biominer-

alization in these tissues. Therefore, those species might be a suitable model for

examining the localization of EMP-like proteins in cap enameloid. However, avail-

able data are limited concerning the ectodermal EMP-like protein in cap enameloid

of Polypterus and gar. Concerning gar, transmission electron microscopy (TEM)-

based immunohistochemistry recently detected EMP-like proteins in enameloid

matrix (Sasagawa etal. in prep). In the present preliminary study, we report the ne

structure of the initial mineralization and immunolocalization of EMP-like proteins

in the enameloid matrix of Polypterus, in addition to gar.

I. Sasagawa et al.

169

18.2 Materials andMethods

The actinopterygian sh Polypterus, Polypterus senegalus (three specimens, total

length 9.5–19 cm), and gars, Lepisosteus oculatus (three specimens, total length

16–55cm), were used in the present study. Local university animal care committees

approved the euthanasia and all other animal procedures.

We subjected the tooth germs during the stages of enameloid formation to TEM

observations and TEM-based immunohistochemical examinations using antibodies

against mammalian amelogenins (AMEL). An afnity-puried, polyclonal anti-

27kDa bovine AMEL antibody (BAA) (Shimokawa etal. 1984) and anti-25kDa

porcine AMEL antiserum (PAA) (Uchida etal. 1989, 1991) were used in the present

study. In previous studies, positive immunoreactivity with the anti-BAA and anti-

PAA has been detected in the collar enamel of teeth and ganoine of ganoid scales in

gar and the collar enamel of teeth in Polypterus, respectively (Sasagawa etal. 2012,

2014, 2016). For the immunohistochemical analyses, jaws containing tooth germs

were placed in 4% paraformaldehyde-0.2% glutaraldehyde xative (0.05 M

4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES) buffer, pH 7.4) for

3h at 4°C.The specimens were then dehydrated with N, N-dimethylformamide and

embedded in LR-White resin (LR-W, London Resin, Reading, UK) at −20°C.The

protein A-gold (PAG) method was employed for immunohistochemical analyses.

Fig. 18.1 (a) Schematic sketch of the structure of erupted teeth in Polypterus (Modied after

Sasagawa et al. 2013). (b–f) Light (b) and transmission electron (c–f) micrographs showing

enameloid formation during the early stage of mineralization (b, c) and the late stage of mineral-

ization (d–f), in Polypterus. (b) Histological section of a tooth germ stained with TB.Mineralization

has not started yet. (c) EDFSs that may originate from MV (arrow). (d) Initial ne crystallites

found on EDFSs (arrow) and collagen brils. (e) Crystallites oriented along the collagen brils. (f)

Slender crystallites form bundles and seem to align in the direction of collagen brils. B bone, CAE

cap enameloid, COE collar enamel, D dentin, DP dental pulp, IDE inner dental epithelium, PO a

process of odontoblast. Bar=50μm (b), 200nm (c, f), 100nm (d, e)

18 Im munolocalization ofEnamel Matrix Protein-Like Proteins intheTooth…

170

For TEM immunohistochemistry, ultrathin sections of the tooth germs obtained

from LR-W resin blocks were mounted on nickel grids. The specimens were oated

on a drop of 1% NGS for 15min and then incubated overnight on a drop of the

relevant antibody or antiserum diluted 1:200–1:400 with BSA-PBS.The sections

were then washed and transferred onto a drop of PAG conjugate (5 nm gold,

EMPAG5, BBI) diluted 1:10 with BSA-PBS for 30min after being treated with 1%

NGS for 10min. These sections were stained with platinum blue (TI blue, Nissin

EM, Tokyo, Japan) and lead citrate (TI-Pb) and often stained with phosphotungstic

acid (PTA) in addition to TI-Pb (TI-Pb-PTA) and then examined in a TEM (JEM-

1010, JEOL). Negative controls were performed by incubating sections in PBS

lacking antibody or antiserum, or in normal rabbit serum (NRS) instead of antibody

or antiserum. For TEM studies, ultrathin sections were mainly stained with lead

citrate alone and examined using a TEM.Semi-thin sections that had been stained

with 0.1% toluidine blue (TB) were also prepared for light microscopy. Details of

the methods were described in previous reports (Sasagawa etal. 2012, 2014, 2016).

18.3 Results

Formation of cap enameloid is divided into three stages, namely, matrix formation,

mineralization and maturation, according to morphological studies. In the present

study, we mainly examined the stage of mineralization (Fig.18.1b).

18.3.1 Initial Mineralization ofEnameloid inPolypterus

In the TEM observation, abundant collagen brils were visible in the enameloid

matrix during the early stage of enameloid mineralization. Many EDFSs and a few

MVs were also observed in the enameloid matrix. There were no crystallites in the

EDFSs, MVs and collagen brils, during the early stage (Fig.18.1c). During the

late stage of mineralization, ne, slender crystallites were found in both the EDFSs

and collagen brils (Fig. 18.1d). In area where mineralization process is more

advanced, many marked slender crystallites accumulated along the collagen brils

(Fig.18.1e) and formed bundles that aligned in the direction of the collagen brils

(Fig.18.1f). Afterwards, the crystallites increased in size, associated with the degen-

eration and removal of collagen brils.

I. Sasagawa et al.

171

18.3.2 Immunohistochemical Localization ofEMP-Like

Proteins inEnameloid Matrix

18.3.2.1 Gar

During the early stage of mineralization, a number of PAG particles were observed

in the enameloid matrix, in which crystallites did not appear yet. Most of the PAG

particles were observed on the collagen brils (Figs.18.2a, c). The EDFSs showed

no immunoreactivity (Fig.18.2b). Only a few PAG particles were seen in predentin.

In the IDE cells, electron-dense granules in the distal cytoplasm often contained

PAG particles (Fig.18.2c). Only a few PAG particles were seen in the negative con-

trol sections (Fig.18.2d, e). During the late stage of mineralization, when many ne

crystallites were visible along collagen brils, only a few PAG particles were found

in the enameloid. During the former stage of matrix formation and the subsequent

stage of maturation, little immunolabelling by the antibodies was found in the tooth

germs (data not shown).

Fig. 18.2 Transmission electron micrographs of immunohistochemistry in gar, PAG method using

an anti-bovine AMEL antibody (BAA) and an anti-porcine AMEL antiserum (PAA). (a) Many

PAG particles are found on the collagen brils, BAA, stained with Ti-Pb-PTA. (b) Few PAG par-

ticles are seen on the electron-dense brous substances, PAA, Ti-Pb. (c) PAG particles are found in

the granules (arrows) in the IDE cells, PAA, Ti-Pb-PTA. (d, e) Control sections incubated in NRS

instead of antibodies (d) or in PBS lacking antibodies (e), Ti-Pb. E enameloid, PO a process of

odontoblast. Bar=500nm (a–e)

18 Im munolocalization ofEnamel Matrix Protein-Like Proteins intheTooth…

172

18.3.2.2 Polypterus

In the enameloid matrix during the early stage of mineralization, many PAG parti-

cles were found along the collagen brils (Fig. 18.3a). The EDFSs exhibited no

immunoreactivity. Immunoreactivity was also detected in the granules of IDE cells

(Fig.18.3b). During the late stage of mineralization, in which many slender crystal-

lites accumulated along the collagen brils, immunolabelling was scarcely found in

the enameloid (data not shown). Only weak labelling was visible in the negative

control sections by NRS (Fig.18.3c, Sasagawa etal. 2012)

18.4 Discussion

Positive immunolabelling by the antibodies against mammalian AMEL was detected

in the enameloid matrix during the early stage of enameloid mineralization. The

results indicated that EMP-like proteins are present in the matrix of enameloid in

Polypterus and gar. Immunoreactivity was also found in the granules of IDE cells,

indicating that EMP-like proteins were secreted from IDE cells. During the stage of

enameloid matrix formation, immunolabelling was hardly found in enameloid

matrix. EMP-like proteins might exist in the matrix just before the appearance of

crystallites along collagen brils in the enameloid mineralization stage.

Immunoreactivity was scarcely detected in the mineralizing enameloid matrix, in

which ne crystallites were deposited along collagen brils, during the late stage of

mineralization, suggesting that the EMP-like proteins had degenerated or had been

removed. EMP-like proteins are probably present for a short period during the stage

of mineralization (Fig.18.4).

PAG particles were mainly observed on collagen brils in the enameloid. It is

possible that the EMP-like proteins are involved in mineralization along the colla-

gen brils in enameloid, because the initial crystallites appeared in collagen brils

in the subsequent substage. On the other hand, EDFSs exhibited no immunolabel-

ling. It is likely that EDFSs are not the structure that contains EMP-like proteins. In

Fig. 18.3 Transmission electron micrographs of immunohistochemistry in Polypterus, PAG

method using an anti-porcine AMEL antiserum (PAA), stained with Ti-Pb-PTA. (a) PAG particles

are found on the collagen brils in the enameloid. (b) PAG particles are also visible in the granules

of the IDE cells (IDE). (c) Control sections incubated in NRS instead of antibodies, in the early

stage of enameloid maturation, Ti-Pb. Bar=500nm (a–c)

I. Sasagawa et al.

173

a previous morphological study using TEM (Sasagawa and Ishiyama 2003), it was

assumed that the EDFSs were the site of initial mineralization in cap enameloid.

However, in the present TEM study, ne crystallites seemed to appear at both the

EDFSs and collagen brils simultaneously. Because this phenomenon was observed

in the enameloid matrix of gar (Sasagawa etal. in prep), this initial mineralization

process in the enameloid seems to be very similar in Polypterus and gar. Even if the

EDFSs are one of the sites of initial mineralization, the EDFSs might not be related

to crystallite formation along the collagen brils in enameloid.

In mammals, AMEL occupies approximately 90% of EMPs. It is assumed that

AMEL plays an important part in producing the structure of the enamel layer and to

encourage crystal growth during amelogenesis. In Polypterus and gar, EMP-like

proteins have been detected in the collar enamel and ganoine by several immunohis-

tochemical studies using anti-mammalian AMEL antibodies (Ishiyama etal. 1999;

Sasagawa etal. 2012, 2014, 2016; Zylberberg et al. 1997). According to recent

genetic analyses, however, gars do not have an AMEL gene, but ameloblastin,

enamelin, and many other secretory calcium-binding phosphoprotein (SCPP) genes

are present and are expressed in both teeth and scales (Qu etal. 2015; Braasch etal.

2016). It is likely that the EMP-like proteins in actinopterygian sh have epitopes

similar to mammalian AMEL, and the anti-mammalian AMEL antibodies may have

cross-reacted with these proteins. It is probable that the EMP-like proteins detected

in enameloid matrix are SCPPs (Kawasaki etal. 2017).

Fig. 18.4 Schematic drawings showing IDE cells during three stages of enameloid formation in

gar (Modied from Sasagawa and Ishiyama 2005a). The long arrow and solid stars indicate posi-

tions of positive immunoreactivity for anti-mammalian AMEL antibodies. BL basal lamina, CAE

cap enameloid, D dentin, IDE inner dental epithelial cells, OD odontoblasts, ODE outer dental

epithelial cells, PC procollagen granule, PD predentin, SG secretory granule, SI stratum interme-

dium, SR stellate reticulum

18 Im munolocalization ofEnamel Matrix Protein-Like Proteins intheTooth…

174

Acknowledgements The authors wish to thank Dr. T.Uchida of Hiroshima University and Dr.

H.Shimokawa of Tokyo Medical and Dental University for donations of the antibodies against

porcine AMEL (TU) and the antibody against bovine AMEL (HS). This study was supported in

part by Grants-in-Aid for Scientic Research No. 16591844 and 21592329 from the Ministry of

Education, Science, Sports, and Culture, Japan, and by a Research Promotion Grant (NDUF-

13-

10, NDUF-14-12, NDU Grants N-15015) from The Nippon Dental University.

Conict of Interest The author declares no conicts of interest associated with this manuscript.

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K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_19

Chapter 19

Geographical andSeasonal Variations

oftheShell Microstructures intheBivalve

Scapharca broughtonii

KozueNishida andTakenoriSasaki

Abstract Cyclical ontogenetic changes of shell microstructures have been observed

in the subfamily Anadarinae (Mollusca: Bivalvia, Arcidae) including fossil taxa.

The changes in the bloody clam Scapharca broughtonii are controlled by tempera-

ture, which uctuates seasonally, and can be used to determine the age of the indi-

viduals and to reconstruct paleoenvironments. In this study, samples of S. broughtonii

from eight localities covering broad geographical regions at various latitudes in

Japan, Korea, and Russia were examined to assess the utility of time series varia-

tions in microstructures for paleoenvironmental and paleoecological studies. All

specimens showed cyclical changes in the relative thickness of the composite pris-

matic and crossed lamellar structures in the outer layer with ontogenetic progres-

sion, and thus, this feature can be used as a proxy for water temperature of their

habitats. Specimens from southern latitudes showed higher annual shell growth

rates than northern specimens, suggesting that low temperatures arrest shell growth

in S. broughtonii and play a key role in determining the longevity and body size in

S. broughtonii. In long-lived individuals from the four northernmost localities, the

relative thickness of the composite prismatic structure tended to decrease as the

individuals aged, which may be a consequence of declining physiological activity,

such as organic matrix secretion.

Keywords Shell microstructure · Geographic variation · Water temperature ·

Growth rate · Age determination · Bivalve · Scapharca broughtonii · Temperate

species

K. Nishida (*)

Ibaraki College, National Institute of Technology, Ibaraki, Japan

Japan Society for the Promotion of Science (JSPS), Tokyo, Japan

e-mail: nishida@gm.ibaraki-ct.ac.jp

T. Sasaki

The University Museum, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

e-mail: sasaki@um.u-tokyo.ac.jp

178

19.1 Introducton

She mcrostructures of mouscs are hghy dversed (Carter 1990), and the she

mcrostructures formed by a snge ndvdua can dffer, dependng on phyogenetc

(Tayor et a. 1969; Shmamoto 1986; Sato and Sasak 2015), crystaographc

(Ubukata 2001; Checa eta. 2009, 2013), and envronmenta (Carter 1980; Kennsh

1980; Lutz and Cark 1984) factors. Recenty, Nshda eta. (2012) reported sea-

sona changes n the reatve thckness of the two mcrostructures (composte prs-

matc and crossed amear structures) n the outer ayer of the boody cam

Scapharca broughton. The composte prsmatc and crossed amear structures of

the outer ayer are formed on the exteror and nteror sdes, respectvey (Fg.19.1),

wth the composte prsmatc structure beng thcker at ower water temperatures

(Nshda eta. 2012). Nshda eta. (2015) observed she mcrostructures n cu-

tured specmens of S. broughton reared at ve dfferent temperatures, demonstrat-

ng expermentay the therma dependency of the mode of she mcrostructura

formaton n ths speces. Cycca changes n mcrostructures wth ontogeny have

been observed n the subfamy Anadarnae (Mousca: Bvava, Arcdae), ncud-

ng foss taxa (Kobayash and Kamya 1968; Kobayash 1976a, 1976b; Nshda

eta. 2012), and can be usefu for age determnaton and temperature reconstruc-

ton. Knowedge on geographca varatons n she mcrostructura formaton n S.

broughton remans mted (Nshda eta. 2012). Thus, sampes of S. broughton

were coected for ths study from eght ocates at varous attudes n Japan,

Russa, and Korea to assess the utty of the cycc thckness uctuaton n she

mcrostructures n paeoenvronmenta and paeoecoogca studes.

Fg. 19.1

K. Nshda and T. Sasak

An optca mcrograph of the acetate pee of rada secton of the outer ayer near the

outer she margn n the specmen SB-IN3-01 coected at ocaty 4. Wth the growth toward to

the rght, uctuatons are observed n the reatve thckness of the composte prsmatc and crossed

amear structures of the outer ayer. Gray arrows ndcate growth breaks. Abbrevatons: CL,

crossed amear ayer; CP, composte prsmatc ayer

179

19.2 Materials andMethods

We examined S. broughtonii shells collected from six sites in Japan (Localities 1, 2,

4, 5, 7, 8), one site in Russia (Locality 3), and one site in Korea (Locality 6)

(Table19.1, Fig.19.2). Of the 12 specimens collected from Localities 2–6 and 8, 9

were collected by dredge operations, and the remaining 3 specimens were likely

collected also by dredging. The specimens at Locality 1 were cultured in a net, and

the specimens at Locality 7 were cultured in a cage. Shell microstructures of those

14 specimens were prepared by the acetate peel method (Kennish etal. 1980), and

then the thickness of the composite prismatic and crossed lamellar structures and

the total thickness of the outer layer were measured at approximately 1-mm inter-

vals following Nishida etal. (2012) with ImageJ/NIH image analysis software (ver-

sion 1.45; http://imagej.nih.gov/ij/). Data of three specimens from Localities 1, 2,

and 7 (SB-MT3, SB-YR-101-1, and SB-KM10b-2, respectively) reported in Nishida

etal. (2012) were used for comparison with the data obtained in this study. According

to Nishida etal. (2012), the age of each individual could be estimated by the number

of the growth break (summer break) intervals and the positive peaks observed in the

relative thickness of the crossed lamellar structure.

19.3 Results

The relative thickness of the composite prismatic and crossed lamellar structures in

the outer layer of each specimen changed cyclically with ontogeny (Figs.19.2 and

19.3). The ratio of the composite prismatic structure thickness to the total outer

layer thickness was 0% at the minimum and had a maximum value as high as

58–80% (Figs.19.2 and 19.3). For all specimens, the intervals between the cycle of

relative thickness uctuation of the two structures shortened with ontogeny, and the

range of uctuation in the relative thickness of the composite prismatic structure

decreased in specimens older than 4years (Figs.19.2 and 19.3). In the specimens

from Vladivostok (SB-RU11–01, SB-RU11-02; Fig.19.2), the relative thickness of

the composite prismatic structure uctuated seasonally during earlier growth stages,

while the uctuations became smaller at later growth stages until the cyclic changes

in the relative thickness became almost indiscernible.

To examine the variations in annual growth of individuals from the same locali-

ties, we compared at least two specimens each for four localities (Localities 1–3, 7;

Figs.19.3 and 19.4). Individuals cultured in the same cage at Locality 7 showed a

similar pattern of microstructural changes (Fig.19.3c, d). In contrast, growth pat-

terns of the individuals cultured at Locality 1 showed considerable variations

(Fig.19.3a, b).

Growth curves for the specimens from the eight localities are shown in Fig.19.2.

The annual shell growth rate was higher in the specimens from southern localities

than in those from northern localities, corresponding to the general increase in water

19 Geographical andSeasonal Variations oftheShell Microstructures intheBivalve…

180

Table 19.1 Specimens of S. broughtonii examined in this study. All specimens are registered at

the Department of Historical Geology and Paleontology, University Museum, The University of

Tokyo (UMUT). Asterisks indicate specimens reported by Nishida etal. (2012); specimens 1, 2a,

2b, 3a, and 3b in Nishida et al. (2012) are identied as specimens SB-MT3, SB-YU101–1,

SB-YR102–4, SB-KM10b-2, and SB-KM10b-3, respectively, in this study

Locality

number

Sampling

site

Sampling

method Depth

Collection

date

Number

specimens

Specimen

number

Collection

number

Locality

Mutsu Bay,

Aomori

Prefecture

Cultured

in net

5–10m 20

September

2010

N=2 SB-MT3* UMUT

RM31012

SB-MT4 UMUT

RM32670

Locality

(2–1) at

38°05’ N,

140°58′ E,

Miyagi

Prefecture,

in the

Pacic

Ocean

Dredge 22–

23m

December

2010

N=3 SB-YR101–1* UMUT

RM31013

SB-YR101–4 UMUT

RM32671

SB-YR101–11 UMUT

RM32672

(2–2) at

38°09’ N,

140°59′ E,

Miyagi

Prefecture,

in the

Pacic

Ocean

Dredge 22–

23m

December

2010

N=3 SB-YR102–2 UMUT

RM32673

SB-YR102–4* UMUT

RM31014

SB-YR102–9 UMUT

RM32674

Locality

Off

Vladivostok,

Sea of Japan

Dredge? – July 2011 N=2 SB-RU11–01 UMUT

RM32675

SB-RU11–02 UMUT

RM32676

Locality

Nanao Bay,

Ishikawa

Prefecture,

Sea of Japan

Dredge 30m 01

November

2011

N=l SB-IN3–01 UMUT

RM32677

Locality

Kohama

Bay, Fukui

Prefecture,

Sea of Japan

Dredge 4–5m 24–27

February

2011

N=2 SB-FK1 UMUT

RM32678

SB-FK2 UMUT

RM32679

Locality

Jinhae-gu,

Sea of

Japan,

Korea

Dredge? – 29 June

2011

N=l SB-KOT-3 UMUT

RM32680

(continued)

K. Nishida and T. Sasaki

181

temperature (Figs.19.1, 19.4 and 19.5). Nishida et al. (2012) reported that shell

growth of the eld-collected specimens of S. broughtonii was probably arrested at

temperatures below 12°C.The length of time in a year when the water temperature

was above 12°C was longer in the south than in the north (Fig.19.2b).

19.4 Discussion

All specimens showed cyclical ontogenetic changes in the relative thickness of the

two structures (composite prismatic and crossed lamellar structures) in the outer

shell layer. Thus, this character of shell microstructure in this species can be applied

as a proxy of water temperature in different geographic regions. The annual shell

growth rate was higher in southern specimens than in northern specimens (Fig.19.5),

probably due to the shorter duration of temperatures below 12°C, a temperature

range in which shell growth is reported to be arrested (Nishida etal. 2012). The

specimens from Locality 8 (water temperature range 16–26°C) probably grew all

year round. On the other hand, the specimens from Locality 4 (0–25°C) may form

shells only for a period of approximately 4months. Thus, low temperatures below

12 °C are suggested to play a key role in the longevity and shell size in S.

broughtonii.

Nishida etal. (2015) suggested that the faster growth at lower temperatures is

achieved by dominantly building the composite prismatic structure, probably as an

adaptive strategy to precipitate shells under cold water environments. However, as

the composite prismatic structure is physically weaker than the crossed lamellar

structure (Taylor and Layman 1972; Currey 1976), it is disadvantageous for main-

taining the shell mechanical strength. Thus, a trade-off between growth and physi-

cal characteristics (e.g., strength) should be considered in investigations of thermal

Table 19.1 (continued)

Locality

number

Sampling

site

Sampling

method Depth

Collection

date

Number

specimens

Specimen

number

Collection

number

Locality

At 33°58’

N, 131°50′

E off

Kudamatsu

city,

Yamaguchi

Prefecture,

in the Seto

Island Sea

Cultured

in cage

10m 22

December

2010

N=2 SB-KM10b-2* UMUT

RM31015

SB-KM10b-3* UMUT

RM31016

Locality

Tachibana

Bay,

Nagasaki

Prefecture

Dredge 22–

23m

January

2011

N=l SB-NT1 UMUT

RM32681

19 Geographical andSeasonal Variations oftheShell Microstructures intheBivalve…

182

Fig. 19.2 The growth curves and changes in the relative thickness of the two structures in the

outer layer at eight localities arranged from north to south along the coasts of Japan, Russia, and

Korea. Arrow heads indicate growth breaks; black, gray, and white areas indicate composite pris-

matic structure, crossed lamellar structure, and missing sections of the outer layer, respectively,

and the growth years are indicated by horizontal bars. The water temperature data are from the

Japan Oceanographic Data Center (JODC, http://www.data.jma.go.jp/obd/stats/etrn/index.php).

Water temperature at each of the eight localities is shown with gray shading on months with water

temperature above 12°C and the number indicating the number of months with water temperature

above 12°C.The growth curve of Locality 7 is for the reference specimen cited from Nishida etal.

(2012)

K. Nishida and T. Sasaki

183

adaptation of microstructures in molluscs. The growth strategy of S. broughtonii

inferred by shell growth patterns and microstructures (e.g., to reach a larger body

size and/or maturity faster) might be important in the growth stage before maturity.

In long-lived specimens from Localities 1–4, the relative thickness of the composite

prismatic structure tended to decrease as the individuals aged (Figs.19.2 and 19.3).

Fig. 19.3 Differences in shell microstructural records between two cultured individuals reared in

the same localities. (a) Specimen SB-MT4 at Locality 1. (b) Specimen SB-MT3 at Locality 1,

reported by Nishida etal. (2012). (c) Specimen SB-KM10b-2 at Locality 7, reported by Nishida

etal. (2012). (d) Specimen SB-KM10b-3 at Locality 7. Arrows indicate growth breaks in the outer

shell surface; black, gray, and white areas indicate composite prismatic structure, crossed lamellar

structure, and missing sections of the outer layer, respectively

Fig. 19.4 Growth curves of the specimens from Localities 1, 2, 3, and 7 drawn based on the inter-

vals of the summer growth breaks and the positive peaks in the thicknesses of the crossed lamellar

structure. Asterisks indicate specimens reported by Nishida etal. (2012); specimens 1, 2a, and 3a

in Nishida etal. (2012) are identied as specimens SB-MT3, SB-YU101–1, and SB-KM10b-2,

respectively, in this study. (a) Specimens SB-MT3 and SB-MT4 from Locality 1. (b) Specimens

SB-YR101–1, SB-YR101–4, SB-YR101–11, SB-YR102–2, and SB-YR102–9 from Locality 2.

SB-KM10b-3 from Locality 7

19 Geographical andSeasonal Variations oftheShell Microstructures intheBivalve…

184

Although the primary factor controlling the relative thickness of the two structures

in the outer layer would be the seasonal changes in water temperature, physiological

factors related to aging may also control microstructural formation in S. broughto-

nii. Palmer (1983) suggested that the cost of shell production is cheaper in organic-

rich shells than in organic-poor shells. Composite prismatic structure in bivalve

shells is richer in organics than is the crossed lamellar structure (Taylor and Layman

1972; Nishida etal. 2015) and, thus, after sexual maturity, a decrease in the volume

of composite prismatic structure in shells may be accompanied by a decline in phys-

iological activity, such as organic matrix secretion. Age-related changes in shell

microstructures may show a trade-off between growth and physiological factors

attributable to aging. At later growth stages of the individuals from Locality 3, the

relative thickness of the composite prismatic structure became thinner with aging

until cyclic changes in the relative thickness were almost indiscernible. Because this

region is in the northern limit for this species, energetic cost might be needed not

only for shell microstructural formation but also other physiological demands.

Differences observed in cultivation experiments may also have some effect.

Patterns of the relative thickness of the two shell structures were more variable in

the specimens from Locality 1, where they were cultured in a net hanging in the

water column above the seaoor than in those from Locality 7, where they were

cultured in a cage resting on the bottom sediment. Yurimoto etal. (2007) reported a

Fig. 19.5 Observed water temperature and estimated annual shell growth rates at eight localities

arranged from north to south. The temperature data are from the Japan Oceanographic Data Center

(JODC, http://www.data.jma.go.jp/obd/stats/etrn/index.php). The average annual seawater tem-

perature at the eight localities approximately ranges from 7.8 to 20.0°C.Annual shell growth rate

was estimated from growth curves in Fig.19.2. Annual growth rates of 1- and 2-year-old speci-

mens at Locality 4 were not estimated because no summer growth break was observed in the shell

surface of the 1-year-old specimen

K. Nishida and T. Sasaki

185

lower monthly shell growth rate in the individuals of Scapharca kagoshimensis cul-

tivated in hanging nets than in those cultivated in cages on the seaoor and attrib-

uted this difference to buffeting of the suspended individuals by waves. Thus, the

specimens from Locality 7 likely experienced less growth stress than those from

Locality 1.

Acknowledgments We thank Kazuyoshi Endo, Toshihiro Kogure, Takanobu Tsuihiji, Hodaka

Kawahata, Atsushi Suzuki, Toyoho Ishimura, and paleobiology seminar members of University of

Tokyo for their suggestions on research methods and their comments; Yuji Kuyama, Makoto Fukui

(Kudamatsu Institute of Mariculture, Yamaguchi Prefecture, Japan), and the other members of this

institute; Shizuka Murakami (Kudamatsu City, Yamaguchi Prefecture, Japan), Hiroyuki Izumo

(Miyagi Federation of Fisheries Cooperative Associations, Yuriage branch), and the other members

of this association; Shoji Ohhasi (Nagasaki Prefecture Fisheries Technology Institute, Nagasaki

Prefecture, Japan), Kei Senbokuya (Ishikawa Prefecture Fisheries Technology Institute, Ishikawa

Prefecture, Japan), and the other members of these institutes for the donated specimens; and the

members of FORTE Co. Ltd. for English editing. This study was supported by Mikimoto Fund for

Marine Ecology and KAKENHI 24654167, 17K14413, 17J11417 funded by JSPS.

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K. Nishida and T. Sasaki

Part V

Biomineralization in Medical

and Dental Sciences

189© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_20

Chapter 20

Enhancement ofBone Tissue Repair

byOctacalcium Phosphate Crystallizing

into Hydroxyapatite InSitu

OsamuSuzuki andTakahisaAnada

Abstract We have reported that octacalcium phosphate (OCP) enhances bone

repair in critical-sized rat calvaria defects, while OCP is gradually crystallized to

apatite structure. Our studies showed that:

1. OCP enhances differentiation of osteoblastic cells in two- and three-dimensional

conditions.

2. OCP enhances osteoclast formation from bone marrow cells (macrophages) in

the co-culture with osteoblasts by raising osteoclast-inducing factor (RANKL).

3. OCP enhances macrophages migration.

4. These cellular responses are brought about associated with the hydrolysis of

OCP toward a nonstoichiometric Ca-decient hydroxyapatite (HA), which is

accompanied by physicochemical changes such as inorganic ion exchanges and

serum proteins adsorption.

Combining OCP with natural polymers, such as collagen and gelatin, improves

not only their moldabilities but also increases the osteoconductivity and the biode-

gradability in vivo. The hydrolysis of OCP may be involved in displaying bone

regenerative capacity of OCP.

Keywords Octacalcium phosphate (OCP) · Hydroxyapatite·Hydrolysis ·

Osteoblasts · Osteoclasts · Bone substitute materials

20.1 Introduction

It has been advocated that octacalcium phosphate (OCP, Ca8H2(PO4)6 · 5H2O)

could be a precursor phase to biological apatite crystals in the bone (Brown

1966) although there is still a controversy about whether OCP is actually present

O. Suzuki (*) · T. Anada

Division of Craniofacial Function Engineering, Graduate School of Dentistry,

Tohoku University, Sendai, Japan

e-mail: suzuki-o@m.tohoku.ac.jp; anada@m.tohoku.ac.jp

190

in bone mineralization (Rey etal. 2014). It has been reported that in a highly

supersaturated calcium and phosphate solution with respect to hydroxyapatite

(HA, Ca10(PO4)6(OH)2), amorphous calcium phosphate (ACP, Ca3(PO4)2·nH2O)

is formed rst, and then it transforms to an OCP-like phase before crystallizing

to the thermodynamically most stable HA phase (Meyer etal. 1978) at physio-

logical pH.ACP has been conrmed in growing chicken embryonic long bone

(Kerschnitzki etal. 2016). The structure of the OCP-like phase has been sug-

gested to resemble HA having a similar characteristics in X-ray diffraction

(XRD) (Meyer etal. 1978). In fact, it has been shown that an apatite structure,

having a solubility similar to OCP, can be synthesized in the presence of uoride

ions (Shiwaku etal. 2012). It has also been shown that ACP is aggregated as a

cluster forms and then transforms to HA via a Ca-decient OCP-like structure

(Habraken etal. 2013) and that growing calcium phosphates involve the common

cluster structure throughout the HA formation (Onuma etal. 2017). We hypoth-

esized that the introduction of synthetic OCP into bone defects should lead to the

enhancement of the initial deposition of bone matrix followed by additional bone

formation (Suzuki etal. 1991). An experiment of onlay graft of OCP in the gran-

ule form onto mouse calvaria, in comparison with HA materials, was conducted

to test the hypothesis and showed that OCP enhances the appearance of bone

tissue around these granules more than those by HA materials (Suzuki et al.

1991). This review article summarizes how the OCP materials work in bone

repair if placed in bone defects and in the vicinity of bone-tissue-related cells

invitro.

20.2 Bone-Bonding Property ofOCP Implanted inBone

Defects

It has been proposed that direct bone bonding of ceramic materials is brought about

through an apatite layer formation on the materials, thereby allowing chemical

bonding between newly formed bone apatite crystals and the apatite formed on the

materials (Kokubo 1991). Such direct bonding has been reported to occur in ceramic

materials, such as glass ceramics (Hench et al. 1973; Kokubo 1991), HA (Aoki

1973), and β-tricalcium phosphate (β-TCP, Ca3(PO4)2) (Kotani et al. 1991). The

property is ascribed to the osteoconduction, which is dened as the bone formation

taking place in orthotopic site (LeGeros 2002). We have found that when the tissue

response was observed using undecalcied sections of the granules of OCP implan-

tation onto mouse calvaria by transmission electron microscopy, newly formed

bone crystals directly bonded to the surface of crystals in the OCP granules (Suzuki

etal. 2008). From this observation, it was apparent that the OCP has an osteocon-

ductive property (Oyane etal. 2012).

O. Suzuki and T. Anada

191

20.3 Hydrolysis fromOCP toCa-Decient HA

inPhysiological Conditions

It is known that OCP is more soluble than β-TCP and HA and less soluble than

dicalcium phosphate dihydrate (DCPD, CaHPO4・2H2O) under neutral pH (Brown

etal. 1981; Chow 2009). It is of interest to learn about whether OCP is actually

converted to HA invivo conditions. It has been expected that OCP is converted

(hydrolyzed) to HA if placed in the invivo environment (Suzuki etal. 1991). There

is a general consensus that OCP is stacked by apatite layer alternatively with

hydrated layers and that the hydrolysis is once initiated; it advances spontaneously

and irreversibly (LeGeros etal. 1989; Suzuki etal. 1995a, b; Tomazic etal. 1989).

The structural changes of OCP have been investigated because the laboratory syn-

thesized OCP is a well-grown crystal (Kobayashi etal. 2014; Sakai et al. 2016;

Suzuki et al. 1991, 2006a, b). Other investigations showed that OCP remained

untransformed in simulated body uid (SBF) even prolonged incubation (Ito etal.

2014) under supersaturated conditions with respect to HA theoretically estimated

(Lu etal. 2005). The implantation of OCP in the bone and subcutaneous tissues

promoted the structural changes in XRD (Suzuki etal. 1991, 1993, 2006b) and in

Fourier transform infrared (FTIR) absorption from that of OCP to apatite structure

with increasing Ca/P molar ratio (Sakai etal. 2016) and carbonate ion containment

(Suzuki etal. 2009) although characteristics of OCP in XRD still remained (Sakai

et al. 2016; Suzuki et al. 1991, 1993, 2006b). One of features in the structural

changes of OCP by its implantation and incubation in physiological conditions is

that the chemical composition of Ca/P molar ratio has never reached to that of a

stoichiometric value (1.67) but tends to go to producing a Ca-deciency, resulting

in the formation of a Ca-decient HA (Sakai etal. 2016; Suzuki etal. 1995b). OCP

hydrolysis can be enhanced with experimentally given higher supersaturated condi-

tions invitro by promoting calcium ion consumption into the crystals and phosphate

ion release from the crystals (Kobayashi etal. 2014; Sakai etal. 2016; Suzuki etal.

2006a), suggesting that invivo environment could be providing such an ionic condi-

tions. It has been reported that human serum is saturated with respect to OCP

(Eidelman etal. 1987), which is not contradict to the proposition that HA can be

grown on OCP template (Miake etal. 1993).

20.4 Osteoblastic Cell Response

It was observed that, when the granules of OCP were implanted onto mouse cal-

varia, the crystals of OCP were accumulated by circulating non-collagenous serum

proteins, including α2HS-glycoproteins and apolipoprotein E (Kaneko etal. 2011;

Suzuki etal. 1993), the ultrastructure of which bears a close resemblance to the tis-

sue structure so-called bone nodules, which is considered as the initial bone deposi-

tion locus in intramembranous bone development (Barradas etal. 2011; Suzuki

20 Enhancement ofBone Tissue Repair byOctacalcium Phosphate Crystallizing…

192

etal. 1991). Osteoblasts then started to form a collagenous bone matrix around the

bone nodule-like OCP-protein complex (Suzuki etal. 1991, 2008), indicating that

OCP acts on a nucleus of bone formation. From these observations, it was hypoth-

esized that OCP may activate osteoblastic cell activity on its surfaces. In order to

test the hypothesis, mouse bone marrow stromal ST-2 cells were inoculated on OCP

particles coated on plastic cell culture plate in comparison with HA materials

(Anada etal. 2008; Suzuki etal. 2006b). mRNAs of osteoblast differentiation mark-

ers, such as alkaline phosphatase (ALP), type I collagen, and osterix, increased with

increasing the dose of OCP (Anada etal. 2008).

20.5 Osteoclastic Cell Response

When OCP is placed in bone defects, OCP shows biodegradable characteristics that

tend to be replaced with new bone (Honda etal. 2009; Imaizumi etal. 2006; Kikawa

etal. 2009; Miyatake etal. 2009; Murakami etal. 2010; Suzuki 2013). It was ascer-

tained that OCP hydrolysis is accompanied by a subtle reduction from a neutral pH

to some extent acidic pH with the hydrolysis (Masuda etal. 2017), which may affect

the cellular responses of immune cells (Hirayama etal. 2016). A histological exami-

nation showed that OCP enhanced macrophage migration (Hirayama etal. 2016)

and multinucleated giant cells appearance around the surfaces (Honda etal. 2009;

Imaizumi etal. 2006; Kikawa etal. 2009; Miyatake et al. 2009; Murakami et al.

2010; Suzuki 2013), where osteoblasts are forming new bone, more than HA

(Suzuki 2013). The multinucleated giant cells were shown to be tartrate-resistant

acid phosphatase (TRAP) positive osteoclast-like cells (Imaizumi etal. 2006), sug-

gesting that OCP biodegrades through phagocytic resorption not by simple chemi-

cal dissolution. It is known that osteoclasts can be formed by the fusion of

macrophages (Asagiri etal. 2007; Kong etal. 1999; Yasuda etal. 1999) so that the

macrophage migration to OCP surfaces may be involved in the formation of

osteoclast-

like cells (Hirayama etal. 2016). An invitro study in fact revealed that

the multinucleated giant cells, expressing osteoclast marker genes such as TRAP

and cathepsin K, can be formed on the surface of OCP but not on the surface of HA

in the co-culture with osteoblasts (Takami etal. 2009). The osteoblasts cultured on

OCP expressed receptor activator of NF-kappaB ligand (RANKL), an osteoclast

differentiation factor (Takami et al. 2009). Calcium ion concentration in culture

medium decreased in the presence of OCP, which corresponds to the change induced

by OCP during its hydrolysis (Suzuki et al. 2006a), while the experimentally

reduced calcium ion concentration induced RANKL mRNA expression of osteo-

blasts (Takami etal. 2009). These results suggest that the physicochemical environ-

ment induced by OCP brings about the biodegradable characteristics of this material

through osteoclast differentiation from macrophages increasing through RANKL

expressions by osteoblasts in the implanted OCP.The mechanism of physicochemi-

cal changes, including the protein adsorption, induced during the hydrolysis from

O. Suzuki and T. Anada

193

OCP to Ca-decient HA, is summarized in Fig.20.1. The osteoblast differentiation

stimulated by OCP, as a nucleus of the initial bone deposition, is summarized in

Fig.20.2.

20.6 Bone Substitute Materials

OCP cannot be sintered with keeping its crystalline phase, unlike HA and β-TCP,

due to the inclusion of large amount of water molecules (Brown etal. 1962). The

granules or the precipitates of OCP were combined with various natural polymers,

8H2(PO4)65H2OCa10-XHx(PO4)6(OH)2-X

AH tneicifed-aCPCO

in site hydrolysi

Overgrowth on surface

Ca2+,F-, CO32- Uptake

PO43- Release

Hydrolysis

Ca/P molar ratio: Increase

HPO4content Decrease

Albumin adsorption affinity Increase

Serum proteins such as ApoE protein: Accumulated

Fig. 20.1 Observed various physicochemical reactions caused during the hydrolysis of synthetic

OCP placed invivo(Suzuki etal. 1991, 2006b, 2008) and invitro(Suzuki etal. 2006a, b) environ-

ments. OCP can be progressively hydrolyzed to Ca-decient HA under a physiological environ-

ment via topotaxial conversion (in situ hydrolysis) and dissolution-reprecipitation (overgrowth of

apatite on an OCP crystal surface) (Brown etal. 1981; Miyake etal. 1993; Suzuki etal. 1995b).

Ca-decient HA can be formed, and chemical composition change and surface reaction are pro-

moted during the hydrolysis (Suzuki etal. 1995b, 2006b; Kaneko etal. 2011)

Mouse calvaria

Osteoblasts

Newly formed bone matrix

(collagen fibers)

Each crystal of OCP

(consisting granule form)

Proteins adsorbed

Fig. 20.2 Conceptual scheme of the beginning of bone formation from the synthetic OCP crystals

implanted in bone tissue that is based on the ultrastructural observation (a) (Suzuki etal. 1991,

1993, 2008) and newly boned bone around OCP granules implanted onto mouse calvaria for

21days (b). Right gure (b), decalcied histological section stained by hematoxylin and eosin.

Asterisks (*): OCP granules, NB new bone; bar = 50 μm. (Reproduced from Fig. 2c in Acta

Biomater 5:1756–1766, 2009 (Kikawa etal. 2009) with permissions from Elsevier Ltd)

20 Enhancement ofBone Tissue Repair byOctacalcium Phosphate Cr ystallizing…

194

such as collagen, gelatin, alginate, and hyaluronic acid, in order to acquire the mold-

ability and improve the handling property (Fuji et al. 2009; Handa et al. 2012;

Kamakura etal. 2006; Shiraishi etal. 2010; Suzuki etal. 2014). OCP/collagen was

made of OCP granules mixed with re-constituted collagen matrix (Kamakura etal.

2006). OCP/gelatins were made of OCP granules mixed with re-constituted gelatin

matrix (Saito et al. 2016) and OCP directly precipitated with gelatin molecules

(Handa etal. 2012). OCP/collagen composite enhanced the bone formation more

than OCP alone when compared in a rat critical-sized calvaria defect (Kamakura

etal. 2006). Bone regeneration was augmented in a dose-dependent manner of OCP

in the collagen matrix (Kawai etal. 2009), which corresponded to the tendency of

increasing osteoblastic cell differentiation invitro (Anada et al. 2008). After pre-

clinical trial (Kawai etal. 2014), the OCP/collagen composite is under a company-

initiative clinical trial in the eld of oral surgery in Japan. The OCP-co-precipitated

gelatin composite showed a highly biodegradable property in critical-sized rat cal-

varia defect (Handa etal. 2012) and also in rabbit tibia defect which is an orthopedic

bone defect model (Chiba etal. 2016). The inclusion of OCP crystals in gelatin was

shown to produce an oriented bone regarding newly formed collagen matrix (Ishiko-

Uzuka et al. 2017). OCP/alginates were made of OCP granules mixed with a

calcium- cross-linked re-constituted alginate matrix (Shiraishi etal. 2010) and OCP

directly precipitated with alginate molecules (Fuji etal. 2009). The combining OCP

with alginate allowed the composite to proliferate osteoblastic cells although the

alginate is a material that does not have a cellular binding motif (Fuji etal. 2009).

OCP/hyaluronic acids (HyAs) were made of OCP granules mixed with HyA medi-

cal products, having different molecular weights (Suzuki etal. 2014). OCP/HyAs

acquired injectability and OCP combined with a lower molecular weight HyA and

with a higher molecular weight HyA enhanced bone augmentation more than OCP

alone when placed on the subperiosteal region of mouse calvaria through an osteo-

clastic resorption of OCP (Suzuki etal. 2014). Thus, the osteoconductive property

and the handling property of OCP could be controlled by those natural polymers

that are combined with OCP.

20.7 Conclusion

It seems likely that the enhancement of bone formation by OCP implantation is

derived from its stimulatory capacity on osteoblastic cell activity during its crystal-

lizing into Ca-decient HA.OCP-based materials could be used as bone substitute

materials in various bone defects.

Acknowledgments This study was supported in part by grants-in-aid (17076001, 23106010

17K19740and 18H02981) from the Ministry of Education, Science, Sports, and Culture of Japan,

the Uehara Memorial Foundation, the Suzuken Memorial Foundation, and the Iketani Science and

Technology Foundation. The author thanks Emeritus Professor and former President of Japan Fine

Ceramics Co. Ltd., S.Ito; Professors R.Kamijo, M.Nakamura, and M.Takami, Showa University;

O. Suzuki and T. Anada

195

Professor T.Katagiri, Saitama Medical School; Professors E.Itoi, S.Kamakura, T.Takahashi, and

Emeritus; Professors M.Sakurai, M.Kagayama, and S.Echigo, Tohoku University; Drs. T.Kawai,

and Y.Shiwaku, Tohoku University; and Dr. Y.Honda, Osaka Dental University.

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20 Enhancement ofBone Tissue Repair byOctacalcium Phosphate Crystallizing…

199© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_21

Chapter 21

The Relationship Between theStructure

andCalcication ofDentin andtheRole

ofMelatonin

HiroyukiMishima, SakiTanabe, AtsuhikoHattori, NobuoSuzuki,

MitsuoKakei, TakashiMatsumoto, MikaIkegame, YasuoMiake,

NatsukoIshikawa, andYoshikiMatsumoto

Abstract The present study aimed to examine the relationship between the struc-

ture and composition of dentin and odontoblasts and the role of melatonin during

the calcication process. The expression of MT1 and MT2 melatonin receptor was

conrmed in the odontoblasts of the control group. In addition, the expression of

MT1 was stronger than that of MT2. A strong expression of MT1 was detected in

the odontoblasts in the melatonin-treated groups. MALDI-TOF MS analysis

revealed that two peaks of 795m/z and 818m/z were found in dentin. These peaks

increased commensurately with the amount of melatonin. The number and size of

calcospherites in predentin increased in proportion to the concentration of melato-

nin. The degree of mineralization increased slightly in the melatonin-treated group

using CMR analysis. Two peaks could be clearly detected in the melatonin-treated

H. Mishima (*)

Department of Dental Engineering, Tsurumi University School of Dental Medicine,

Yokohama, Japan

e-mail: mishima-h@tsurumi-u.ac.jp

S. Tanabe

Central Clinical Engineering Section, Osaka City General Hospital, Osaka, Japan

e-mail: mrc.skww02@icloud.com

A. Hattori

Department of Biology, College of Liberal Arts and Sciences, Tokyo Medical and Dental

University, Ichikawa, Japan

e-mail: ahattori.las@tmd.ac.jp

N. Suzuki

Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa

University, Housu-gun, Japan

e-mail: nobuos@staff.kanazawa-u.ac.jp

M. Kakei

Tokyo Nishinomori Dental Hygienist College, Tokyo, Japan

e-mail: mitsuo-kki@jcom.home.ne.jp

200

group at nighttime using X-ray diffraction analysis. Melatonin may participate in

the dentin composition and the calcication mechanism of dentin.

Keywords Dentin · Melatonin · Melatonin receptor · Calcospherite · Calcication

· Circadian rhythm

21.1 Introduction

In the hierarchy of internal biological rhythms, there are the circadian rhythm (cir-

cadian cycle: about 24h), the circalunar rhythm (about 28days), and the circannual

rhythm (about 1year) (Koukkari and Sothern 2006; Mishima etal. 2013; Pfeffer

etal. 2012). There is also circadian rhythm in the concentrations of Ca and P of the

blood. It is reported that Ca will deposit on teeth and bones because blood pH tends

to be alkaline and blood Ca value decreases at night (Ishida etal. 1983). It became

clear that circadian rhythm is shown in oral tissues including salivary glands

(Papagerakis etal. 2014).

Melatonin is the synchronization factor of circadian rhythm (Hattori 2017;

Koukkari and Sothern 2006). One of the effects of melatonin is the adjustment of

circadian rhythm, and it serves as the transmission material of the information at the

nighttime. The melatonin synthesis changes between day and night. In the daytime,

the melatonin amount decreases (Hattori 2017; Koukkari and Sothern 2006; Pfeffer

etal. 2012). Melatonin is also associated with tooth development (Kumasaka etal.

2010; Tachibana et al. 2014). Melatonin may play an essential role in regulating

bone growth (Roth etal. 1999; Satomura etal. 2007). When melatonin was admin-

istered in aged rats, the bone mass increased, and the internal structure of the bone

was reinforced (Tresguerres etal. 2014). Antiaging effect of melatonin is expected

(Hattori etal. 2006; Hattori 2017). Many of the physiological functions of melato-

nin are exerted via two melatonin receptors (MT1 and MT2) of the cell membrane

T. Matsumoto

Department of Laboratory Diagnosis, University Hospital, Nihon University School of

Dentistry at Matsudo, Matsudo, Japan

e-mail: matsumoto.takashi@nihon-u.ac.jp

M. Ikegame

Department of Oral Morphology, Science of Functional Recovery and Reconstruction,

Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Advanced Research

Center for Oral and Craniofacial Sciences, Okayama University, Okayama, Japan

e-mail: ikegame@md.okayama-u.ac.jp

Y. Miake

Department of Histology and Developmental Biology, Tokyo Dental College, Tokyo, Japan

e-mail: miake@tdc.ac.jp

N. Ishikawa · Y. Matsumoto

Applied Life Science Course, Faculty of Agriculture, Kagawa University, Takamatsu, Japan

e-mail: s18g604@stu.kagawa-u.ac.jp; myoshiki@med.kagawa-u.ac.jp

H. Mishima et al.

201

(Pfeffer etal. 2012; Hattori 2017). MT1 is involved in the transmission of optical

information and works on hypnosis, and MT2 is involved in synchronizing the cir-

cadian rhythm (Liu etal. 2013). MT1 is also involved in tooth development and

formation (Kumasaka et al. 2010; Mishima et al. 2014; Tachibana et al. 2014).

Melatonin may regulate various physiological functions in the body and is

synchronized with diurnal change (Hattori 2017; Koukkari and Sothern 2006;

Shimozuma etal. 2011). We have reported that an increase or decrease in the mela-

tonin secretion inuences the incremental line and formation of dentin (Mishima

et al. 2013, 2014, 2015). The present study aimed to examine the relationship

between the structure and composition of dentin and odontoblasts and the role of

melatonin during the calcication process.

21.2 Materials andMethods

21.2.1 Ethics

These animal experiments were conducted with the approval of the animal labora-

tory ethics committee of the Meikai University School of Dentistry (Approval No.

A 1019, A 1105, A 1221, A1525) and Kagawa University (Approval No. 17636).

Animal experiments were conducted in compliance with the animal experiment

implementation regulations of the Meikai University School of Dentistry and

Kagawa University.

21.2.2 Materials

In this experiment, 5-, 6-, and 7-day-old SD rats were used. These rats were divided

into three groups: (1) a control group (0.5% alcohol-containing water), (2) a low-

concentration group (0.5% alcohol +20μg/ml melatonin-containing water), and (3)

a high-concentration group (0.5% alcohol +100μg/ml melatonin-containing water).

Before and after childbearing, melatonin was administered for 8days at the 5-day-

old rats, 9days at the 6-day-old rats, and 10days at the 7-day-old rats. The slaughter

was carried out at midday and midnight. The samples taken during the day were

classied as daytime specimens, and specimens taken at night were classied as

nighttime specimens.

21 The Relationship Between theStructure andCalcication ofDentin andtheRole…

202

21.2.3 Methods

These samples were removed together with the jaw bones around the incisors. These

were xed in 10% neutral buffered formalin solution. The demineralized specimens

were decalcied for 1week with a 0.5% EDTA decalcication solution. The demin-

eralized specimens were made into the sliced continuous sections with the thickness

of about 4μm with a microtome. The sliced sections were stained with HE staining

for the analysis of calcospherites and dentin structure. Immunostaining of the mela-

tonin receptor was performed, and the localization of the melatonin receptor in the

tissue was searched. The melatonin receptors were used, the melatonin 1a (MT1,

Biorbyt, Cat# orb11085, USA) and melatonin 1b (MT2, LifeSpan BioSciences,

Cat# LS-A934, USA). The immunohistochemistry procedure was carried out

according to the method of Mishima etal. 2016. The staining sections were observed

and analyzed using the light microscopy (ECL-IPSE80i, Nikon, Japan).

On the ground sections, one single-side polishing and double-side polishing

were carried out with a grinding stone, a diamond lapping lm (nal particle size

3μm), and a diamond paste (nal particle size 0.25 μm). These ground sections

were observed and analyzed by a light microscopy (Eclipse 80i, Nikon, Japan), a

polarizing microscopy (Eclipse LV100N, Nikon), a scanning electron microscope

(SEM, JSM-6500F, JEOL, Japan), a contact microradiography (CMR, Softex CMR-

K, Japan), an atomic force microscopy (AFM, Nanosurf Easyscan, Nanosurf, AG,

Switzerland), X-ray diffraction method (RINT2500, Rigaku, Japan), and a mass

spectrometry (matrix-assisted laser desorption ionization time-of-ight mass spec-

trometer: MALDI-TOF MS, ultraeXtreme-KG1, Bruker, USA). Measurements of

the number of calcospherites in predentin and the expression concentration of mela-

tonin receptor in odontoblasts were performed using image analysis software

WinROOF (MITANI, Japan). The measurement and analysis procedure were car-

ried out according to the method of Mishima etal. (2014, 2016). The labial dentin

covered the enamel, and lingual dentin covered the thin cementum in incisor. We

observed and analyzed both dentins. The statistical signicance between the control

and melatonin-treated groups was assessed by Bonferroni correction and by Tukey’s

test. In all cases, the selected signicance level was p<0.05 and p<0.01. The Ca,

P, and Mg concentrations in blood serum of rats in the control group and melatonin-

treated groups were analyzed using a SPOT-Chem blood analysis (SPOTCHEM EZ

SP-443, ARKRAY, Japan).

21.3 Results

The number of calcospherites increased in the melatonin-treated group of 6-day-old

rats in the nighttime specimens (Fig.21.1). A signicant difference was observed

between the control group and the high-concentration group (Fig.21.1, *p<0.05).

However, no clear difference was found between the control group and the

H. Mishima et al.

203

melatonin- treated groups in the daytime specimens. A signicant difference was

observed between the daytime and nighttime specimens in the control group

(Fig.21.1, **p<0.05). The expression of MT1 and MT2 melatonin receptor was

conrmed in the odontoblasts of the control group. In addition, the expression of

MT1 was stronger than that of MT2. As compared with the control group (Fig.21.2c),

the strong expression of MT1 was detected in the melatonin-treated groups in the

nighttime specimens (Fig.21.2a, b). A signicant difference was observed between

the daytime and nighttime specimens in the high-concentration group (Fig.21.3,

*p<0.05). The difference in interference color (arrows) was found for the layers of

incremental lines in the high-concentration group in the nighttime specimens

(Fig.21.4a). No change in interference color was observed in the control and low-

concentration group (Fig.21.4b, c). By the backscattered electron image of SEM,

the diameter of calcospherites of the control group was 10–18μm (Fig.21.5a), and

ControlLow High

DayNight

Number of calcospherites

Fig. 21.1 Number of calcospherites in predentin. These data were obtained from the result of HE

staining. In the nighttime specimens, calcospherites increased in the melatonin-treated group.

6-days old. Lingual dentin. *p<0.05, **p<0.05

Fig. 21.2 Immunohistochemistry micrographs of the expression of MT1 melatonin receptor at the

nighttime. The strong expression of MT1in odontoblasts was detected in the high groups. 6 days

old. Labial dentin. (a) High group, (b) low group, (c) control group. O odontoblast. Scale bar,

100μm

21 The Relationship Between theStr ucture andCalcication ofDentin andtheRole…

204

that of calcospherites of the high group was 13–25μm (Fig. 21.5b). The size of

calcospherites in predentin increased in proportion to the concentration of melato-

nin. By the CMR images, calcospherites increased in the high-concentration group

as compared with the control group (Fig.21.6, arrow). On the degree of mineraliza-

tion (mineral-volume %), the control group was 29.00%, the low-concentration

group was 32.29%, and the high-concentration group was 32.61%. The degree of

mineralization increased slightly in the melatonin-treated group. On the AFM

images of ground section, the crystal surfaces of dentin were observed (Fig.21.7a–

c). The crystal surface of dentin regularly arranged with globular uniform size in the

high-concentration group (Fig.21.7c). The diameter on crystal surface in the high-

concentration group was 161–258nm. On the results of X-ray diffraction method,

ControlLow High

Saturation

DayNight

Fig. 21.3 Expression concentration of MT1 melatonin receptor in odontoblasts. The expressions

of MT1 of the low and high group were somewhat stronger than that of the control group. The

vertical axis represents saturation (range, 0–255). *p<0.05

Fig. 21.4 Micrographs of polarizing microscopy of labial ground dentin. The difference in the

interference color was found in the layer of incremental lines of the high group (arrows). 6 days

old. (a) High group, (b) low group, (c) control group. Scale bar, 100μm

H. Mishima et al.

205

one peak of crystal plane index was detected in the control group (Fig. 21.8a).

However, two peaks of crystal plane indices were detected in the high-concentration

group in the nighttime specimens (7-days old) (Fig.21.8b). The low-angle plane

index is (002), and the other plane index is one in which three plane indices (211),

(112), and (300) overlap.

MALDI-TOF MS analysis of the ground sections revealed that two peaks of

795m/z and 818m/z were found in dentin. As the dose of melatonin increased, the

detected intensity increased in the nighttime specimens (Fig.21.9). There was no

clear difference in daytime specimens. Figure21.10 shows the results of Ca, P, and

Mg concentration in blood serum using the SPOT-Chem blood analysis. A signi-

cant difference was observed between day and night in the control group on the

concentration of P (Fig.21.10, *p<0.01). However, the concentration of P in the

melatonin-treated group did not differ between day and night. The difference was

Fig. 21.5 Calcospherites in predentin using the backscattered electron image of SEM.The size of

calcospherites in predentin increased in proportion to the concentration of melatonin. 7 days old.

(a) Control group, (b) high group. Arrows: calcospherites. Scale bar, 10μm

Fig. 21.6 CMR images of the transverse sections of incisor dentin. Calcospherites increased in the

high group. The degree of mineralization increased slightly in the high group. The difference in

thickness of dentin on (a) and (b) is due to the difference in the cutting region. 6 days old. (a)

Control group, (b) high group. Arrows show calcospherites. Scale bar, 100μm

21 The Relationship Between theStructure andCalcication ofDentin andtheRole…

206

observed between day and night in the high-concentration group on the concentra-

tion of Ca, but there was no signicant difference (p=0.08). The concentration of

Ca and P in the blood serum changed by melatonin administration. Regarding the

concentration of Mg, there was no change.

21.4 Discussion

From HE staining and SEM observation, the number and size of calcospherites in

predentin increased in the melatonin-treated group. By CMR analysis, the calcica-

tion was higher in the melatonin-treated groups. Ca and P content of dentin were

higher in the melatonin-treated group (Mishima etal. 2013, 2014, 2015). The distri-

bution density of Ca and P was greater in the melatonin-treated group (Mishima

etal. 2015). The width of dentinal tubules was narrower than that of the control

(Mishima etal. 2015). It is likely that the formation of peritubular dentin was pro-

moted by melatonin (Mishima etal. 2015). From the X-ray diffraction and AFM

X*0 m 2 µm

Y*0 m 2 µm

Topography – Line fit

Topography range Line fit 369 nm

X*0 m 2 µm

Y*0 m 2 µm

Topography – Line fit

Topography range Line fit 275 nm

X*0 m 2 µm

Y*0 m 2 µm

Topography – Line fit

Topography range Line fit 733 nm

abc

Fig. 21.7 AFM images of longitudinal ground section of dentin. The crystal surface of dentin

arranged irregularly both control and low groups. The crystal surface of dentin regularly arranged

with uniform size in the high group. 7 days old. (a) Control group, (b) low group, (c) high group.

Length of X axis and Y axis, 2μm

Fig. 21.8 X-ray diffraction patterns of dentin. Two peaks could be clearly detected in the high

group at the nighttime. 7 days old. (a) Control group, (b) high group. ※1 shows (002) and ※2

shows (211), (112), and (300)

H. Mishima et al.

207

analyses, it was considered that the crystallinity and orientation of the apatite crystal

of dentin improved by administration of melatonin. We think that odontoblasts were

activated by melatonin administration and promoted the dentin calcication.

From the result of the polarizing microscopy, it is considered that melatonin

causes a change in the structure of dentin apatite crystals and collagen bers. The

Korff’s bers were more clearly distributed in the melatonin-treated group of

nighttime and daytime specimens (Mishima et al. 2016). It is considered that the

odontoblasts were activated by melatonin, and the secretion of collagen bers was

promoted. Mature dentin is made up of approximately 70% inorganic material, 20%

Fig. 21.9 Results of MALDI-TOF MS analysis. Two peaks of 795m/z and 818m/z are found in

dentin at nighttime. As the dose of melatonin increased, the detected intensity increased. 6 days

old. The vertical axis represents intensity (0–2500), and the horizontal axis represents m/z

(793–893)

Ca PMg

ControlLow High

DayNight

ControlLow High

DayNight

ControlLow High

DayNight

mg/d L

Fig. 21.10 Ca, P, and Mg concentration in blood serum using the SPOT-Chem blood analysis.

From the left gure, the results of Ca, P, and Mg are shown. 7 days old. The vertical axis represents

mg/dL. *p<0.01

21 The Relationship Between theStructure andCalcication ofDentin andtheRole…

208

organic material, and 10% water by weight. The organic phase of dentin is com-

posed mainly of type I collagen (Nanci 2003). In mass spectrometry, the peaks of

type I collagen are detected at 653–960m/z in the bone (Schweitzer et al. 2009).

The MALDI-TOF MS analysis revealed that two peaks of 795m/z and 818 m/z

were found in dentin. Two peaks are considered to be the peptides in which the type

I collagen has been resolved. These detected peaks intensity increased in the

melatonin- treated groups. Thus, it is estimated that the peptides which a large

amount of collagen was metabolized and resolved increased in the melatonin-

treated group. We thought that administration of melatonin promotes collagen

secretion of odontoblasts and the collagenation of dentin.

A strong expression of MT1 was detected in the odontoblasts in the melatonin-

treated groups. MT1 is considered to be involved in the formation and development

of teeth (Mishima etal. 2015). Kumasaka etal. (2010) and Tachibana etal. (2014)

report the expression of MT1in odontoblasts. Melatonin is thought to affect physi-

ological functions of odontoblasts in the daytime and nighttime. It was suggested

that the expression of MT1 depends on the amount of melatonin and that melatonin

is efciently incorporated into odontoblasts. Melatonin administration changed the

behavior of Ca and P concentration in serum at night. In summary, the present study

suggests that melatonin changed the blood composition in the body and inuenced

the structure, calcication, and crystallinity of dentin.

Acknowledgments The work was supported by JSPS KAKENHI Grant Number15K11034. This

study was performed under the cooperative research program of the Center for Advanced Marine

Core Research (CMCR), Kochi University (16A006, 16B006, 17A009, 17AB009). Regarding the

image of AFM, we were aided by Dr. E.Yoshida of Tsurumi University School of Dental Medicine.

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21 The Relationship Between theStructure andCalcication ofDentin andtheRole…

211© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_22

Chapter 22

Fabrication ofHydroxyapatite

Nanobers withHigh Aspect Ratio via

Low-Temperature Wet Precipitation

Methods Under Acidic Conditions

MasahiroOkada, EmilioSatoshiHara, andTakuyaMatsumoto

Abstract HAp nanobers (or whiskers) have been attracted considerable attention

for their application as adsorbents and reinforcing llers owing to their unique mor-

phologies. However, fabrication of HAp nanobers has been limited to high-

temperature and/or long-term methods. Herein, we report that HAp nanobers with

more than 5μm in length (aspect ratio >100) can be easily obtained by a simple wet

precipitation method without additives at relatively low temperature (80°C) under

acidic conditions (initial pH of 6.5 and nal pH of 3.9), without pH control during

the precipitation.

Keywords Hydroxyapatite · Nanober · Wet chemical precipitation · Acidic

condition · Crystal growth

22.1 Introduction

Hydroxyapatite (HAp) is recognized as a major inorganic component of human

hard tissues (bones and teeth). Synthetic HAp, a type of bioceramics, exhibits bio-

compatibility (i.e., nontoxicity) (Lawton et al. 1989; Abdel-Gawad and Awwad

2010) and excellent cell adhesion properties (Dorozhkin 2010). Therefore, HAp and

its composites with polymers or metals have been widely used in orthopedic and

dental tissue engineering elds (Choi etal. 2010; Honda et al. 2010; Okada and

Matsumoto 2015). Other important applications of HAp include their use as drug

delivery carriers (Matsumoto etal. 2004; Bouladjine et al. 2009; Tomoda etal.

2010) and in liquid chromatographic packingmaterials (Kawasaki 1991) by utiliz-

ing the favorable adsorption capacity of the HAp surface for biomolecules, such as

M. Okada (*) · E. S. Hara · T. Matsumoto

Department of Biomaterials, Graduate School of Medicine, Dentistry and Pharmaceutical

Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, Japan

e-mail: m_okada@cc.okayama-u.ac.jp; gmd421209@s.okayama-u.ac.jp;

tmatsu@md.okayama-u.ac.jp

212

cell adhesion proteins (Kilpadi etal. 2001; Lee etal. 2010). HAp belongs to a hex-

agonal crystal system and possesses different properties on its a and c planes, and

hence, its morphology strongly affects protein adsorption properties (Kilpadi etal.

2001; Lee etal. 2010). Therefore, the control of HAp morphology is of fundamental

importanceto improve its adsorption properties.

HAp nanobers (or whiskers) have been attracted considerable attention, owing

to their unique morphologies, for application as adsorbents and reinforcing compo-

nents in biomedical composites (Qi etal. 2017). Nevertheless, previous reports have

demonstrated the synthesis of HAp nanobers only at high temperature, during long

periods, or by adding additives, such as hydrothermal methods (e.g., 180–200°C)

(Sadat-Shojai etal. 2012; Chen and Zhu 2016), homogeneous precipitation methods

(e.g., 72h) (Aizawa etal. 2005; Zhan etal. 2005), and wet precipitation methods

with surfactants (Liu etal. 2002; Chen and Zhu 2016). Therefore, synthesis of HAp

nanobers at mild conditions could reduce costs and enable more diverse applica-

tions of HAp. However, the synthesis of HAp nanobers at low temperature without

additives is still challenging.

Herein, we report that HAp nanobers could be easily obtained by a simple wet

precipitation method without additives at relatively low temperature (80°C) under

acidic conditions, without pH control during the precipitation. The formation pro-

cess of the HAp nanobers by the simple wet precipitation method was also

evaluated.

22.2 Materials andMethods

Unless otherwise mentioned, all materials were of reagent grade and were pur-

chased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All materials

were used as received. Milli-Q water (Millipore Corp., Bedford, MA, USA) with a

specic resistance of 18.2 × 106Ω·cm was used.

An aqueous solution of Ca(NO3)2·H2O (10g/L = 42.3mM; 160mL) was poured

into a 500-mL conical ask equipped with an inlet for nitrogen and a magnetic stir-

rer. After the reactor was heated at a predetermined temperature (30, 50, or 80°C),

initial pH of the solution was adjusted to 6.5 (i.e., acidic condition) or 10.0 (i.e.,

alkaline condition) by adding a 28% ammonia solution. After the temperature and

the initial pH were equilibrated for 5 min, an aqueous solution of (NH4)2HPO4

(101.6mM; 40mL; pH 8.0) was added at a feed rate of 8.0mL/h into the conical

ask, and the resultant mixture was stirred for another 12h at a constant tempera-

ture, with variations within 0.1°C. During the reaction, although the pH was not

controlled, the changes in pH were recorded with a pH meter (HM-31P; pH resolu-

tion, 0.01; DKK-TOA Corp., Tokyo, Japan) at a time interval of 2min. The resulting

product was then centrifugally washed three times with distilled water and then

dried at room temperature under reduced pressure for 1day.

The dispersed sample was dried on an aluminum stub and coated using an

osmium coater Neoc-Pro (Meiwafosis Co. Ltd., Tokyo, Japan) before particle

M. Okada et al.

213

morphology observation by scanning electron microscopy (SEM) using a JSM-

6701F microscope (JEOL Ltd., Tokyo, Japan) operated at 5 kV or the sample

wasdried on a collodion-coated grid before particle morphology observation and

electron diffraction measurement by transmission electron microscopy (TEM)

using a JEM- 2100F microscope (JEOL Ltd.) operated at 200kV.The number-aver-

aged size (N = 50) was determined from SEM photographs with image analysis

software (Image J; National Institutes of Health, Bethesda, MD, USA). Fourier-

transform infrared (FTIR) spectra were obtained using an IRAfnity-1S system

(Shimadzu Corp., Kyoto, Japan) with a KBr pellet method at a resolution of 4cm−1

with 32 scans. Product identication was also conducted by X-ray diffraction

(XRD) measurements (RINT2500HF; Rigaku Corp., Tokyo, Japan) equipped with

a Cu-Kα radiation source.

22.3 Results andDiscussion

In the case of alkaline conditions (i.e., initial pH = 10.0), the nal pH after the reac-

tion decreased to 8.2–9.3, and HAp crystals were obtained as shown in Fig.22.1a

(i–iii). The crystal morphologies varied by changing the temperature (Fig.22.2a–c);

i.e., more elongated HAp crystals were obtained by increasing the reaction tempera-

ture, which is consistent with previous reports (Sadat-Shojai etal. 2013; Okada and

Matsumoto 2015). Note that the size distributions of HAp crystals formed in the

Fig. 22.1 (a) XRD patterns of the products synthesized by wet precipitation methods under dif-

ferent initial pH and temperature conditions: (i) pH 10.0, 30°C; (ii) pH 10.0, 50°C; (iii) pH 10.0,

80°C; (iv) pH 6.5, 30°C; (v) pH 6.5, 50°C; (vi) pH 6.5, 80°C. (b) A phase diagram of the products

after hydrolysis of α-tricalcium phosphates at different pH and temperature conditions (Monma

1980; Monma etal. 1981) and the pH changes during wet precipitation methods at different initial

pH and temperature conditions: (open squares) pH 6.5, 30°C; (open triangles) pH 6.5, 50°C;

(open circles) pH 6.5, 80°C. Abbreviations: HAp hydroxyapatite, OCP octacalcium phosphate,

DCPD dicalcium phosphate dihydrate

22 Fabrication ofHydroxyapatite Nanobers withHigh Aspect Ratio via…

214

alkaline conditions were broad (i.e., polydispersed), which should be due to long

particle nucleation stage. In other words, the saturated solution concentration of

HAp is too small at alkaline conditions (Matsumoto et al. 2007), and hence the

particle nuclei formed throughout the feeding period (5h) of phosphate ion solution

into the calcium ion solution.

In the case of acidic conditions (i.e., initial pH = 6.5), the nal pH signicantly

dropped to 5.5–3.9 after the reaction, as shown in Fig.22.1b. Plate-like dicalcium

Fig. 22.2 SEM photographs of the products synthesized by wet precipitation methods under dif-

ferent initial pH and temperature conditions: (a) pH 10.0, 30°C; (b) pH 10.0, 50°C; (c) pH 10.0,

80°C; (d) pH 6.5, 30°C; (e) pH 6.5, 50°C; (f) pH 6.5, 80°C.The insets show magnied images

M. Okada et al.

215

phosphate dihydrate (DCPD) and/or octacalcium phosphate (OCP) were formed at

lower temperatures of 30°C and 50°C (Figs.22.1a (iv, v) and 22.2d–e), which is

almost consistent with the phase diagram (Fig.22.1b) reported from the data of the

hydrolysis products of α-tricalcium phosphate (α-TCP) (Monma 1980; Monma

etal. 1981). Interestingly, pure HAp crystals were obtained at 80°C even in the

acidic condition (nal pH = 3.9), which is not consistent with the phase diagram for

α-TCP hydrolysis (Fig.22.1b). The HAp crystals formed under the acidic condition

at 80°C showed a ber-like morphology, with the long axis being parallel to c axis

of HAp (Fig.22.3), and were longer than those formed under the alkaline condition

at the same temperature.

In order to check the formation process of the HAp nanobers under the acidic

condition at 80°C, a part of precipitation was collected during the reaction as shown

in Fig.22.4. From the XRD measurements (Fig.22.4c), only HAp crystals were

detected throughout the reaction even at the end of the feeding and ripening periods.

From SEM observation (Fig.22.4b), needle-like crystals of around 500nm in length

were formed at 30min, and they elongated into ber-like crystals with more than

10μm in length and around 50–100 nm in width (i.e., aspect ratio >100) at 1300

min.

Taken together, these results indicate that needle-like HAp was rstly precipi-

tated at the initial pH of 6.5 at 80°C, which is consistent with the phase diagram

(Fig.22.1b). The rstly formed HAp acted as a seed crystal during the following

feeding and ripening periods even at acidic conditions. The unexpected stability of

enamel apatite (i.e., ribbon-like apatite crystal elongated extremely in its c-axis

direction) at acidic conditions has been also reported; that is, enamel apatite did not

transformed to DCPD or other calcium phosphates even at pH 4 for 2months due to

a decrease in the ion product of enamel apatite with the decrease in pH (Larsen and

Jensen 1989). The acidic condition would be preferable for preventing both

secondary nuclei formation (due to an increase in the saturated solubility of HAp

Fig. 22.3 (a) TEM image of the product synthesized by the wet precipitation method at initial pH

6.5 and 80°C and (b) electron diffraction pattern of the area highlighted by a dotted circle in the

image (a). The incident electron beam direction in the diffraction pattern was parallel to [1–10]

zone of HAp crystal

22 Fabrication ofHydroxyapatite Nanobers withHigh Aspect Ratio via…

216

(Matsumoto etal. 2007) and CO32− contamination (due to a decrease in the saturated

solubility of CO2), which are known to inhibit HAp crystal growth (i.e.,

elongation).

The HAp nanobers prepared by the simple wet precipitation method without

additives at relatively low temperature (80°C) have potential applications as adsor-

bents and reinforcing components in biomedical composites because of their unique

morphologies (Qi etal. 2017). The preparation method described here accompanies

with the crystal growth of HAp (without secondary nuclei formation, under appro-

priate conditions). Further optimization of this low-temperature wet precipitation

method could enable (1) the preparation of more uniform size distribution of HAp

nanocrystals for the pre-prepared seed crystals with controlled initial size and num-

ber of seeds (or by developing a modied wet precipitation method with a stepwise

pH control from alkaline to acidic condition during the reaction) and (2) the devel-

opment of brush-like HAp coating for the substrates pre-coated with HAp seed

crystals. Note that brush-like HAp coating of titanium substrate showed superior

osteoconductivity compared with other morphologies (i.e., needle-like, plate-like,

net-like, and spherical) of HAp (Kuroda and Okido 2012). Nevertheless, brush-like

HAp coating has been limited to some metallic substrates because the previously

developed methods require electroconductive and thermally stable substrates due to

their coating conditions [e.g., electrochemical and/or high temperature conditions

such as above 140°C (Kuroda and Okido 2012) for wet methods and above 300°C

(Teshima etal. 2012) for dry methods]. The fabrication of brush-like HAp coated

substrate at low temperature and its application will be reported in the near future.

Fig. 22.4 (a) A variation of pH during the wet precipitation method at initial pH of 6.5 and 80°C.

(b) SEM photographs, (c) XRD patterns, and (d) FTIR spectra of the part of precipitation collected

during the wet precipitation method at 30, 60, 300 (end of feeding), and 1300min

M. Okada et al.

217

Acknowledgments This work was supported partly by the Japan Society for the Promotion of

Science KAKENHI (grant numbers: JP16H05533, JP15K1572307, and JP25220912), the

Matching Planner Program (MP27115663113) from Japan Science and Technology Agency, and

the Cooperative Research Project of Research Center for Biomedical Engineering, Ministry of

Education, Culture, Sports, Science and Technology of Japan.

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

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Commons license, unless indicated otherwise in a credit line to the material. If material is not

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statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

M. Okada et al.

219© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_23

Chapter 23

Physico-chemical Characterisation

oftheProcesses Involved inEnamel

Remineralisation by CPP-ACP

KeithJ.Cross, N.LailaHuq, BoonLoh, Li-MingBhutta, BillMadytianos,

SarahPeterson, DavidP.Stanton, YiYuan, CoralieReynolds, GlenWalker,

PeiyanShen, andEricC.Reynolds

Abstract Casein phosphopeptides derived from tryptic digests of milk caseins

spontaneously assemble with calcium and phosphate ions at high pH to form casein

phosphopeptide-amorphous calcium phosphate complexes (CPP-ACP). These com-

plexes have been shown to be able to repair lesions in tooth enamel (biohydroxyapa-

tite– HA) both invitro and invivo (specically white spot lesions in the early stages

of tooth decay). In order to better understand the processes involved in enamel rem-

ineralisation, the chemical equilibria between the CPP and calcium and phosphate

ions as a function of pH were investigated. Furthermore, a thin-enamel slab tech-

nique was developed with enhanced sensitivity to monitor the diffusion of radio-

opaque ions into individual lesions over a period of days to weeks.

Keywords Enamel · CPP-ACP · Remineralisation · Hydroxyapatite · Diffusion ·

NMR · Model

23.1 Introduction

Dental caries is initiated by the action of plaque odontopathogenic bacteria that fer-

ment dietary sugars and starches, thus producing organic acids that demineralise the

subsurface of enamel hydroxyapatite (Robinson etal. 2000). Since enamel caries is

K. J. Cross · N. L. Huq · B. Loh · L.-M. Bhutta · B. Madytianos · S. Peterson · D. P. Stanton

Y. Yuan · C. Reynolds · G. Walker · P. Shen · E. C. Reynolds (*)

Oral Health Cooperative Research Centre, Melbourne Dental School,

Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne,

Melbourne, VIC, Australia

e-mail: keith.cross@unimelb.edu.au; laila@unimelb.edu.au; billym@student.unimelb.edu.au;

speterson1@student.unimelb.edu.au; dstanton@unimelb.edu.au; yuay@unimelb.edu.au;

coralie@unimelb.edu.au; gdwalker@unimelb.edu.au; peiyan@unimelb.edu.au;

e.reynolds@unimelb.edu.au

220

essentially a chemical process, at early stages of dental caries, the hydroxyapatite

mineral loss is reversible. Any products that prevent enamel demineralisation and

promote remineralisation are described as exhibiting anticaries activity. The princi-

pal components of dairy products associated with their anticariogenic activity are

multi-phosphorylated caseins complexed with calcium and phosphate. Enzymic

hydrolysis of caseins yields phosphopeptides known as casein phosphopeptides

(CPP). These peptides, with their multiple phosphoseryl residues, bind relatively

large quantities of calcium and phosphate ions in an amorphous, bioavailable form

(Reynolds etal. 1995). The resulting complexes are known as casein phosphopeptide-

amorphous calcium phosphate (CPP-ACP). The two dominant, self-assembling

peptides are β-CN(1-25) and αS1-CN(59-79) forming 20–30% by mass of the total

CPP (Fig. 23.1). These bovine casein-derived peptides all contain the cluster

sequence motif -(Ser(P)-)3(Glu-)2.

The aim of this study was to investigate the interactions between the peptides and

crystalline and non-crystalline mineral components during the remineralisation

process.

23.2 Materials andMethods

23.2.1 Materials

Extracted human third molars were obtained from patients attending the Melbourne

Dental School with ethics approval (1340048). Enamel slices (~300μ thick) were

prepared from these teeth for examination.

[1] Arg1-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-

(Ser(P)-)3(Glu-)2-Ser-Ile-Thr-Arg25 β-CN(1-25)

[2] Gln59-Met-Glu-Ala-Glu-Ser(P)-Ile-(Ser(P)-)3(Glu-)2-Ile-Val-Pro-Asn-Ser(P)-Val-

Glu-Gln-Lys79 αS1-CN(59-79)

[3] Asn46-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-(Ser(P)-)3(Glu-)2-Ser(P)-Ala-Glu-

Val-Ala-Thr-Glu-Glu-Val-Lys70 αS2-CN(46-70)

[4] Lys1-Asn-Thr-Met-Glu-His-Val-(Ser(P)-)3(Glu-)2-Ser-Ile-Ile-Ser(P)-Gln-Glu-Thr-

Tyr-Lys21 αS2-CN(1-21)

Fig. 23.1 Sequences of the predominant tryptic phosphopeptides of CPP-ACP in three-letter

code. The cluster sequence motif critical to calcium and phosphate binding is highlighted in red

K. J. Cross et al.

221

23.2.2 Ion-Binding Studies

To evaluate the inuence of pH on the calcium and phosphate ion equilibria, solu-

tions of CPP-ACP, αS1-CN(59-79)-ACP, and β-CN(1-25) were subjected to pH titra-

tions. Calcium and phosphate ion concentrations were determined using

microltration using previously described protocols, (Cross etal. 2005) modied to

use a Dionex ion analyser.

23.2.3 Nuclear Magnetic Resonance Studies

NMR spectra were acquired at 599.741MHz on a Varian Unity Inova spectrometer

as described previously (Cross etal. 2016). Solution-phase diffusion measurements

were performed using the sLED experiment as described previously (Altieri etal.

1995).The amplitude of the NMR signal is a function of the applied magnetic eld

gradient and the Stokes-Einstein radius of the diffusing species

(S∝exp(−αDG2),where D is the diffusion coefcient, G is the magnetic eld

gradient, and α depends on experimental values. The diffusion coefcient is related

to the hydrodynamic radius by D=kBT/6πρR where kB is the Boltzmann constant, T

the absolute temperature, ρ the solution viscosity, and R the hydrodynamic radius of

the spherical particles.

23.2.4 Remineralisation Studies

The novel technique utilised thin slabs of enamel (~300μ thick) cut from human

third molars that allowed sound mineral portions of the slabs to be used as controls

in measuring the time course of remineralisation of articial lesions. The technique

is an extension of that previously described (Cochrane etal. 2008), with reminerali-

sation of individual slabs being assessed after 0, 2, 3, 6, 12, 15, and 20days immer-

sion in a remineralisation solution at either pH 5·5 or pH 7·0. Diffusion of

radio-opaque ions into the articially prepared lesions was monitored by TWIM

(Thomas etal. 2006). Acid-resistant nail polish was used to dene the remineralisa-

tion zone and applied three times to prevent leakage during the soaking in the rem-

ineralisation solutions. Remineralising solutions consisted of 1% solutions of either

CPP-ACP or β-CN(1-25)-ACP prepared at either pH 7.0 or 5.5 to compare the

effects of neutral and acidic pH.

Lesion-sections were subjected to transversal wavelength-independent microra-

diography (TWIM) at days 0, 2, 3, 6, 12, 15, and 20 to visualise the time depen-

dence of mineral ion uptake during enamel remineralisation. Microradiographs

acquired on day 0 were used as control images for each lesion-section. The

23 Physico-chemical Characterisation oftheProcesses Involved inEnamel…

222

lesion- sections remained soaked in the remineralisation solutions except when

being X-rayed.

DOSY experiments were performed using the sLED technique to determine the

relative rates of diffusion of the complexes using either the integrated aromatic or

aliphatic signals of a β-CN(1-25)-ACP sample.

SDS-PAGE of CPP cross-linked using glutaraldehyde was conducted to analyse

the multimerisation of CPP as described previously (Cross etal. 2016).

23.3 Results

23.3.1 The CPP-ACP Complexes Exist inEquilibria withBoth

Bound andFree Calcium andPhosphate Ions

When prepared at pH 9, the CPP-ACP complexes have most of the calcium and

phosphate peptide bound. However, these complexes exist in solution in equilib-

rium with free ionic calcium and phosphate. Figure23.2 shows sigmoidal pH titra-

tion curves for β-CN(1-25)-ACP representative of results obtained in this study. The

pKa values for Ca2+ and Pi binding are 5.983 ± 0.038 and 6.302 ± 0.067,

respectively.

Further pH titrations were performed on 1% and 2% β-CN(1-25)-ACP com-

plexes prepared with varying Ca/Pi ratio and varying peptide:Ca ratio. Plots of the

bound calcium and phosphate concentrations against pH revealed sigmoidal curves

whose shape remained independent of the Ca/Pi ratio. For the complexes prepared

at ratios of Ca/Pi ranging from 1.6 to 1.51, and 12–15 Ca/peptide, the pH dependence

Fig. 23.2 Representative pH titration curves for a laboratory-prepared sample of β-CN(1-25)-

ACP. The solid-line curves are t to the Henderson-Hasselbalch formula yielding effective pKa

values of 6.302 ± 0.067 for the phosphate ion curve and 5.983 ± 0.038 for the calcium ion curve.

Note that at low pH, the peptides bind residual calcium ions but no phosphate ions

K. J. Cross et al.

223

of the bound calcium and phosphate were similar. Further analysis using αS1- CN(59-

79)-ACP revealed a well-dened phase of ACP stabilised by the CPP (Fig.23.3a–

c). Figure 23.3d shows a representative powder X-ray diffraction pattern of

CPP-ACP that is consistent with an amorphous phase of calcium phosphate.

23.3.2 The CPP-ACP Complexes Are Small Readily Diffusible

Species

The sLED experiment yields a diffusion-dependent signal whose functional depen-

dence on the applied, magnetic-eld gradient is dependent on the hydrodynamic

radius of the molecule being studied. Figure 23.4a shows a plot of sLED signal

intensity against the applied magnetic eld gradient. This provides the ratio of

hydrodynamic radii of the β-CN(1-25)-ACP complex relative to that of water. The

Fig. 23.3 (a) A plot illustrating the linear relationship between CPP-bound inorganic phosphate

and CPP-bound calcium in excess of that bound at low pH (denoted by ν0Ca). (b) A plot of CPP-

bound inorganic phosphate against ACP ion activity product. This illustrates that only an amor-

phous calcium phosphate phase predicts a one-to-one functional dependence of calcium phosphate

ion activity product and the activity of phosphate (shown here) or calcium. (c) A plot illustrating

that the best t between the ion activity product and the phosphate (or calcium) activity occurs with

a slightly calcium-rich, non-stoichiometric ACP phase having the formula Ca3.0425(PO4)2(OH)0.085.

The lower curve is for calcium-decient ACP phases and does not have a maximum at realistically

achievable compositions. (d) X-ray powder diffraction image of a CPP-ACP sample demonstrat-

ing the broad peaks expected from an amorphous solid

23 Physico-chemical Characterisation oftheProcesses Involved inEnamel…

224

measured hydrodynamic radii of the β-CN(1-25)-ACP complex range from 1.53 ±

0.03nm at pH 6 to 1.92 ± 0.08nm at pH 9. The deviation from the tted curves at

high-magnetic-eld-gradient values ts a two-component model that is consistent

with the formation of aggregates.

SDS-PAGE of CPP cross-linked using glutaraldehyde, in the presence of either

calcium ions (Fig.23.4b) or calcium and phosphate ions (Cross et al. 2016), sug-

gests that the complexes contain up to six CPP peptides. Figure 23.4c shows a

model of the CPP-ACP complex consistent with the results of these experiments.

Fig. 23.4 (a) A representative plot of data from the sLED NMR experiment (Altieri etal. 1995)

that uses magnetic eld gradients to measure the relative rates of diffusion of various protonated

species. The water signal (blue) can be compared with either the integrated aromatic (green) or

aliphatic (red) signals of a CPP-ACP sample. The data from both peptide curves provided an inter-

nal consistency check. The Stokes-Einstein equation relates the hydrodynamic radius of a particle

and its diffusion constant, allowing the determination of the hydrodynamic radius of the complexes

given the known radius of water. The calculated hydrodynamic radii of the CPP-ACP complexes

ranged from 1.53 ± 0.03nm at pH 6 to 1.92 ± 0.08nm at pH 9. The deviation from the tted curves

at high-magnetic-eld-gradient values ts a two-component model consistent with the formation

of aggregates. (b) A representative SDS-PAGE gel featuring the cross linking of the CPP using

glutaraldehyde, observed in the presence of calcium ions. The multimerisation was also observed

in the presence of calcium and phosphate ions suggesting that the complexes contain up to six CPP

peptides. (c) A model of the CPP-ACP complex consistent with these and various other experi-

ments. The peptide is depicted as sticks within a translucent van der Waals surface, and the ACP is

shown as a pale grey van der Waals surface

K. J. Cross et al.

225

23.3.3 Both CPP-ACP andβ-CN(1-25)-ACP Complexes

Release Mineral Ions that Remineralise Demineralised

Enamel Lesions

Blocks of enamel from third molars with demineralised lesions were prepared that

yielded multiple uniformly demineralised lesion-sections that would enable inter-

tooth and intra-tooth comparisons (Fig.23.5a). The sound enamel regions provided

the experimental control regions for the experiments. Acid-resistant nail polish was

used to dene the remineralisation zone. Microradiography was used to visualise

the time dependence of mineral ion uptake, in enamel remineralisation experiments.

Figure23.5b shows a sampleX-ray micrograph withthe demineralised zone (arrow)

and the adjacent sound enamel (star).

Fig. 23.5 Key features of the developed method to study the time dependence of remineralisation

of enamel using thin slabs of enamel (~300μ) cut from human third molars. The sound enamel

regions provide the experimental control regions for the experiments. (a) Thin slab of enamel cut

from human third molar and prepared with acid-resistant nail polish dening the remineralisation

zone. (b) X-ray micrograph showing demineralised zone (arrow) and adjacent sound enamel (star).

ions into the lesion. Remineralisation occurs within a few days. This plot of X-ray opacity against

lesion depth enables the calculation of As being the area under the sound enamel curve, Am repre-

senting the areas under the individual ‘remineralised’ enamel curves, and Ad being the area under

the ‘demineralised’ enamel curve (day 0) to determine extent of remineralisation (seeFig. 23.6)

23 Physico-chemical Characterisation oftheProcesses Involved inEnamel…

226

Figure 23.5c shows a plot of the time-dependent, X-ray opacity of a specic

enamel slab after demineralisation at day 0 and following remineralisation until day

20. The X-ray data showed a diffusion-dependent increase in electron density, inter-

preted as mineralisation.

A time-dependent uptake of mineral was observed in the presence of both CPP-

ACP and β-CN(1-25)-ACP at both pH values. The calculated data from each sample

was tted to the time-dependent part of a diffusion equation of the form

RR kt

=´

maxtanh( )

where ‘k’ represents the rate constant for diffusion and ‘t’ represents the elapsed

time taken in days. The Pearson correlation coefcients for the non-linear curve t

conrmed that the data were consistent with a simple diffusion model for reminer-

alisation. Figure 23.6 illustrates time-dependent plots of the calculated extent of

remineralisation (Rf) for four representative sections. The data is consistent with

mineral ions from CPP-ACP and β-CN(1-25)-ACP diffusing into and interacting

with the enamel crystals. A recent study of the interaction of CPP-ACP with either

enamel or saliva-coated enamel (Huq et al. 2018) indicates that chemical

Fig. 23.6 Plot of

time-dependent

remineralisation for four

lesion-sections. Each data

point represents the extent

of remineralisation

calculated by the ratio

AmAd

AsA

, where

Am is the area under the

‘remineralised’ enamel

curve, Ad is the area under

the ‘demineralised’ enamel

curve (day 0), and As is the

area under the sound

enamel curve. Statistically

signicant differences in

the extent of

remineralisation are

observed between different

samples and may be due to

biological differences: the

lowest Rf of 0.17 ± 0.04 (in

the graph above) differs

signicantly from the

maximum Rf of 0.28 ± 0.04

at a p=0.025

K. J. Cross et al.

227

equilibrium is established within a few hours; thus, the rate-limiting step for enamel

remineralisation is the rate of diffusion of ions into the subsurface enamel.

23.4 Discussion

In this study, a variety of methods have been used to characterise the complexes

formed by the casein phosphopeptides with calcium and phosphate ions. CPP bind

calcium and phosphate to form stable CPP-ACP complexes in alkaline solution.

These studies show that the ratio of bound calcium to bound phosphate is constant

and independent of pH in the range of pH 7–9. Furthermore, the ion activity product

ts a single curve for a calcium-rich, non-stoichiometric calcium phosphate phase.

DOSY experiments using the sLED sequence demonstrated that the complex has a

small but signicant variation in size with pH.These experiments further revealed

the ability of the small complexes to aggregate as concentrations were increased.

The model of the CPP-ACP complex with all amino acids in the peptides interacting

with the ACP surface is consistent with earlier ndings that the peptide length inu-

ences the extent of binding to calcium and phosphate ions (Cross etal. 2005).

The importance of small readily diffusible CPP-ACP complexes is conrmed by

the experiments using thin slabs of human enamel with articial lesions that mimic

early carious lesions. To enable the time-dependent monitoring, the current remin-

eralisation procedures (Shen etal. 2011) required extensive improvements. The use

of the single section for all-time points required a thicker section to withstand

repeated handling. To accommodate the increased thickness, TWIM was used

instead of the commonly used transverse microradiography (TMR) technique, both

methods being validated techniques for monitoring carious lesions (Thomas etal.

2006). In addition, the acid-resistant nail varnish was reapplied three times to pre-

vent leakage during the soaking in remineralisation solutions. To improve the

signal-

to-noise ratio, an additional 500-μ-thick aluminium lter was used with an

optimal X-ray tube voltage of 30kV.

We observed statistically signicant differences in the extent of remineralisation

within the multiple lesion-sections derived from the same individual tooth. These

differences were attributed to varying microporosities of the demineralised lesion-

sections of each individual tooth.

In conclusion, the CPP-ACP complexes were characterised to be a small readily

diffusible species that can release the mineral ions on contact with demineralised

enamel lesions. Furthermore both CPP-ACP and β-CN(1-25) complexes were able

to remineralise within a few days in invitro experiments.

Acknowledgements This study was funded by the Oral Health Cooperative Research Centre and

NHMRC. Extracted human third molars were obtained from patients attending the Melbourne

Dental School with ethics approval (1340048).

23 Physico-chemical Characterisation oftheProcesses Involved inEnamel…

228

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dient NMR self-diffusion measurements. JAm Chem Soc 117(28):7566–7567

Cochrane NJ, Saranathan S, Cai F, Cross KJ, Reynolds EC (2008) Enamel subsurface lesion rem-

ineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and uo-

ride. Caries Res 42(2):88–97. https://doi.org/10.1159/000113161

Cross KJ, Huq NL, Palamara J, Perich JW, Reynolds EC (2005) Physicochemical characteriza-

tion of casein phosphopeptide-amorphous calcium phosphate nanocomplexes. JBiol Chem

280(15):15362–15369

Cross KJ, Huq NL, Reynolds EC (2016) Casein phosphopeptide-amorphous calcium phosphate

nanocomplexes: a structural model. Biochemistry 55(31):4316–4325. https://doi.org/10.1021/

acs.biochem.6b00522

Huq NL, Cross KJ, Myroforidis H, Stanton DP, Chen YY, Ward BR, Reynolds EC (2018)

Molecular interactions of peptide encapsulated calcium phosphate delivery vehicle at enamel

surfaces. Proc BIOMIN XIV

Reynolds EC, Cain CJ, Webber FL, Black CL, Riley PF, Johnson IH, Perich JW (1995)

Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the

rat. JDent Res 74(6):1272–1279

Robinson C, Shore RC, Brookes SJ, Strafford S, Wood SR, Kirkham J (2000) The chemistry of

enamel caries. Crit Rev Oral Biol Med 11(4):481–495

Shen P, Manton DJ, Cochrane NJ, Walker GD, Yuan Y, Reynolds C, Reynolds EC (2011) Effect of

added calcium phosphate on enamel remineralization by uoride in a randomized controlled in

situ trial. JDent 39(7):518–525. https://doi.org/10.1016/j.jdent.2011.05.002

Thomas RZ, Ruben JL, de Vries J, ten Bosch JJ, Huysmans MC (2006) Transversal wavelength-

independent microradiography, a method for monitoring caries lesions over time, validated

with transversal microradiography. Caries Res 40(4):281–291

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

K. J. Cross et al.

229© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_24

Chapter 24

Molecular Interactions ofPeptide

Encapsulated Calcium Phosphate Delivery

Vehicle at Enamel Surfaces

NoorjahanLailaHuq, KeithJohnCross, HelenMyroforidis,

DavidPhillipStanton, Yu-YenChen, BrentRobertWard,

andEricCharlesReynolds

Abstract Phosphorylated peptides derived from milk caseins, known as casein

phosphopeptides (CPP), self-assemble and encapsulate the calcium and phosphate

mineral in the form of amorphous calcium phosphate (ACP), thus forming CPP-

ACP nanocomplexes that are nontoxic and biocompatible. The biomedical applica-

tion is the repair of tooth surfaces (enamel) at early stages of tooth decay. These

nanocomplexes release calcium and phosphate ions to rebuild demineralised HA

crystals in enamel subsurface lesions. The topical application of CPP-ACP at the

tooth surface initiates a series of interactions at the enamel mineral hydroxyapatite

surface and at the enamel salivary pellicle that are not well understood. In this study,

we have shown that the β-casein (1-25) peptide binds reversibly to Ca2+, Mg2+, Mn2+,

La2+, Ni2+, and Cd2+ metal ions. In contrast, binding to Sn2+, Fe2+, and Fe3+ ions

resulted in ion-induced aggregation. The casein peptides as well as the mineral ions

dissociate from the CPP-ACP complexes to adsorb to both the uncoated and saliva-

coated mineral surface with the mineralisation increasing monotonically with

increasing pH.Furthermore, SEM of the CPP-ACP revealed images of spherical

particles surrounded by ACP mineral. In conclusion, the enamel remineralisation

process involves an array of interactions between the peptide and mineral ions of the

CPP-ACP delivery vehicle and the tooth enamel mineral with its salivary pellicle.

Keywords Enamel · CPP-ACP · Saliva · SEM · Hydroxyapatite · Mineralisation

N. L. Huq · K. J. Cross · H. Myroforidis · D. P. Stanton · Y.-Y. Chen · B. R. Ward

E. C. Reynolds (*)

Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute of

Molecular Science and Biotechnology, The University of Melbourne,

Melbourne, Victoria, Australia

e-mail: laila@unimelb.edu.au; keith.cross@unimelb.edu.au; dstanton@unimelb.edu.au;

yyyc@unimelb.edu.au; brentrw@unimelb.edu.au; e.reynolds@unimelb.edu.au

230

24.1 Introduction

Dental caries is the destruction of tooth surfaces by acid generated by plaque odon-

topathogenic in a complex chemical process (Robinson etal. 2000). The reversibil-

ity of enamel hydroxyapatite mineral loss at early stages of dental caries has led to

the development of various anticariogenic approaches to repair enamel lesions

(Cochrane etal. 2010). One oral therapeutic consists of phosphorylated peptides

derived from milk caseins, known as casein phosphopeptides (CPP) that self-

assemble and encapsulate the calcium and phosphate mineral in the form of amor-

phous calcium phosphate (ACP) (Reynolds etal. 1995).

At the tooth surface, the mechanism of the remineralisation process by CPP-ACP

including the changes in the chemical equilibria of the complexes and the enamel

lesions is unclear. Above the enamel hydroxyapatite are further zones of chemical

complexity, including the layer of salivary proteins known as the acquired enamel

pellicle and the outermost layer of oral biolm. Our long-term goal has been to

study the molecular interactions between CPP-ACP complexes (Cross etal. 2016)

and enamel hydroxyapatite, salivary proteins (Huq etal. 2016), and the oral biolm

(Dashper etal. 2016) that would occur during topical application of CPP-ACP in the

oral cavity. In this study, we investigate the interactions between peptide and crys-

talline and non-crystalline mineral components during the remineralisation process

by the peptide encapsulated delivery vehicle CPP-ACP.

24.2 Materials andMethods

The sequences of four bovine casein-derived tryptic phosphopeptides containing

the cluster sequence motif -(Ser(P)-)3(Glu-)2 are shown in Fig.24.1. The two domi-

nant, self-assembling peptides are β-CN(1-25) and αS1-CN(59-79) forming 20–30%

by mass of the total CPP (Fig.24.1).

[1] Arg1-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-

(Ser(P)-)3(Glu-)2-Ser-Ile-Thr-Arg25 β-CN(1-25)

[2] Gln59-Met-Glu-Ala-Glu-Ser(P)-Ile-(Ser(P)-)3(Glu-)2-Ile-Val-Pro-Asn-Ser(P)-Val-

Glu-Gln-Lys79 αS1-CN(59-79)

[3] Asn46-Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-(Ser(P)-)3(Glu-)2-Ser(P)-Ala-Glu-

Val-Ala-Thr-Glu-Glu-Val-Lys70 αS2-CN(46-70)

[4] Lys1-Asn-Thr-Met-Glu-His-Val-(Ser(P)-)3(Glu-)2-Ser-Ile-Ile-Ser(P)-Gln-Glu-Thr-

Tyr-Lys21 αS2-CN(1-21)

Fig. 24.1 The sequences of the four major casein tryptic phosphopeptides are depicted using the

three-letter code with the motif -(Ser(P)-)3(Glu-)2 underlined

N. L. Huq et al.

231

24.2.1 Adsorption Studies

An assay was developed to determine the binding of the CPP-ACP components 1

and 2 (Fig.24.1) to enamel using an invitro model with synthetic HA used as a

substitute for dental enamel (Huq etal. 2016). The standard curve was derived from

peak heights of the RP-HPLC proles of puried peptides with concentrations rang-

ing from 10 to 1000μg/ml in 25 mM NaCl and 25 mM imidazole buffer (pH 7).

CPP of the same concentrations were incubated with end-over-end rotation for

1–4h at 37°C with 2 mg of HA. The samples were centrifuged at 10,000 g for

15 min to pellet peptide bound to HA. The supernatants analysed by RP-HPLC

provided the unbound peptide concentration. The partitioning of the free and

peptide-

bound ions was determined followed by measurements of calcium and

phosphate ions (Cross etal. 2005). The saliva collection procedures and approval

were as recently described (Huq etal. 2016).

24.2.2 Chemical Equilibria Studies

Test and control solutions were added to 2mg HA.After mixing thoroughly, the

samples were incubated for 2h at RT with end-over-end rotation. To remove the HA

crystals, the samples were centrifuged at 10,000g. The HA crystal-free superna-

tants as well as the original solutions were subjected to ion quantitation for the ions

in both peptide-free and peptide-bound states. In a second study, the HA crystals

were pre-equilibrated with water at the 3 pH values. Following centrifugation, the

calcium, phosphate, H+, and OH− ions released from the uncoated and saliva-coated

HA crystals into the supernatants were measured. These supernatants were used to

prepare the 0.2% CPP-ACP solutions. The partitioning of the ions associated with

the peptide complexes and those free in solution was determined. These superna-

tants were then added to the pre-equilibrated HA.The concentrations of total, free,

and CPP-bound calcium and phosphate ions and pH values of the 0.2% CPP-ACP

solutions prepared at pH 5.5, 7.0, and 8.5 before and after incubation with crystalline-

uncoated HA and saliva-coated HA were measured.

The dissociation study was performed by adding varying amounts of HA to 1%

CPP-ACP solution at pH 7 and 5.5 and monitoring the supernatant prole by

RP-HPLC.

24.2.3 SEM Studies

SEM of a 5% CPP-ACP solution was performed using 1kV with 25pA current with

a T1 detector in a Teneo VS instrument (FEI). Images at 104, 5 × 104, and 8 × 104

magnication were obtained.

24 Molecular Interactions ofPeptide Encapsulated Calcium Phosphate Delivery…

232

24.3 Results

24.3.1

β-Casein (1-25) Peptide Demonstrates aRange

ofInteractions withMetal Ions

We selected one of the principal components of CPP β-CN(1-25) (1, Fig.24.1) for

exposure to different metal ions to examine the recovery of peptide 1 by RP-HPLC

at pH 2. A single peak with identical retention times on the chromatogram (Fig.24.2)

was observed for peptide 1 incubated with Ca2+, Mg2+, Mn2+, La2+, Ni2+, and Cd2+

ions. This was indicative of reversible ion binding. However, peptide 1 incubated

with Sn2+, Fe2+, and Fe3+ ions, eluted as multiple broadened peaks indicative of ion-

induced aggregation. This study conrms that β-CN(1-25) is capable of releasing

the bound calcium ions at low pH without experiencing permanent aggregation.

24.3.2 The Predominant Peptides oftheCPP-ACP Complex

Bind toUncoated HA andSaliva-Coated HA

To determine if the pure peptides derived from CPP directly interact with enamel,

adsorption studies were performed with enamel substitute HA and saliva-coated

HA.We analysed the individual adsorption patterns of the two peptides 1 and 2 with

Fig. 24.2 RP-HPLC chromatogram depicting the recovery elution proles of β-CN(1-25) incu-

bated with Ca2+, Sn2+, Fe2+, and Fe3+ ions. The binding to Ca2+ ions by β-CN(1-25) is reversible; this

is pivotal to the delivery of mineral by CPP-ACP to the enamel

N. L. Huq et al.

233

HA for Langmuir and Freund-type binding. Both peptides bound to HA surfaces

according to a Langmuir-type adsorption model with peptide 1 (K=323 ± 149ml/

μmol) having a higher afnity for HA than peptide 2 (K=49 ± 22ml/μmol). The

adsorption prole of bound versus total showed an intermediate and nal plateau

indicating biphasic processes. At lower peptide concentrations, both peptide 1

(K=463 ± 200ml/μmol) and peptide 2 (K=194 ± 122ml/μmol) had a greater afn-

ity for the saliva-coated HA surface, than their respective afnities for an uncoated

HA surface.

24.3.3 Casein Peptides Dissociate fromtheCPP-ACP Complex

toBind toHA

The levels of soluble peptides from the CPP-ACP complexes at pH values 7.0 and 5.5

were monitored by RP-HPLC.Addition of HA caused a reduction of the soluble levels

of peptides conrming that the multi-phosphorylated [1-4] and mono- phosphorylated

peptide β-CN(33-48) dissociate from the ACP to adsorb onto HA.Figure24.3 shows

a representative time-dependent loss by 1% CPP-ACP at pH 5.5.

Fig. 24.3 RP-HPLC analysed loss of soluble casein phosphopeptides from CPP-ACP at pH 5.5

after addition of HA at 0 min. The multi-phosphorylated [1-4] and mono-phosphorylated

β-CN(33- 48) peptides dissociate from the ACP to adsorb onto the crystalline HA

24 Molecular Interactions ofPeptide Encapsulated Calcium Phosphate Delivery…

234

24.3.4 The Chemical Equilibria ofHA Surfaces Is Altered

bythe Addition oftheIndividual Peptides andPeptide-

ACP Complexes

The changes in chemical equilibria of HA surfaces were monitored during binding

studies of crystalline HA exposed to pure peptides 1 or 2 (both without Ca2+) or

β-CN(1-25)-ACP.The addition of HA to a solution of either of the two pure pep-

tides 1 or 2 dissolved at 120mg/l concentrations resulted in the post-addition mea-

surement of 244 ± 11mM and 124 ± 5mM calcium, respectively, in the peptide

solutions. This was interpreted as a peptide adsorption-mediated release of calcium

ions from the crystalline HA into the solution (Misra 1997). In contrast, albumin

(120mg/l), used as a control, elicited a much lower adsorption-mediated release of

57 ± 16mM calcium from HA.The initial Ca2+ concentration in a 240mg/l solution

of the β-CN(1-25)-ACP complex was reduced after the addition of HA.The Ca2+

reduction from 355 ± 67 to 246 ± 68mM was attributed to the calcium ions in the

amorphous phase previously associated with the peptide 1 now binding to or pre-

cipitating with crystalline HA.

24.3.5 The CPP-ACP Complexes Exhibit apH-Dependent

Release ofMineral Ions toUncoated andSaliva-Coated

HA Crystals

The movement of calcium, phosphate, and hydroxyl ions to and from the uncoated

and saliva-coated HA crystals during exposure to CPP-ACP was monitored during

binding studies (Table24.1). Table24.1 shows a net loss of total calcium and phos-

phate ions from the CPP-ACP on binding to HA at pH 5.5, 7.0, and 8.5. This loss

represented transfer of mineral from the amorphous phase of ACP to the crystalline

HA.The net transfer was greatest at the highest pH 8.5.

While the CPP-ACP complexes prepared at pH 9 have most of the calcium and

phosphate peptide bound, these complexes exist in solution in equilibrium with free

ionic calcium and phosphate (Cross etal. 2005). The values in brackets indicate the

proportion of free ions (Table24.1). The proportion of mineral ions bound within

complexes was reduced after incubation with both uncoated and saliva-coated

HA. This was attributed to the dissociation of peptides from the complexes to

remain free in solution and/or to adsorb to HA.The reductions in pH experienced

by the CPP-ACP solutions following exposure to HA were attenuated in the pres-

ence of saliva-coated HA (Table24.1).

The degree of saturation of the calcium phosphate solutions was calculated using

the solubility products at 37°C of hydroxyapatite (HA) (Ksp~10−117), octacalcium

phosphate (OCP) (Ksp~10−96), dicalcium phosphate dihydrate (DCPD) (Ksp~10−6),

and ACP (Ksp~10−25) (Cross et al. 2005). The calcium and phosphate ions were

N. L. Huq et al.

235

saturated with respect to the HA phase for both total and unbound fractions at all pH

values (Table24.2). In summary, these studies have conrmed that mineral ions are

released from CPP-ACP that adsorb onto the crystalline HA and that mineralisation

increases monotonically with increasing pH.

Table 24.1 Summary of the concentrations of total, free, and CPP-bound calcium and phosphate

ions and pH values of the 0.2% CPP-ACP solutions prepared at pH 5.5, 7.0, and 8.5 before and

after incubation with crystalline HA and saliva-coated HA

Solution [calcium] (μM) before incubation with HA

Total 5261±32 5285 ± 17 5225 ± 9

Free 4801 ± 50 (91%) 1847 ± 49.12 (35%) 365 ± 9 (7%)

CPP-bound 460 ± 60 3438 ± 52 4860 ± 13

Initial pH pH 5.55 pH 6.99 pH 8.5

Supernatant after incubation with HA

Total 5001 ± 43 4201 ± 105 3573 ± 128

Free 4807 ± 92 (96%) 2066 ± 36 (49%) 524 ± 35 (15%)

CPP-bound 194 ± 102 2134 ± 111 3049 ± 133

HA-bound 260 ± 54 1084 ± 106 1652 ± 128

Final pH pH 5.32 pH 6.85 pH 7.89

Supernatant after incubation with saliva-coated HA

Total 5042 ± 51 4224 ± 51 3620 ± 52

Free 4908 ± 169 (97%) 2058 ± 50 (49%) 527 ± 21 (15%)

CPP-bound 134 ± 177 2166 ± 72 3093 ± 56

HA-bound 219 ± 60 1061 ± 54 1605 ± 53

Final pH pH 5.55 pH 6.88 pH 8.04

Solution [phosphate] (μM) before incubation with HA

Total 3699 ± 6 3744 ± 21 3739 ± 16

Free 3354 ± 184 (91%) 1420 ± 73 (38%) 386 ± 3 (10%)

CPP-bound 346 ± 185 2324 ± 76 3353 ± 15

Initial pH pH 5.55 pH 6.99 pH 8.5

Supernatant after incubation with HA

Total 3461±35 2930 ± 42 2485 ± 89

Free 3223 ± 175 (93%) 1599 ± 44 (55%) 627 ± 29 (25%)

CPP-bound 238 ± 179 1331 ± 61 1858 ± 94

HA-bound 238 ± 36 814 ± 47 1254 ± 90

Final pH pH 5.32 pH 6.85 pH 7.89

Supernatant after incubation with saliva-coated HA

Total 3427 ± 37 2757 ± 53 2443 ± 50

Free 3191 ± 50 (93%) 1475 ± 59 (54%) 518 ± 41 (21%)

CPP-bound 236 ± 62 1281 ± 79 1925 ± 65

HA-bound 272 ± 37 987 ± 57 1296 ± 53

Final pH pH 5.55 pH 6.88 pH 8.04

24 Molecular Interactions ofPeptide Encapsulated Calcium Phosphate Delivery…

236

24.3.6 SEM Reveals Images ofNon-crystalline Morphology

forCPP-ACP Complexes

Preliminary SEM images of 5% CPP-ACP solution show areas with a non- crystalline

morphology expected for colloidal complexes(Fig. 24.4).

Table 24.2 Degree of saturation of calcium phosphate solutions before and after incubation of

0.2% CPP-ACP at different pH with HA or saliva-coated HA

A Total fraction: total calcium and phosphate in 0.2% CPP-ACP

Mineral

Original pH Final pH HA DCPD TCP OCP Acid ACP Basic ACP

pH 5.55 pH 5.55 4.05 0.92 0.465 1.41 0.21 0.07

pH 6.99 pH 6.99 36.40 2.86 4.21 7.45 1.78 0.65

pH 8.5 pH 8.5 152.45 2.67 15.07 16.23 5.94 2.43

After incubation with HA

pH 5.55 pH 5.32 2.60 0.68 0.29 0.99 0.13 0.05

pH 6.99 pH 6.85 26.20 2.27 2.99 5.67 1.27 0.46

pH 8.5 pH 7.89 74.96 2.43 7.81 10.52 3.17 1.24

After incubation with saliva-coated HA

pH 5.55 pH 5.55 3.89 0.87 0.44 1.36 0.20 0.07

pH 6.99 pH 6.88 26.90 2.25 3.06 5.74 1.30 0.47

pH 8.5 pH 8.04 86.06 2.38 8.81 11.28 3.55 1.41

B Unbound fraction: calcium and phosphate in 0.2% CPP-ACP not associated with

peptide complex

Mineral

Original pH Final pH HA DCPD TCP OCP Acid ACP Basic ACP

pH 5.55 pH 5.55 3.78 0.85 0.43 1.32 0.19 0.07

pH 6.99 pH 6.99 18.48 1.35 1.97 3.83 0.83 0.31

pH 8.5 pH 8.5 31.80 0.50 2.63 3.59 1.04 0.45

After incubation with HA

pH 5.55 pH 5.32 2.50 0.65 0.28 0.95 0.13 0.04

pH 6.99 pH 6.85 16.62 1.37 1.795 3.637 0.76 0.28

pH 8.5 pH 7.89 23.81 0.73 2.19 3.514 0.89 0.36

After incubation with saliva-coated HA

pH 5.55 pH 5.55 3.76 0.836 0.427 1.309 0.19 0.07

pH 6.99 pH 6.88 16.91 1.34 1.82 3.64 0.77 0.29

pH 8.5 pH 8.04 26.56 0.68 2.38 3.64 0.96 0.40

(A) Calcium and phosphate in total fraction; (B) calcium and phosphate in unbound fraction.

Values in bold indicate saturation with respect to the phase

N. L. Huq et al.

237

Fig. 24.4 SEM images of CPP-ACP at increasing magnication, taken using a T1 (BSE) detector

with beam deceleration in a Teneo VS Instrument (FEI) for enhanced surface details. Two μl of 5%

CPP-ACP, pH 7.24, was deposited onto a silicon substrate on an aluminium stub, and the excess

liquid was removed after 1min. CPP appeared to have formed spherical particles that were sur-

rounded by ACP.Landing energy, 1keV; stage bias, −2kV

24 Molecular Interactions ofPeptide Encapsulated Calcium Phosphate Delivery…

238

24.4 Discussion

The observed biomimicry of CPP that spontaneously self-assemble at pH 9 forming

complexes with calcium phosphate ions offers a functional advantage for the stable

storage and delivery of mineral ions. This paper describes interactions of the peptide

and mineral components of this delivery vehicle with enamel substitute HA.

In addition to the adsorption of puried casein phosphopeptides onto uncoated

HA, these studies conrmed that casein phosphopeptides also adsorbed onto the

saliva-coated HA being a mimic of the enamel salivary pellicle. Previously, we have

demonstrated that the casein phosphopeptides interact with salivary proteins includ-

ing those forming the enamel pellicle (Huq etal. 2016). Collectively, these studies

conrm that during topical application of a formulation with CPP-ACP complexes,

the peptides are not simply inert carriers of mineral. Instead, they are capable of

adsorbing to the crystalline enamel surface that has been recently brushed as well as

interacting with the salivary pellicle that forms on the enamel surface.

Since HA is not an inert material, the molecular events at the HA surface are of

interest. The casein phosphopeptides elicited an adsorption-mediated release of cal-

cium ions from HA into the solution. This is consistent with previous studies docu-

menting the release of ions from HA during adsorption of proteins and amino acids

(Misra 1997; Pearce 1981). In contrast when HA was introduced to the peptide-

ACP complexes in solution, there was a net loss of calcium and phosphate ions from

the solution indicating a net gain of calcium and phosphate ions by the crystalline

HA.These studies conrm that the mineral ions of the amorphous phase of ACP

that is stabilised by the peptides transfer to the crystalline HA monotonically with

increasing pH.Furthermore, during topical application, the dissociation of the CPP-

ACP complexes and the subsequent migration of ions to the enamel surface are not

hindered by the enamel salivary pellicle.

In conclusion, within the oral environment, the enamel remineralisation process

involves a complex interplay between the peptide and mineral ions of the CPP-ACP

delivery vehicle and the tooth enamel mineral with its salivary pellicle.

Acknowledgements We would like to thank Mr. Roger Curtain from the Bio21 Advanced

Microscopy Facility, the University of Melbourne for his assistance in SEM imaging.

References

Cochrane NJ, Cai F, Huq NL, Burrow MF, Reynolds EC (2010) New approaches to enhanced

remineralization of tooth enamel. JDent Res 89:1187–1197

Cross KJ, Huq NL, Palamara JE, Perich JW, Reynolds EC (2005) Physicochemical characteriza-

tion of casein phosphopeptide-amorphous calcium phosphate nanocomplexes. JBiol Chem

280:15362–15369

Cross KJ, Huq NL, Reynolds EC (2016) Casein phosphopeptide-amorphous calcium phosphate

nanocomplexes: a structural model. Biochemistry 55:4316–4325

N. L. Huq et al.

239

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

24 Molecular Interactions ofPeptide Encapsulated Calcium Phosphate Delivery…

241© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_25

Chapter 25

Preparation ofRandom andAligned

Polycaprolactone Fiber asTemplate

forClassical Calcium Oxalate Through

Electrocrystallization

LazyFarias, NicoleButto, andAndrónicoNeira-Carrillo

Abstract The aim of this study was to evaluate the effect of random and oriented

electrospun polycaprolactone (PCL) ber meshes on conductive indium tin oxide

(ITO) electrode on the in vitro electrocrystallization (EC) of calcium oxalate

(CaOx). For that, random and aligned PCL bers were prepared through at and

rotating collectors and directly collected on conductive ITO support that was used

as organic solid template for controlling the invitro EC of CaOx. Our ndings

revealed that electrospun PCL surface topology induced preferentially the nucle-

ation and crystal growth of CaOx along on individual aligned PCL bers during the

EC of CaOx. Scanning electron microscopy (SEM), energy dispersive X-ray spec-

troscopy (EDX), chronoamperometry, and X-ray diffraction (XRD) spectroscopy of

CaOx crystals show that the morphological orientation of PCL ber meshes acted

as selective good nucleation site at PCL surface controlling their CaOx crystal mor-

phologies and the crystallographic orientation of crystals inducing the coexistence

of dehydrated CaOx (COD) and monohydrated CaOx (COM) crystals as the unique

polymorphism.

Keywords Electrospun bers · Electrocrystallization · Calcium oxalate (CaOx) ·

Indium zinc oxide (ITO) · Polycaprolactone (PCL) · Dehydrated CaOx (COD) ·

Monohydrated CaOx (COM)

L. Farias · N. Butto · A. Neira-Carrillo (*)

Department of Biological and Animal Sciences, School of Veterinary and Animal Sciences,

University of Chile, Santiago, Chile

e-mail: nbutto@veterinaria.uchile.cl; aneira@uchile.cl

242

25.1 Introduction

Biological crystallization or biomineralization is the process by which living organ-

isms from bacteria to eukaryotes cells form hierarchical hybrid biogenic minerals

(Lowenstam and Weiner 1989; Estroff 2008). Its role in nature is diverse such as

protection, motion, storage, optical and gravity sensing, defense, detoxication, etc.

(Mann 2000). They are highly organized from molecular level to the nano- and

macroscale, with intricate nano-architectures that ultimately make up a myriad and

remarkable properties and complex shape of different functional soft and hard tis-

sues (Sumper and Brunner 2006; Guru and Dash 2014; Neira-Carrillo etal. 2015a,

b). These properties can inspire mimetic strategies intending to design nanomateri-

als based on mineral controlled crystallization concept. Biological crystallization,

however, also occurs in a pathological manner in nature, e.g., concretions, gall-

stones (Wang etal. 2006; Xie etal. 2015), and the mineralization of CaOx within

the urinary tract often called urolithiasis (Khan and Canales 2009). Therefore,

biominerals are outstanding materials not only for understanding the biomineraliza-

tion concept but also for novel conned-materials synthesis and design, avoiding

undesirable pathological biomineralization. Composite biogenic nanomaterials are

also of increasing interest to materials scientists who seek novel materials syntheses

such as brillary hydrogel, platelet or ber structures, and crystalline matrices and/

or interfaces with similar crystalline forms to those produced by nature. There is an

abundant diversity of chemical compositions and structures for minerals such as

carbonates, silicates, phosphates, oxalate, oxides, etc. (Pai and Pillai 2008; Neira-

Carrillo etal. 2010, 2015a, b). In general, invitro study of inorganic minerals can

be performed by using additives or organic substrates through different experimen-

tal methodologies.

With this in mind, random and oriented electrospun polycaprolactone (PCL)

ber meshes on indium tin oxide (ITO) support were prepared through electrospin-

ning and used as an organic template for controlling the invitro electrocrystalliza-

tion (EC) of CaOx. Electrospinning is a nanofabrication technique, in which the

organic polymer bers orientation can be topologically controlled at the surface of

PCL meshes. Electrospinning involves the application of an electric eld to a drop

of polymer solution that is deformed and forced to be ejected to a metallic plate col-

lector in which the arrangement of bers can be controlled with random (Kishan

and Cosgriff-Hernandez 2017) or aligned (Lee etal. 2017) bers orientation.

Therefore, in order to study the effect of PCL surface topology as organic solid

template for controlling the invitro EC of CaOx, random and aligned PCL bers

were directly collected on ITO glass electrode by using at and rotating collectors.

The use of EC has been documented for CaOx (Neira-Carrillo etal. 2015a, b) and

for other inorganic minerals such as calcium carbonate crystals (Pavez etal. 2004;

Buttlo etal. 2017; Sanchez etal. 2017).

L. Farias et al.

243

25.2 Materials andMethods

The invitro EC of CaOx on PCL electrospun ber meshes was carried out onto

conductive ITO electrode at 9mA using 18% PCL solution (Mw, 80,000, Sigma-

Aldrich) in organic ethyl acetone/acetate 3:1 (v:v) solvents in an electrospinning

instrument (Fluidnatek® LE-10). Random and aligned PCL ber meshes were spun

on a xed metal at (30 × 30cm) and rotary (10cm in diameter) collectors, respec-

tively. The control of PCL surface topology was achieved by using the following

parameters: 16kV, solution ow rate of 1200μl/h, 15min, nozzle-collector distance

from between 15 and 18cm, and rotating speed of 2000 rpm. The modied ITO-

containing PCL ber meshes were immersed in an electrocrystallization solution

composed of sodium oxalate (Sigma-Aldrich®), calcium nitrate (MERCK®), and

ethylenediaminetetraacetic acid tetrasodium salt (Sigma-Aldrich®) and put into an

electrochemical cell. The potenciostat-galvanostat (Epsilon-BASi) instrument and

the Epsilon EC-USB program were used for performing all the EC of CaOx assays.

The SEM-EDX surface morphology of the resultant CaOx crystals was examined

using a scanning electron microscope (Jeol JSM-IT300LV, JEOL USA Inc., USA)

connected to an energy dispersive X-ray detector for elemental analysis with

computer-

controlled software, the Aztec EDX system (Oxford Instruments,

Abingdon, UK). Powder X-ray diffraction (PXRD) was performed by using a

Siemens D-5000X X-ray diffractometer with Cu-Ka radiation (graphite monochro-

mator) and an ENRAF Nonius FR 590. The crystal structure of CaOx was deter-

mined by using Cu-Ka radiation (40 kV), steps of 0.2°, and the geometric

Bragg-Brentano (θ–θ) scanning mode with an angle (2θ) range of 5–70°. The

DiffracPlus program was used as a data control software.

25.3 Results

25.3.1 Preparation ofPCL Fibers andChronopotentiometric

Curve ofCaOx

Random and oriented electrospun polycaprolactone (PCL) ber meshes were pre-

pared by using electrospinning directly deposited on ITO support as working elec-

trode for conducting the in vitro EC of CaOx. Therefore, EC of CaOx using

surface-modied ITO PCL ber meshes with controlled topology was used as solid

template, and their electrochemical potentiometric behavior follows at room tem-

perature for 5min. We observed a notorious difference in the behavior of electro-

chemical curves during the EC of CaOx when both surface-modied ITO PCL ber

meshes were used indicating a different mechanism of CaOx crystallization

25 Preparation ofRandom andAligned Polycaprolactone Fiber asTemplate…

244

(Fig.25.1). This can be rationalized by increasing the formed CaOx material when

the EC was performed at constant applied current of 9mA.Thus, when the invitro

EC of CaOx in the presence of aligned PCL ber mesh was performed (Fig.25.1☆),

the electrochemical potential reached a maximum value of 2.2V at 1.5min, keeping

this constant value until the end of the test. Meanwhile when the PCL ber meshes

with random distribution were used as template (Fig. 25.1□), the potential (V)

showed a progressive increase behavior of 2.35V until the end of the experiment.

The same potentiometric curve was also observed in absence of surface-modied

ITO as control essays reaching ca. of 2.30V until the end of the experiment.

25.3.2 SEM, EDX, andXRD Characterization ofCaOx

Obtained byEC Method

The morphological aspect and distribution of CaOx crystals grown on modied- ITO

support were carried out by scanning electron microscopy (SEM). SEM analysis

showed that crystals grown on surface-modied ITO with randomly PCL bers

were found into network-PCL ber mesh forming conglomerate crystalline parti-

cles. Here, classic bypyramidal morphology of COD crystals was observed

(Fig. 25.2a). On the other hand, CaOx crystals formed on surface-modied ITO

with aligned PCL bers grown along and surrounding on PCL bers (inset,

Fig. 25.2b). Here, we also observed several COD crystals with distinct

012345

1,7

1,8

1,9

2,0

2,1

2,2

2,3

2,4

Potenal (V)

Time (min)

Fig. 25.1 Chronopotentiometric curves for EC of CaOx crystals on ITO electrode substrate.

Without additive as control (■) and in the presence of random PCL ber (□) and aligned PCL

ber (☆) meshes

L. Farias et al.

245

morphological COD with slight crystalline shape of CaOx crystals (Fig. 25.2b).

Regular and ower-like COD crystals morphologies were also observed in other

part deposited on aligned PCL ber samples. This result indicated that the growth of

crystal faces normal to the [100] direction was selectively inhibited by changing the

topological surface of PCL bers as template on the surface-modied ITO support.

The energy dispersive X-ray spectroscopy (EDX) detector on CaOx crystals was

also used, demonstrating the characteristic elemental and chemical composition of

CaOx crystals (Fig.25.3) and to the conductive ITO support. The SEM and EDX

studies of CaOx were carried out by using “analysis” as observation condition and

the “charge-up reduction mode” as observation mode. The weight percent (wt.-%)

concentration of elements at the crystal surface was automatically calculated

Fig. 25.2 SEM images of CaOx crystals obtained through EC method on surface-modied ITO

with (a) random PCL ber and (b) with aligned PCL bers meshes

Fig. 25.3 EDX measurements of CaOx crystals obtained through EC method on surface-modied

ITO with (a) random PCL ber and (b) aligned PCL ber meshes. The colors assigned to each

element were arbitrarily selected in the EDX measurements

25 Preparation ofRandom andAligned Polycaprolactone Fiber asTemplate…

246

through EDX software. In order to determine the internal lattice of crystalline CaOx

products, X-ray diffraction (XRD) analysis was also performed. Figure25.4 shows

XRD pattern of CaOx crystals obtained by EC in the presence of surface-modied

ITO with random PCL bers (Fig.25.4a) and surface-modied ITO with aligned

PCL bers (Fig.25.4b). In general, the XRD spectra demonstrated the coexistence

of COD and COM polymorphism; the crystalline peaks showed slight difference in

the intensity of the main reections of COD and COM, which are ascribed at

2θ=14.3°, 20.0°, 21.3°, 32.2°, 37.2°, 40.2°, and 50.7° and 2θ=15.0°, 23.0°,67.0°,

and 30.2°, respectively. It was noted that the portion of COD crystals in the resultant

product on surface-modied ITO became dominant. The current type of CaOx poly-

morphism designations is in agreement with COM and COD crystals obtained by

using acid-rich biopolymers as additive (Jung etal. 2005).

25.4 Discussion

In vitro electrocrystallization on controlled surface-modied ITO with PCL ber

meshes as classical crystallization of CaOx was performed. Thus, EC of CaOx on

ITO working electrode with random and aligned electrospun PCL bers was used

as solid template to evaluate the topology effect onto ITO electrode on the morphol-

ogy, distribution, and polymorphism of the CaOx crystals. Random and aligned

Fig. 25.4 XRD spectra of CaOx crystals obtained through EC on surface-modied ITO with (a)

random PCL bers mesh, black line, and (b) aligned PCL bers mesh, green line. Diffraction

peaks show crystalline polymorphs of monohydrate (COM) and dihydrate (COD) CaOx

L. Farias et al.

247

PCL bers were obtained by using at and rotary collectors through electrospinning

technique, respectively. In summary, CaOx crystals were effectively electrodepos-

ited, and a clear difference in the distribution, morphology, and crystal growth of

CaOx crystals was observed. We suggest that the active surface topology of PCL

ber meshes can act as good nucleation point and in particular on the aligned sur-

face of each PCL bers inducing a favorable site for the invitro crystallization of

CaOx. In addition, polymorphism of CaOx can be selectively controlled onto sur-

face-modied ITO, although the absence of chemical functionality on PCL ber as

template. Here, we observed a coexistence in the polymorphism of calcium oxalate;

however, the COD crystals were the predominant particles on the surface of the ITO

substrates.

Acknowledgments The authors are grateful to Project Fondecyt 1171520 and 1140660, Program

U-Redes, Vice-Presidency of Research and Development, University of Chile.

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H, Arias JL (2015a) Sulfonated polymethylsiloxane as an additive for selective calcium oxalate

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calcium oxalate morphology through electrocrystallization as an electrochemical approach for

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Sánchez M, Vásquez-Quitral P, Butto N, Díaz-Soler F, Yazdani-Pedram M, Silva JF, Neira-Carrillo

A (2017) Effect of alginate from Chilean Lessonia nigrescens and MWCNTs on CaCO3 crys-

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

L. Farias et al.

Part VI

Bio-inspired Materials Science and

Engineering

251© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_26

Chapter 26

Dysprosium Biomineralization

by Penidiella sp. Strain T9

TakumiHoriike, HajimeKiyono, andMitsuoYamashita

Abstract Biomineralization approaches have gained signicant attention as a

means to recover rare earth elements from acidic mine drainage and industrial liquid

wastes. We isolated an acidophilic fungus, Penidiella sp. strain T9, that accumulates

dysprosium (Dy) from acidic model drainage during growth. To develop the appli-

cation of biomineralization by the strain T9, we elucidated the localization and the

chemical structure of biomineralized Dy and performed to establish the labo-scale

bioprocess for selective recovery of Dy. High-magnication scanning electron

microscopic analysis showed that the strain T9 formed a mineralized Dy (T9-Dy)

layer with 1.0 μm thickness over the cell surface, along with some intracellular

nano-micro meter-sized Dy particles. X-ray photoelectron spectrometry and X-ray

absorption ne structure analyses showed that the chemical composition of T9-Dy

corresponded to DyPO4. X-ray diffraction analysis did not yield any spectrum from

T9-Dy. Therefore, we concluded that the strain T9 accumulates and mineralizes Dy

as an amorphous DyPO4. Dysprosium desorption rate from T9-Dy was 100% using

0.3M hydrochloric acid. Furthermore, after desorption process, the strain T9 grows

again in the new medium and retains the Dy accumulation ability. Thus, the strain

T9 has a potential as a bioaccumulator for Dy recovery from acidic drainage through

biomineralization.

Keywords Biomineralization · Bioaccumulation · Drainage · Dysprosium ·

Acidophilic microorganism · Metal-biotechnology

T. Horiike · M. Yamashita (*)

Shibaura Institute of Technology, Saitama, Japan

e-mail: i030589@shibaura-it.ac.jp; yamashi@shibaura-it.ac.jp

H. Kiyono

Shibaura Institute of Technology, Tokyo, Japan

e-mail: h-kiyono@shibaura-it.ac.jp

252

26.1 Introduction

Rare earth elements (REE) are indispensable ingredients in the manufacturing of

high-tech products (US Geological Survey 2015). In particular, dysprosium (Dy)

has increased its global demand because to use in making heat resistant magnets for

high-tech products; nevertheless, the supply of Dy is limited. Acidic mine drainage

and industrial liquid waste containing low concentration of Dy have attracted atten-

tion as a new Dy resource (Protano and Riccobono 2002). Therefore, it is important

to develop new recovery techniques from Dy-containing drainage using as a new

resource.

Recently, biotechnological approaches for metal resource recycling have been

emphasized (Zhuang etal. 2015). Biomineralization and bioaccumulation are stud-

ied for recovering targeting metal from mine drainage and industrial waste

(Nancharaiah etal. 2016). An REE-bioaccumulating acidophilic fungus, Penidiella

sp. strain T9 was isolated and able to recover approximately 50% (w/v) soluble Dy

from acidic model drainage during growth (Horiike and Yamashita 2015). To exploit

this capability on an industrial scale, the rate of accumulation and reaction time

must be improved. To start addressing these issues, we have explored the localiza-

tion and the chemical structure of Dy-containing compounds following their bioge-

netic solidication by the strain T9. To develop the application of biomineralization

by the strain T9, we elucidated the localization and the chemical structure of

biomineralized Dy and performed to establish the labo-scale bioprocess for selec-

tive recovery of Dy.

26.2 Materials andMethods

26.2.1 Media andCultivation Conditions

The Basal salt medium (BSM) was prepared in accordance with a recent study

(Horiike and Yamashita 2015). For Dy bioaccumulation tests, BSM was prepared at

pH 2.5 with 20mM potassium hydrogen phthalate-hydrochloric acid (HCl) buffer.

Cultivation was carried out at 30°C on a rotary shaker at 120rpm.

26.2.2 Dy Bioaccumulation by Strain T9

The Dy accumulation test was performed in accordance with a recent study (Horiike

and Yamashita 2015). The strain T9 was cultivated in 50mL of BSM containing

100mg/L Dy (as DyCl3) and cultivated on a rotary shaker at 120rpm at 30°C for

7days. After 7days of cultivation, the strain T9 cells were centrifuged (15,900× g,

20min, 4°C) and then collected by ltration using an Omnipore membrane lter

T. Horiike et al.

253

(0.2-μm pore size; Merck Millipore, MA, USA). The cell pellet on the lter was

washed twice with sterile saline water (isotonic solution).

26.2.3 Analytical Methods

To characterize Dy precipitate prepared by the strain T9, scanning electron

microscopy- energy dispersive X-ray spectroscopy (SEM-EDX), X-ray absorption

ne structure (XAFS) analyses, and X-ray diffractometry (XRD) were conducted

with the detail procedure and the standard materials prepared according to reference

(Horiike etal. 2016).

26.2.4 Purication ofDy fromDy Precipitate

Dy precipitate prepared by the strain T9 after 3days cultivation was used. Each of

HCl and ethylenediamine-N, N, N′, N′-tetraacetic acid (EDTA), as solubilizing

reagents of Dy-precipitated compounds in the strain T9, was prepared at 300mM,

30mM, and 3.0 mM, respectively. The nal concentration of Dy in the solution

containing HCl or EDTA was 500mg/l (3.0mM Dy) from Dy precipitate, and then

the mixture was reacted at 30°C on a rotary shaker at 120rpm for 3h. The concen-

tration of Dy in the solution was determined using inductively coupled plasma-

atomic emission spectrometry (ICP-AES).

26.3 Results andDiscussion

26.3.1

Localization ofDy-Precipitated Compounds

inPenidiella sp. Strain T9

To clarify the localization of Dy-precipitated compounds in the strain T9, sections

of T9 cells were observed using SEM-EDX after the Dy bioaccumulation test

(Fig.26.1). Bright dots and bright regions were observed in the cell (1.0μm diam-

eter) and on the cell surface (1.0μm thickness), respectively. Three points, bright

dot (Spot 1), bright region (Spot 2), and cytoplasm as background (Area 3), were

further analyzed using EDX (Fig.26.1b–d). Specic high peaks corresponding to

Dy, P, and O at spots 1 and 2 were observed; these peaks were absented from area 3

(background). Peaks of C and N were found at all three points. From these results,

we conclude that the strain T9 mainly incorporates a layer of Dy on the cell surface

and nano-micro sized particles (bright dots) of Dy in the cytoplasm. Furthermore,

since P and O were also detected in Dy-precipitated material, we infer that the Dy

26 Dysprosium Biomineralization by Penidiell a sp. Strain T9

254

compounds also contain phosphate. High-magnication SEM-EDX analysis

showed that the strain T9 formed a precipitated Dy (T9-Dy) layer with 1.0-μm

thickness over the cell surface and some nanometer-sized particles of Dy in the

cytoplasm (data not shown). The T9-Dy layer consisted of coagulated nanometer-

sized particles.

26.3.2 Chemical Structure ofDy-Precipitated Compounds

To determine the chemical state of T9-Dy, Dy LIII-edge spectrum in XANES of

T9-Dy, DyPO4 (chemical precipitate), CMC-Dy (Dy-binding carboxymethyl cellu-

lose), and CP-Dy (Dy-binding cellulose phosphate) was carried out (Horiike etal.

2016). The strength of the peak across the samples was as follows: DyPO4 ≦ T9-Dy

< CMC-Dy ≦ CP-Dy. This indicates that the strength of peak depends on both the

Fig. 26.1 SEM-EDX analyses of a cross section of the Dy compounds from the strain T9. (a)

Back scattered electron (BSE) image of a cross section of strain T9 with Dy. The image reveals

nanoparticles and precipitates in cytoplasmic regions (i.e., Spot 1) and the cell wall (i.e., Spot 2),

respectively. Bar, 10μm. EDX spectra in (b) Spot 1, (c) Spot 2, and (d) Area 3. Area 3 corresponds

to a background cytoplasmic region. (Horiike etal. 2016)

T. Horiike et al.

255

ligand and structure around Dy. The Dy LI-edge spectrum in XANES is shown

(Horiike etal. 2016). The full width at half maximum values of Peak B was as fol-

lows: DyPO4 ≒ T9-Dy < CMC-Dy ≒ CP-Dy. A shoulder (C) of Peak B (at ~9072eV)

was observed in the spectra of T9-Dy and DyPO4. From the results, we conclude

that the chemical bond and the local structure around Dy in the T9-Dy are closer to

that of DyPO4 than that of either CMC-Dy or CP-Dy. To identify the ligand-bonding

environment in T9-Dy, Dy LIII-edge spectrum in FT-EXAFS was carried out

(Horiike etal. 2016). Peak E corresponds to P and C, and Peak F corresponds to Dy

or P around Dy. From the FT-EXAFS results, the ligand-bonding environment in the

T9-Dy appears to be very similar to that observed in DyPO4.

To characterize the crystalline phase of T9-Dy, XRD analysis was performed.

XRD patterns of wet and dry T9-Dy indicated a halo peak at 30° and indistinct

peaks, respectively (data not shown), showing that they are amorphous. The posi-

tions of the distinct peaks are the same as those reported for DyPO4·1.5H2O (ICDD

20-0385). Taken together, these data indicate that the crystalline phase of the T9-Dy

is clearly different from that of chemically precipitated DyPO4.

The acidophilic fungus Penidiella sp. strain T9 accumulates and incorporates Dy

in a form that corresponds to DyPO4. The precipitated Dy formed from nano- to

micro-meter sized particles in the cytoplasm and aggregates on the cell surface.

Therefore, we suggest that the strain T9 must have an active uptake mechanism for Dy

into the cells and an ability to sorb Dy into phosphate groups present in the cell wall.

These ndings highlight the importance of phosphoric acid to improve Dy recovery

by the strain T9, which has the available capacity in the cells to accumulate Dy.

26.3.3 Purication ofDy fromDy-Precipitated Compounds

To purify Dy from T9-Dy, an examination of Dy solubilization from T9-Dy pre-

pared by 3days cultivation was performed using a mineral acid (HCl) or EDTA

(Fig. 26.2). One hundred percent of Dy contained in T9-Dy was dissolved by

300 mM HCl at 30 min incubation, but 26% and 5% of Dy were dissolved by

30mM and 3.0mM HCl, respectively, at 3h. The results suggested that the purica-

tion of Dy from microbial Dy precipitant using the strain T9 was easier than chemi-

cal Dy precipitant, which was not dissolved by HCl. Ninety six percent, 100%, and

95% of Dy from T9-Dy were dissolved in 300mM, 30mM, and 3.0mM of EDTA,

respectively, at 3 h. Thus, 300mM HCl and 30 mM EDTA were more effective

chemicals to purify Dy from T9-Dy.

26 Dysprosium Biomineralization by Penidiell a sp. Strain T9

256

26.3.4 Reuse oftheStrain T9 toRecover Dissolved Dy

The reusability of the strain T9in the REE accumulation-desorption cycle is impor-

tant to develop an economical REE recovery process. The cells of T9 strain after the

Dy desorption were cultivated in a new BSM, and then Dy accumulation test was

carried out reusing the T9 cells. The dissolved Dy in the culture were decreased less

than 50% after 3days of cultivation using the cells treated with EDTA or HCl in

desorption (Fig.26.3). These results indicated that the cells of the strain T9 grew

again in the new medium and retained the Dy accumulation ability after desorption

Fig. 26.2 Dy desorption

rate from T9-Dy using

EDTA or HCl. White bars,

using EDTA; black bars,

using HCl

Fig. 26.3 Time course of

the Dy concentration

during cultivation in BSM

using the strain T9 after

desorption. Broken lines,

cells after desorption using

EDTA; solid lines, cells

after desorption using HCl.

Concentrations of

desorption reagents;

300mM (solid circle),

30mM (solid triangle),

3.0mM (solid square)

T. Horiike et al.

257

process using HCl or EDTA.Taken together, the strain T9 has a great potential as a

bioaccumulator to develop a continuous recycling system of Dy. Mineral acids,

such as HCl, HNO3, and H2SO4, and chelating agents, such as EDTA and NTA

(nitrilotriacetic acid), have lethal effects on most of microbial cells (Tsezos 1984).

Since the strain T9 has ability to tolerate chemicals like HCl and EDTA, the strain

has strong potential to be developed as the REE-accumulator for REE recovery

cycle.

Acknowledgments XAFS analysis was performed under the approval of the Photon Factory

Program Advisory Committee. This study was supported partially by the Japan Oil, Gas and

Metals National Corporation and the Kurita Water and Environment Foundation.

References

Horiike T, Yamashita Y (2015) A new fungal isolate, Penidiella sp. strain T9, accumulates the rare

earth element dysprosium. Appl Environ Microbiol 81:3062–3068

Horiike T, Kiyono H, Yamashita M (2016) Penidiella sp. strain T9 is an effective dysprosium

accumulator, incorporating dysprosium dysprosium phosphate compounds. Hydrometallurgy

166:260–265

Nancharaiah YV, Mohan SV, Lens PNL (2016) Biological and bioelectrochemical recovery of

critical and scarce metals. Trends Biotechnol 34:137–155

Protano G, Riccobono F (2002) High contents of rare earth elements (REEs) in stream waters of a

Cu–Pb–Zn mining area. Environ Pollut 117:499–514

Tsezos M (1984) Recovery of uranium from biological adsorbents-desorption equilibrium.

Biotechnol Bioeng 26:973–981

U.S. Geological Survey (2015) Mineral commodity summaries 2015. U.S.Geological Survey,

Virginia

Zhuang WQ, Fitts JP, Ajo-Franklin CM, Maes S, Alvarez-Cohen L, Hennebel T (2015) Recovery

of critical metals using biometallurgy. Curr Opin Biotechnol 33:327–335

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

26 Dysprosium Biomineralization by Penidiell a sp. Strain T9

259© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_27

Chapter 27

Various Shapes ofGold Nanoparticles

Synthesized by Glycolipids Extracted

fromLactobacillus casei

YugoKato, FumiyaKikuchi, YukiImura, EtsuroYoshimura,

andMichioSuzuki

Abstract Gold nanoparticles have particular properties distinct from bulk gold crys-

tals. The gold nanoparticles are used in various applications in optics, catalysis, and

drug delivery. Although many reports on microbial synthesis of gold nanoparticles

have appeared, the molecular mechanism of gold nanoparticle synthesis in microor-

ganisms is unclear. Previously we reported that the amounts of diglycosyl diacylg-

lycerol (DGDG) and triglycosyldiacylglycerol (TGDG) bearing unsaturated fatty

acids were much reduced after formation of gold nanoparticles. DGDG puried from

L. casei induced the synthesis of gold nanoparticles invitro. These results suggested

that glycolipids, such as DGDG, play important roles in reducing Au(III) to Au(0). In

this paper, we reported that the concentration change of DGDG induced various

shapes of gold nanoparticles invitro. Our work will lead to the development of novel

and efcient methods to synthesize metal nanoparticles using microorganisms.

Keywords Gold nanoparticle · Lactobacillus casei · Glycolipid

Y. Kato · F. Kikuchi · Y. Imura · M. Suzuki (*)

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life

Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

e-mail: amichiwo@mail.ecc.u-tokyo.ac.jp

E. Yoshimura

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life

Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Department of Liberal Arts, The Open University of Japan, Chiba, Japan

e-mail: ayoshim@mail.ecc.u-tokyo.ac.jp

260

27.1 Introduction

Gold nanoparticles (containing a few tens of gold atoms) have various unique prop-

erties. A gold nanoparticle solution is wine-red in color, because of surface plasmon

resonance (SPR) (Jin 2010; Zhang etal. 2010). Over the past two decades, such

nanoparticles have found novel applications in general industry, chemistry, biology,

and medicine. Antibody-bearing nanoparticles are useful labeling agents in electron

microscopy and can reveal the detailed locations of organic molecules within cell

organelles (Bendayan and Garzon 1988). Gold nanoparticles attached to DNA frag-

ments can detect DNA-DNA interactions (which trigger color changes) (Storhoff

etal. 2000). This technology is used to diagnose viral infections (Wang etal. 2001;

Cao etal. 2002). When synthesizing gold nanoparticles, the appropriate selections

of reducing agents active on gold ions, and dispersing agents that hold the particle

size in the nanometer range, are important. Industrially, large amounts of gold

nanoparticles are synthesized in the reaction of gold with citric acid under condi-

tions of high temperature and pressure (Frens 1973). Novel methods using macro-

molecular polymers or biomacromolecules, such as proteins and DNA, have been

used to develop more functional gold nanoparticles (controlled in terms of shape) at

low nancial and energy costs, in the absence of unwanted by-products (Selvakannan

etal. 2004; Xie etal. 2009; Liu etal. 2011). Recently, many methods using micro-

organisms to synthesize metallic nanoparticles have been reported. For example,

some previous works about synthesize gold nanoparticles using by Rhodobacter

capsulatus (Feng etal. 2008), silver nanoparticles using by Fusarium oxysporum

(Ahmad etal. 2003), CdS quantum dots using by Escherichia coli (Sweeney etal.

2004) were reported. In such processes, the microorganisms are cultured at normal

temperature under normal pressure and do not produce any toxic by-product. Such

benets suggest that the use of microorganisms to synthesize gold nanoparticles

will be important in the future. To improve the efciency of such synthesis, it is

essential to clarify the molecular mechanisms involved. Recently, we reported that

diglycosyl diacylglycerol (DGDG) plays important roles in reducing Au(III) to

Au(0) by Lactobacillus casei (Kikuchi etal. 2016). This report shows the function

of DGDG for the synthesis of gold nanoparticles.

27.2 Materials andMethods

We extracted lipid from L. casei (strain JCM1134, purchased from RIKEN Microbe

Division) according to the Bligh and Dyer method (Bligh and Dyer 1959). We

extracted thin layer chromatography (TLC) analysis. After spotting of samples in

the origin point, plates were transferred to TLC chambers saturated with the chro-

matographic solvent (chloroform/methanol/acetic acid, 65:25:10). DGDG was

extracted from TLC plate and dissolved in ethanol. To conrm DGDG, we mea-

sured mass spectra on a matrix-assisted laser-desorption ionization–time-of-ight

Y. Kato et al.

261

mass spectrometer (ultraex MALDI-TOF/TOF, Bruker). The sample solution was

mixed with 500mM 2, 5-dihydroxybenzoic acid in chloroform/methanol (1:1) solu-

tion as the matrix and dried on the plate.

DGDG extraction from the TLC plate dissolved in 40μL ethanol was applied to

960μL of auric acid solution (nal concentration of K[AuCl4]: 250μM). The mix-

ture solution was incubated at 37°C for 24h. Forty microliters of ethanol without

DGDG was mixed with 960 μL of auric acid solution (nal concentration of

K[AuCl4]: 250μM) as a negative control experiment. UV/VIS spectra of the super-

natant were measured using UV/VIS spectroscopy photometer (V-550 spectropho-

tometer, JASCO). To examine the formation of gold nanoparticles, we used

transmission electron microscopies (TEM) observation and energy dispersive X-ray

spectrometry (EDS). TEM analyses were performed using a JEOL JEM-2000EX

TEM operated at 200 kV, and EDS analyses were performed using a JEOL

EX-24025JGT.

27.3 Results

27.3.1 Extraction ofDGDG fromL. casei

L. casei cells were suspended in chloroform/methanol solution to extract lipids, and

these extracts were subjected to TLC.The extracted sample separated from the TLC

was analyzed by MALDI-TOF MS.The spectrum of MALDI-TOF MS showed four

major peaks at 939.6, 953.6, 967.6, and 981.6 (m/z) and certain isotope peaks

(Fig.27.1a). These peaks showed the different chain length of the unsaturated fatty

acids in DGDG.The chemical structure of DGDG was shown in Fig.27.1b. R1 and

R2 mean the alkyl chains containing one double bond in each chain.

Fig. 27.1 (a) MALDI-

TOF- MS spectrum of the

extract from TLC. (b)

The schematic structure

of DGDG.R1 and R2 showed

the alkyl chains of with one

double bonding

27 Various Shapes ofGold Nanoparticles Synthesized by Glycolipids Extracted…

262

27.3.2 Reaction ofAuric Acid Solution withDGDG

Puried DGDG from L. casei was solubilized in ethanol to mix with auric acid.

Then, the solution was incubated at 37°C for 24h. The colors of auric acid solution

without DGDG kept the transparent yellow. On the other hand, the color of auric

acid solution with DGDG became violet (Fig.27.2a). The absorbance spectrum of

each solution at wavelengths from 400 to 700nm was measured (Fig.27.2b). The

intensity and wavelength of peak top varied by the DGDG concentration. The high

concentration of DGDG showed the smaller wavelength indicating that the high

concentration of DGDG synthesize the small nanoparticles. On the other hand, the

low concentration of DGDG induced the bathochromic shift indicating that low

concentration of DGDG induced the bigger nanoparticles.

0.2

0.4

0.6

0.8

400 500 600 700

Absorbance

Wavelength(nm)

0 μg/ml 5 μg/ml 20 μg/ml

80 μg/ml 160 μg/ml

Fig. 27.2 (a) Auric acid solution (0.25mM K[AuCl4]) with DGDG puried from the TLC plate

(1) 5.0μg/mL, (2) 20μg/mL, (3) 80μg/mL, (4) 160μg/mL, (5) 0μg/mL), (b) UV/VIS spectra after

24h. Light blue line, 0 μg/mL; orange break line, 5.0μg/mL; black break line, 20 μg/mL; light

blue dotted line, 80μg/mL; dark blue dotted and break line, 160μg/mL

Y. Kato et al.

263

27.3.3 Observation Nanoparticles by TEM

To investigate morphology of nanoparticles, the sample of each solution was sub-

jected to TEM (Fig.27.3a). TEM observations showed that black dots correspond-

ing to gold nanoparticles were synthesized in all conditions. TEM observation of the

condition with 5.0μg/mL DGDG showed sphere shape of nanoparticles (Fig.27.3a-

1). 20 μg/mL DGDG synthesized small nanoparticles and rod shape of gold

(Fig. 27.3a-2). 80 μg/mL DGDG induced the small and triangle shape of gold

(Fig. 27.3a-3). 160 μg/mL DGDG synthesized the hexagonal shape of gold

(Fig.27.3a-4). EDS showed that the various shapes of nanoparticles were composed

of Au (sphere shape (Fig. 27.3b-1), rod shape (Fig. 27.3b-2), triangle shape

(Fig.27.3b-3), and hexagonal shape (Fig.27.3b-4).

27.4 Discussion

In the present study, incubation of DGDG with auric acid invitro succeeded to pro-

duce gold nanoparticles, suggesting that DGDG has a function of both reducing and

dispersing agents. The carboxylic groups of citric acid bind to the surface of Au(0)

to create nanoparticles (Frens 1973). The degradation products of DGDG may inter-

act with the Au(0) surface to stabilize nanoparticles. On the other hand, the size of

particles synthesized by L. casei was 30nm on average (Kikuchi etal. 2016). The

gold nanoparticles synthesized by DGDG were slightly larger than those synthe-

sized by L. casei cells, suggesting that the ability of dispersing capacity of DGDG

is not enough to make the 30nm nanoparticles. The high concentration of DGDG

induced the various shape of gold crystals suggesting that DGDG also affected the

crystal growth of gold. However, the other dispersing agents within L. casei cells

may also play roles in inhibiting Au(0) aggregation and crystal growth. Further

work is thus needed to reveal exactly how L. casei forms gold nanoparticles.

Identication of the key organic molecules may allow modication of L. casei

genes, permitting such recombinants to promote the synthesis of gold nanoparticles

more effectively. In the future, the modied recombinant L. casei strains may be

used for the applications in metal recycling and phytoremediation.

27 Various Shapes ofGold Nanoparticles Synthesized by Glycolipids Extracted…

100

200

300

400

500

600

700

800

02468101214

counts

Energy (keV)

200

400

600

800

1000

02468101214

counts

Energy (keV)

100

150

200

250

300

350

02468101

214

counts

Energy (keV)

100

150

200

250

300

0246810 12

counts

Energy (keV)

Fig. 27.3 (a) TEM image of gold nanoparticles synthesized by DGDG (1, 5.0 μg/mL; 2, 20 μg/

mL; 3, 80 μg/mL; 4, 160 μg/mL; DGDG). Arrow A indicates the rod shape of gold, arrow B indi-

cates the triangle shape of gold, arrow C indicates the hexagonal shape of gold. (b) EDS spectra of

nanoparticles of (a) (1, sphere shape; 2, rod shape; 3, triangle shape; 4, hexagonal shape). The

arrows showed the characteristic X-ray peaks of Au. X showed the peak of Cu from the copper grid

265

References

Ahmad A etal (2003) Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium

oxysporum. Colloids Surf B Biointerfaces 28:313–318

Bendayan M, Garzon S (1988) Protein G-gold complex: comparative evaluation with protein

A-gold for high-resolution immunocytochemistry. JHistochem Cytochem 36:597–607

Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purication. Can JBiochem

Physiol 37:911–917

Cao YC, Jin R, Mirkin CA (2002) Nanoparticles with Raman spectroscopic 21 ngerprints for

DNA and RNA detection. Science 297:1536–1540

Feng Y et al (2008) Diversity of aurum bioreduction by Rhodobacter capsulatus. Mater Lett

62:4299–4302

Frens G (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold

suspensions. Nature Phys Sci 241:20–21

Jin R (2010) Quantum sized, thiolate-protected gold nanoclusters. Nano 2:343–362

Kikuchi F etal (2016) Formation of gold nanoparticles by glycolipids of Lactobacillus casei. Sci

Rep 6:34626

Liu CL etal (2011) Insulin-directed synthesis of uorescent gold nanoclusters: preservation of

insulin bioactivity and versatility in cell imaging. Angew Chem Int Ed Eng 50:7056–7060

Selvakannan P etal (2004) Water-dispersible tryptophan-protected gold nanoparticles prepared by

the spontaneous reduction of aqueous chloroaurate ions by the amino acid. JColloid Interface

Sci 269:97–102

Storhoff JJ etal (2000) What controls the optical properties of DNA-linked gold nanoparticle

assemblies? JAm Chem Soc 122:4640–4650

Sweeney R et al (2004) Bacterial biosynthesis of cadmium sulde nanocrystals. Chem Biol

11:1553–1559

Wang J, Xu D, Kawde AN, Polsky R (2001) Metal nanoparticle-based electrochemical stripping

potentiometric detection of DNA hybridization. Anal Chem 73:5576–5581

Xie J, Zheng Y, Ying JY (2009) Protein-directed synthesis of highly uorescent gold nanoclusters.

JAm Chem Soc 131:888–889

Zhang Q, Xie J, Yu Y, Lee JY (2010) Monodispersity control in the synthesis of monometallic and

bimetallic quasi-spherical gold and silver nanoparticles. Nanoscale 2:1962–1975

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

27 Various Shapes ofGold Nanoparticles Synthesized by Glycolipids Extracted…

267© The Author(s) 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_28

Chapter 28

Octacalcium Phosphate Overgrowth

onβ-Tricalcium Phosphate Substrate

inMetastable Calcium Phosphate Solution

MayumiIijima andKazuoOnuma

Abstract The effects of the particle size of β-tricalcium phosphate (β-Ca3(PO4)2;

β-TCP) on octacalcium phosphate (Ca8(HPO4)2(PO4)4·5H2O; OCP) overgrowth on a

β-TCP substrate were evaluated under physiological conditions by using two types

of substrate; one composed of micrometer-sized particles (micro-TCP substrate)

and one composed of nanometer-sized particles (nano-TCP substrate). When the

β-TCP substrate was immersed in a simple calcium phosphate solution, it was

quickly covered with OCP.The morphology and size of the OCP crystals, as well as

the structure, thickness, and crystal density of the overgrown OCP layer, depended

on the β-TCP particle size. In case of the micro-TCP substrate, OCP crystals grew

directly on the micrometer-sized particles. In case of the nano-TCP substrate, string-

like (S) precipitates initially deposited, and then ake-like (F) crystals formed on

them. Plate-like (PL) OCP crystals grew on the ake-like crystals; as a result, a

three-layer structure (S-layer/F-layer/PL-layer) was formed. Small amounts of tiny

OCP crystals and HAp-nanobers precipitated in the micro-TCP substrate, whereas

only HAp-nanobers precipitated in the nano-TCP substrate. Thus, various types of

OCP-overgrown layers were fabricated on β-TCP scaffold. These ndings will

facilitate the structural design of OCP-coating layers on a β-TCP scaffold.

Keywords Octacalcium phosphate · Coating · Wet chemical method · Overgrowth

· β-tricalcium phosphate · Scaffold · Particle size

28.1 Introduction

Both β-TCP and OCP have been proven to have promising osteoconductive charac-

teristics. β-TCP has been applied in the form of granules and three-dimensional

(3D) scaffolds (Hench and Polak 2002; Karageogiou and Kaplan 2005; Wang etal.

M. Iijima · K. Onuma (*)

Biomaterial Research Group, Health Research Institute Central 6, National Institute of

Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan

e-mail: mayumi-iijima@aist.go.jp; k.onuma@aist.go.jp

268

2015). The biocompatibility and degradability of conventional micrometer-sized

β-TCP powder have been improved by the use of nanometer-sized β-TCP (Zhang

etal. 2008; Kato etal. 2016). On the other hand, OCP is a metastable phase of HAp

and tends to transform into HAp spontaneously (Brown etal. 1962). The intrinsic

properties of OCP are hypothesized to be responsible for its excellent performance

invivo; moreover, implanted OCP granules have provided cores for nucleating mul-

tiple osteogenic sites (Suzuki etal. 1991, 2006). The combined usage of β-TCP with

its better formability and OCP with its better osteoconductivity would boost the

potential of both materials as a bone graft substitute. A practical way to achieve this

is coating β-TCP scaffolds with OCP.

The purpose of the present study was to examine OCP formation on a β-TCP

substrate in a simple calcium phosphate solution and to evaluate the effect of the

particle size of the β-TCP substrate on the overgrowth of OCP under physiological

conditions. To achieve this, micrometer- and nanometer-sized β-TCP particles were

used to form β-TCP substrate.

28.2 Materials andMethods

Reagent-grade β-TCP powder (Wako, Ltd.) and atomized powder were molded into

substrate composed with micrometer-sized particles (0.5–3μm) (micro-TCP sub-

strate, Fig.28.1a) and with nanometer-sized particles (<100nm) (nano-TCP sub-

strate, Fig.28.1b).

Each substrate section was immersed in a calcifying solution (5 mM CaCl2,

5mM K2HPO4+KH2PO4, 50 mM CH3COONa, pH 6.2, 37 ± 0.5 °C) for required

Fig. 28.1 SEM images of the surfaces (a1, b1) and cross sections (a2, b2) of (a) micro-TCP sub-

strate and (b) nano-TCP substrate before immersion

M. Iijima and K. Onuma

269

periods. After the reaction terminated, the substrate was rinsed in Mill-Q water and

in 99.5% ethanol and dried in air. The crystals in the overgrown layer and inside the

substrate were characterized using powder XRD, microbeam XRD, thin-lm in-

plane XRD, FE-SEM, and TEM.

28.3 Results andDiscussion

28.3.1 Early Stage ofOvergrowth

On the micro-TCP substrate (Fig.28.2), crystal growth started with the formation of

sparse island-like aggregates of string-like precipitates, which gradually grew into

small akes (1–10min) and subsequently increased in size (−30min). Plate-like

OCP crystals grew directly on the β-TCP particles and completely covered the sub-

strate surface after 40min. Thin-lm in-plane XRD of the 40min overgrown layer

exhibits a shoulder peak at 4.7° (2θ), which corresponds to the (100) peak of OCP.It

became strong after 60min. On the nano-TCP substrate (Fig.28.2), particles with a

size of 10–20nm precipitated after 1min. These particles fused into strings (3min),

which subsequently formed root-like structure (5min). The top part of the root-like

deposits grew into small akes (10min), which completely covered the substrate

surface. Thirty minutes overgrown layer exhibits a shoulder peak at 4.7° (2θ).

Subsequently, plate-like OCP crystals grew on the thin layer of the akes (60min).

Fig. 28.2 Time-resolved SEM observations and thin-lm in-plane XRD measurements of TCP

substrate after 1–60 min immersion. Characteristic XRD peaks of OCP (JCPDF 26-1056) and

β-TCP (JCPDF 09-0169) are labeled. An arrow indicates shoulder peak of (100)OCP at 4.7° (2θ)

28 Octacalcium Phosphate Overgrowth onβ-Tricalcium Phosphate Substrate…

270

28.3.2 Later Stage ofOvergrowth

Figure 28.3a, b, respectively, show powder XRD and thin-lm in-plane XRD pro-

les of the micro-TCP substrate after 20h. Both XRD proles of the nano-TCP

substrate are almost the same. In the powder XRD prole, the (002) and (004) peaks

are strong, while the other peaks are very weak. In the thin-lm in-plane XRD pro-

le, the peaks in the a-axis direction, (100), (200), and (010), are strong, while the

peak in the c-axis direction, (002), is weak. This is due to the c-axial orientation of

the OCP crystals on the substrate. The overgrown crystals are identied as OCP by

combined analysis of both XRDs.

On both substrates, OCP crystals grew in the same manner. As the immersion

period increased, the length of OCP crystals in the c-axis direction increased:

4–6μm after 60min, 10μm after 3h, 15–20μm after 5h, and 66–70μm after 20h.

In the case of the nano-TCP substrate, plate-like OCP crystals (PL) grew on the

layer of small ake-like crystals (F-layer), under which the layer of string-like pre-

cipitates (S-layer) had formed, and as a result, a three-layer structure was observed

(Fig.28.3d3). On the contrary, in the case of the micro-TCP substrate, OCP crystals

grew directly on β-TCP particles, and no such structure was observed (Fig.28.3c3).

Inside of the substrates, tiny OCP crystals (Fig.28.3c3) and small amounts of

HAp-nanobers precipitated in the micro-TCP substrate (Fig.28.4a, c), whereas

Fig. 28.3 (a) Powder XRD, (b) thin-lm in-plane XRD proles of micro-TCP substrate and SEM

images of (c) micro-TCP substrate and (d) nano-TCP substrate after 20h immersion. (c1, d1) top

view and (c2, d2) cross-sectional view. (c3) and (d3) are higher magnications of (c2) and (d2)

M. Iijima and K. Onuma

271

only HAp-nanobers were formed in the nano-TCP substrate (Fig.28.4b). HAp-

nanobers were formed much more in the nano-TCP substrate than in the micro-

TCP substrate, and they were localized around particles less than 500nm in the

micro-TCP substrate (Iijima and Onuma 2017). Thus, it was concluded that nano-

TCP particles induced the formation of HAp-nanobers (Onuma and Iijima 2017).

Thus, varying the particle size of the β-TCP had great effect on the early stage of

overgrowth and precipitates inside the substrates. Various types of OCP-coating lay-

ers were formed on β-TCP substrate. There is a general consensus that the physical

properties of the coating layer, i.e., its thickness and topography, affect the invivo

performance of the coated material (Curtis and Wilkinson 1997; Anselme and

Bigerelle 2011). These ndings will facilitate the structural design of OCP-coating

layers on a β-TCP scaffold.

Acknowledgments This study was supported by a Grant-in-Aid for Scientic Research from the

Japan Society for the Promotion of Science (JSPS KAKENHI C; 16K04954).

References

Anselme K, Bigerelle M (2011) Role of materials surface topography on mammalian cell response.

Int Mater Rev 56:243–266

Brown WE, Smith JP, Lehr JR, Fraier AW (1962) Crystallographic and chemical relation between

octacalcium phosphate and hydroxyapatite. Nature 196:1050–1055

Curtis A, Wilkinson C (1997) Topographical control of cells. Biomaterials 18:1573–1583

Hench LL, Polak JM (2002) Third-generation biomedical materials. Science 295:1014–1017

Iijima M, Onuma K (2017) Particle-size-dependent octacalcium phosphate overgrowth on

β-tricalcium phosphate substrate in calcium phosphate solution. Ceram Int. https://doi.

org/10.1016/j.ceramint.2017.10.167

Karageogiou V, Kaplan D (2005) Porosity of 3D biomaterial scaffolds osteogenesis. Biomaterials

26:5474–5491

Fig. 28.4 2θ-intensity converted microbeam XRD prole of (a) micro-TCP substrate and (b)

nano-TCP substrate after 20h immersion. Characteristic XRD peaks of OCP (JCPDF 26-1056)

and ΗΑp (JCPDF 09-0432) are labeled, respectively, in (a, b). (c) HR-TEM image of a HAp-

nanober formed inside of the micro-TCP substrate and its FFT

28 Octacalcium Phosphate Overgrowth onβ-Tricalcium Phosphate Substrate…

272

Kato A, Miyaji H, Ogawa K, Momose T, Nishida E, Murakami S, Yoshida T, Tanaka S, Sugaya T

(2016) Bone-forming effects of collagen scaffold containing nanoparticles on extraction sock-

ets in dogs. Jpn JConserv Dent 59:351–358

Onuma K, Iijima M (2017) Nanoparticles in β-tricalcium phosphate substrate enhance modula-

tion of structure and composition of an octacalcium phosphate grown layer. Cryst Eng Comm

19:6660–6672

Suzuki O, Nakamura M, Miyasaka Y, Kagayama M, Sakurai M (1991) Bone formation on syn-

thetic precursors of hydroxyapatite. Tohoku JExp Med 164:37–50

Suzuki O, Kamakura S, Katagiri T, Nakamura M, Zhao B, Honda Y, Kamijo R (2006) Bone for-

mation enhanced by implanted octacalcium phosphate involving conversion into Ca-decient

hydroxyapatite. Biomaterials 27:2671–2681

Wang L, Hu YY, Wang Z, Li X, Li DC (2015) Flow perfusion culture human fetal bone cells in large

beta-tricalcium phosphate scaffold with controlled architectures. Ceram Int 41:2654–2667

Zhang F, Chang C, Lin K, Lu J(2008) Preparation, mechanical properties and invitro degradabil-

ity of wollastonite/tricalcium phosphate macroporous scaffolds from nanocomposite powders.

JMater Sci Mater Med 19:167–173

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

M. Iijima and K. Onuma

Part VII

Biominerals for Environmental and

Paleoenvironmental Sciences

275© Springer Nature Singapore Pte Ltd. 2018

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_29

Chapter 29

Coral-Based Approaches toPaleoclimate

Studies, Future Ocean Environment

Assessment, andDisaster Research

AtsushiSuzuki

Abstract Global warming causes serious harm to the Earth’s environment. A more

sophisticated and accurate climate model can be developed by reconstructing cli-

matic change since the Industrial Revolution and for other past periods of global

warming. Coral skeletons are an important archive of past climate changes, and

advances in the ability to read sea surface temperature and salinity in the coral

record have been made by applying state-of-the-art technology. Coral skeletal cli-

matology has been successfully applied to characterize both the recent global warm-

ing trend in the Western Pacic and the mid-Pliocene warming that occurred 3.5

million years ago, and it has also been used to investigate biological and environ-

mental issues such as ocean acidication and coral bleaching, which is caused by

unusually high seawater temperatures. Coral skeletal climatology methods have

also been used to study Porites boulders cast ashore by historical tsunamis; such

studies have high social value from the perspective of regional disaster prevention.

Nevertheless, aspects of coral skeletal climatology still need clarication, including

the basic mechanism by which seawater temperature is recorded in coral skeletons,

and further research on biomineralization will improve predictions of the future

responses of marine calcifying organisms to ocean acidication.

Keywords Coral · Global warming · Ocean acidication · Coral bleaching ·

Tsunami

29.1 Introduction

Annual banding in the skeletons of modern corals was rst described by Ma (1934),

then a PhD student at Tohoku University, after a eld trip to the northern Ryukyu

Islands of Japan (Fig.29.1). The annual bands of coral skeletons became the subject

A. Suzuki (*)

Geological Survey of Japan, National Institute of Advanced Industrial Science and

Technology (AIST), Tsukuba, Ibaraki, Japan

e-mail: a.suzuki@aist.go.jp

276

of active research in the 1990s, but only recently has the research developed into the

eld of coral skeletal climatology (see Suzuki 2012). Corals provide rich archives

of past climatic variability in tropical regions, where instrumental records are rela-

tively few. In this review, I explain why the coral skeleton is such an excellent

archive of past global climate change and describe some of the major ways in which

coral skeletal analyses have been successfully applied to biological and environ-

mental issues, including coral bleaching events and ocean acidication, as well as to

paleo- tsunami research.

29.2 Coral Skeletal Climatology

Geochemists have found useful climate proxies in the coral skeleton. For example,

the strontium/calcium (Sr/Ca) ratio is a good, and pure, proxy for sea surface tem-

perature; that is, the skeletal Sr/Ca ratio is controlled only by seawater temperature.

In contrast, the oxygen isotope ratio (δ18O) is a mixed proxy for both seawater tem-

perature and salinity, and the uranium/calcium (U/Ca) ratio is a mixed proxy for

seawater temperature and pH.By a combined analysis of two proxies, Sr/Ca and

δ18O or Sr/Ca and U/Ca, referred to as a “dual proxy method,” it is possible to

extract past salinity variation (McCulloch etal. 1994) or past seawater pH from the

coral skeletal record (Fig.29.2).

Two examples of twentieth century coral oxygen isotope records from coral reefs

in Japan are shown in Fig.29.3. Fluctuations of δ18O in corals from Ishigaki Island

(124°E, 24°N), which is very close to Taiwan (Mishima etal. 2010), and Chichijima

Island (142°E, 27°N) in the Ogasawara island chain, due south of Tokyo (Felis etal.

2009), record seasonal variations of seawater temperature. In addition, both curves

show a shift toward more negative values with time, indicating a long-term seawater

temperature increase. Moreover, by applying the dual proxy method, the Ogasawara

Fig. 29.1 First published

illustration of annual

banding in modern corals.

(Reprinted from Ma 1934

with permission from the

Institute of Geology and

Paleontology Sendai,

Tohoku University)

A. Suzuki

277

abc

Fig. 29.2 Coral climate proxies that have been developed by geochemists: (a) oxygen isotope

ratio (δ18O); (b) Sr/Ca ratio; and (c) U/Ca ratio. The δ18Oc and δ18Ow denote oxygen isotope ratio

of coral skeleton and seawater, respectively. The skeletal Sr/Ca ratio is controlled only by seawater

temperature, whereas δ18O is a mixed proxy for seawater temperature and salinity and the U/Ca

ratio is a mixed proxy for seawater temperature and pH.Through a combined analysis of Sr/Ca and

δ18O (U/Ca), the seawater salinity (pH) variation can be extracted. Ideal temperature dependency

of δ18O and Sr/Ca ratio proposed by Gagan etal. (2012) are shown in panels (a, b), respectively

Fig. 29.3 Times series of coral δ18O records from Ishigaki Island in the southern Ryukyus

(Mishima etal. 2010) and Chichijima Island in the Ogasawara Islands (Felis et al. 2009) in the

Western Pacic

29 Coral-Based Approaches toPaleoclimate Studies, Future Ocean Environment…

278

corals were found to record a long-term freshening of seawater (decrease in salinity)

in the region. The long-term warming trend revealed by Ishigaki coral can be attrib-

uted to anthropogenic climate change.

Conditions during the Pliocene warm period, about 4.6–3 million years ago, are

thought to be similar to the climate conditions expected to result from global warm-

ing in the near future. Watanabe et al. (2011), who compared analysis results

obtained by the same method between modern corals and well-preserved fossil cor-

als from Luzon Island, the Philippines, showed that El Niño occurred on about the

same cycle during the Pliocene warm period as at present. Their study is an example

of the successful application of coral skeletal climatology to the distant past.

29.3 Application toEnvironmental Issues

Coral skeletal climatology can also be applied to the investigation of biological and

environmental issues such as coral-bleaching events and ocean acidication.

Coral bleaching at a scale unseen before occurred in coral reefs around the

Ryukyu Islands in August 1998, and another major coral bleaching event occurred

in the southern Ryukyu Islands, especially around Ishigaki Island, in summer 2016.

Suzuki etal. (2003) examined skeletal records of bleached corals and observed an

abrupt rise, corresponding to the bleaching period, in the δ18O prole analyzed at

high resolution along the growth axis of the skeleton. They interpreted this jump to

reect a cessation of coral skeletal growth for a few months immediately after

bleaching. As global warming progresses and high seawater temperatures occur

more frequently, environmental conditions can be expected to further inhibit coral

growth.

Another good proxy for the pH of seawater, or, more precisely, that of the calci-

fying uid of the organism, is the boron isotope ratio of the coral skeleton. Kubota

et al. (2017) conducted high-precision boron isotope measurements of two coral

cores collected from Kikai Island (Ryukyu Islands) and Chichijima Island

(Ogasawara Islands) and reported that the ratios from the two islands decreased over

the long term, indicating decreasing pH. Interestingly, in both cases, the rate of

decline increased in the latter half of the twentieth century. Although seawater pH

changes have been observed by shipboard measurements since 1985, the coral

record conrms the existence of an ocean acidication trend in the Western Pacic.

29.4 Application toDisaster Research

The 2011 Tohoku-oki earthquake (Great East Japan Earthquake) occurred on 11

March 2011, and the tsunami generated by the earthquake caused major damage to

the Pacic coasts of the Tohoku and Kanto regions of Japan. To mitigate the effects

of future tsunamis, it is urgent to reevaluate past tsunami damage throughout Japan.

A. Suzuki

279

Coral skeletal climatology methods have been applied to the analysis of Porites

boulders cast ashore by past tsunamis (Suzuki etal. 2008; Fig.29.4). By applying

radiocarbon dating and coral skeletal climatological techniques to Porites boulders

scattered along the eastern coast of Ishigaki Island, southern Ryukyus, Araoka etal.

(2010) demonstrated that some of the boulders, at least, were washed ashore by the

Meiwa tsunami in 1771. Araoka etal. (2013) extended this approach to neighboring

islands in the southern Ryukyus. They selected non-eroded Porites coral boulders

along shorelines for radiocarbon dating, because they retain characteristics that

make it possible to determine the probable timing of their deposition by tsunamis.

Their results demonstrate that the southern Ryukyu Islands have repeatedly experi-

enced tsunami events since at least 2400years ago, with a recurrence interval of

about 150–400years. Their study demonstrates that by reliably dating large num-

bers of coral boulders, it is possible to ascertain the timing, recurrence interval, and

magnitude of past tsunamis in a location where few survey sites exist that include

sandy tsunami deposits.

29.5 Future Directions

Several points still need clarication, including the basic mechanisms by which

climatological factors such as seawater temperature are recorded in the chemical

and isotope compositions of coral skeletons. Further, the inuence of the coral

growth rate on coral climate proxies such as δ18O is still problematic (Fig.29.5).

Special attention needs to be paid to diagenetic alteration of coral proxy signals. In

addition to the geochemical methods, culture experiments should be conducted and

molecular biological methods should be applied to clarify the biological mechanism

of calcication. Recent papers have recognized that coral primary polyps are

particularly suitable for biomineralization studies because of their small size and

simple form (Iwasaki etal. 2016; Ohno et al. 2017). An integrated approach that

brings various perspectives to bear on these problems is needed, because coral

biomineralization reects synergetic effects (Fig.29.6).

Fig. 29.4 (a) Aerial photograph of the fringing coral reef on the eastern shore of Ishigaki Island,

Japan (from the Geospatial Information Authority of Japan). (b) A tsunami boulder composed of a

massive Porites coral on the reef at. This coral was dated to about AD 1771 (Araoka etal. 2010).

29 Coral-Based Approaches toPaleoclimate Studies, Future Ocean Environment…

280

Acknowledgments This paper is based on joint research with H.Kawahata, A.Iguchi, Y.Ohno,

M.Inoue, D. Araoka, T.Watanabe, and many other collaborators. To all of them, I express my

sincere gratitude. This study was supported by KAKENHI grant 15H02813 and 18H03366to AS.

Fig. 29.5 Inuence of the skeletal growth rate on the skeletal oxygen isotope ratio (δ18O) as

reported in the literature (The image of the coral skeleton has been reprinted from McConnaughey

1989 with permission from Elsevier). Hayashi etal. (2013) reported a relatively small growth rate

dependency of skeletal δ18O values, but most previous studies have reported considerable depen-

dence of climate proxies on the skeletal growth rate(Felis etal. 2003; Suzuki etal. 2005)

GW & OA

experiments

Calcif

ication

mechanism

Paleoclimate

/ proxy evaluation

Fig. 29.6 Graphical summary of the integrated approach used by the author’s research group.

Research is conducted from various perspectives simultaneously because we expect synergetic

effects for better understanding biomineralization of corals. GW global warming, OA ocean

acidication

A. Suzuki

281

References

Araoka D, Inoue M, Suzuki A, Yokoyama Y, Edwards RL, Cheng H, Matsuzaki H, Kan H,

Shikazono N, Kawahata H (2010) Historic 1771 Meiwa tsunami conrmed by high-resolu-

tion U/Th dating of massive Porites coral boulders at Ishigaki Island in the Ryukyus, Japan.

Geochem Geophys Geosyst 11:Q06014

Araoka D, Yokoyama Y, Suzuki A, Goto K, Miyagi K, Miyazawa K, Matsuzaki H, Kawahata H

(2013) Tsunami recurrence revealed by Porites coral boulders in the southern Ryukyu Islands,

Japan. Geology 41:919–922

Felis T, Pätzold J, Loya Y (2003) Mean oxygen-isotope signatures in Porites spp. corals: inter-

colony variability and correction for extension-rate effects. Coral Reefs 22:328–336

Felis T, Suzuki A, Kuhnert H, Dima M, Lohmann G, Kawahata H (2009) Subtropical coral reveals

abrupt early 20th century freshening in the western North Pacic Ocean. Geology 37:527–530

Gagan MK, Dunbar GB, Suzuki A (2012) The effect of skeletal mass accumulation in Porites on

coral Sr/Ca and δ18O paleothermometry. Paleoceanography 27:PA1203

Hayashi E, Suzuki A, Nakamura T, Iwase A, Ishimura T, Iguchi A, Sakai K, Okai T, Inoue M,

Araoka D, Murayama S, Kawahata H (2013) Growth-rate inuences on coral climate proxies

tested by a multiple colony culture experiment. Earth Planet Sci Lett 362:198–206

Iwasaki S, Inoue M, Suzuki A, Sasaki O, Kano H, Iguchi A, Sakai K, Kawahata H (2016) The role

of symbiotic algae in the formation of the coral polyp skeleton: 3-D morphological study based

on X-ray microcomputed tomography. Geochem Geophys Geosyst 17:3629–3637. https://doi.

org/10.1002/2016GC006536

Kubota K, Yokoyama Y, Ishikawa T, Suzuki A, Ishii M (2017) Rapid decline in pH of coral calci-

cation uid due to incorporation of anthropogenic CO2. Sci Rep 7:7694

Ma TYH (1934) On the growth rate of reef corals and the sea water temperature in the Japanese

Islands during the latest geological times. Science reports of the Tohoku Imperial University

2nd series. Geology 16(3):165–189

McConnaughey T (1989) 13C and 18O isotopic disequilibrium in biological carbonates: I.Patterns.

Geochim Cosmochim Acta 53:151–162

McCulloch MT, Gagan MK, Mortimer GE, Chivas AR, Isdale PJ (1994) A high resolution Sr/

Ca and 18O coral record from the Great Barrier Reef, Australia, and the 1982–1983 El Niño.

Geochim Cosmochim Acta 58:2747–2754

Mishima M, Suzuki A, Nagao N, Ishimura T, Inoue M, Kawahata H (2010) Abrupt shift toward

cooler condition in the earliest 20th century detected in a 165 year coral record from Ishigaki

Island, southwestern Japan. Geophys Res Lett 37:L15609

Ohno Y, Iguchi A, Shinzato C, Inoue M, Suzuki A, Sakai K, Nakamura T (2017) An aposymbiotic

primary coral polyp counteracts acidication by active pH regulation. Sci Rep 7:40324

Suzuki A (2012) Paleoclimate reconstruction and future forecast based on coral skeletal climatol-

ogy –understanding the oceanic history through precise chemical and isotope analyses of coral

annual bands. Synthesiology 5:78–87

Suzuki A, Gagan MK, Fabricius K, Isdale PJ, Yukino I, Kawahata H (2003) Skeletal isotope micro-

proles of growth perturbations in Porites corals during the 1997–1998 mass bleaching event.

Coral Reefs 22:357–369

Suzuki A, Hibino K, Iwase A, Kawahata H (2005) Intercolony variability of skeletal oxygen and

carbon isotope signatures of cultured Porites corals: temperature controlled experiments.

Geochim Cosmochim Acta 69:4453–4462

29 Coral-Based Approaches toPaleoclimate Studies, Future Ocean Environment…

282

Suzuki A, Yokoyama Y, Kan H, Minoshima K, Matsuzaki H, Hamanaka N, Kawahata H (2008)

Identication of 1771 Meiwa Tsunami deposits using a combination of radiocarbon dating

and oxygen isotope microproling of emerged massive Porites boulders. Quat Geochronol

3:226–234

Watanabe T, Suzuki A, Minobe S, Kawashima T, Kameo K, Minoshima K, Aguilar YM, Wani R,

Kawahata H, Sowa K, Nagai T, Kase T (2011) Permanent El Niño during the Pliocene warm

period not supported by coral evidence. Nature 471:209–211

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

A. Suzuki

283

Chapter 30

An Elemental Fractionation Mechanism

Common toBiogenic Calcium Carbonate

KotaroShirai

Abstract Biological modulation of element incorporation presents a major hurdle

in the interpretation of geochemical data as an environmental proxy, detailed under-

standing and quantitative evaluation of the mechanism of elemental fractionation

both being essential for reliable reconstruction of an environment. Biogenic calcium

carbonate has a specic skeletal microstructure, which is strongly controlled by

biomineralization. Since primary processes are more likely reected on a smaller

spatial scale, elemental distribution patterns associated with skeletal microstructure

should provide unique information on biological elemental uctuations, which can-

not be determined from large-scale analysis. To study elemental fractionation mech-

anisms, microscale elemental distribution patterns have been studied in coral

skeletons and bivalve and foraminiferal shells and the skeletal microstructure, sulfur

distribution, and organic features compared. The microanalytical studies revealed

two characteristic patterns that were common to all studied biogenic calcium car-

bonates, even though the specimens examined represented different phyla: (1) sig-

nicant compositional heterogeneities that could not be explained by changes in the

ambient environment and (2) a strong correlation of “metal/Ca” ratios with all or

some of sulfur distribution, skeletal microstructure, and organic character. Based on

these common features, I propose a mechanism of elemental fractionation, com-

monly applicable to biogenic calcium carbonates and involving both composition

and/or concentration of organics in the calcifying uid, that facilitates preferential

elemental incorporation into biogenic calcium carbonate.

Keywords Biogenic calcium carbonate · Proxy · Vital effect · Geochemistry ·

Trace element · Coral · Bivalve shell · Foraminifera · Sclerosponge

K. Shirai (*)

Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Chiba, Japan

e-mail: kshirai@aori.u-tokyo.ac.jp

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_30

284

30.1 Introduction

Mg/Ca and Sr/Ca ratios in biogenic calcium carbonate are widely used as proxies

for estimating past seawater temperatures (e.g., Henderson 2002; Lea 2003).

However, such ratios are also more or less affected by biological processes, so-

called vital effects (Cohen and McConnaughey 2003). For example, the Sr/Ca ratio

in a coral skeleton (aragonite) and Mg/Ca ratio in a foraminiferan test (calcite)

reect temperature relatively precisely (Lea 2003), whereas Mg/Ca in the former

and Sr/Ca in the latter, and both ratios in bivalve shells (both aragonite and calcite),

are susceptible to biological modulation (Schöne 2013). Although the vital effect is

considered to be species-specic, it remains a major hurdle to interpreting geo-

chemical data as an environmental proxy, as the ultimate mechanism governing

elemental fractionation, a detailed understanding and quantitative evaluation of

which is essential for reliable past environment reconstruction, is still unclear.

A fundamental question concerns the common existence or otherwise of an ele-

mental fractionation mechanism in any biogenic calcium carbonate.

Biomineralization can be interpreted as inorganic mineralization strongly controlled

by soluble and insoluble organic materials, as well as physiological control, such as

that exerted by pH, physical structure, space regulations, and ion transportation

(e.g., Marin etal. 2008). Thus, the relationship between micrometer-scale elemental

distribution and microstructure may provide a unique opportunity to investigate the

mechanism of element fractionation by biological processes. The aim of the present

study was to examine a common, cross-phylum fractionation mechanism based on

microscale elemental distribution in coral skeletons and bivalve and foraminiferal

shells, comparing elemental distribution, skeletal/shell microstructure, sulfur distri-

bution, and organic features.

30.2 Materials andMethods

The examined samples included the reef building branching coral Acropora nobilis

(Shirai etal. 2008a), deep sea solitary coral Caryophyllia ambrosia ambrosia (Shirai

etal. 2005), ocean quahog Arctica islandica (Shirai etal. 2014), deep sea hydrother-

mal mussel Bathymodiolus platifrons (Shirai etal. 2008b), and planktonic foramin-

ifera Globorotalia menardii (Kunioka etal. 2006). Samples were cleaned, embedded

in epoxy resin, sectioned, polished, metal coated where necessary, and analyzed by

electron probe microanalysis (EPMA) or high lateral resolution secondary ion mass

spectrometry (NanoSIMS). The microstructure was observed by SEM or optical

microscope following etching/staining by Mutvei’s solution (Schöne etal. 2005).

Specic details of sample origin and preparation and analytical methods are included

in the above-cited references.

K. Shirai

285

30.3 Resuts

Eementa dstrbuton and skeeta/she mcrostructure of the examned sampes

are shown n Fgs.30.1, 30.2, 30.3, 30.4, and 30.5. Regardess of mneraogy and

phyum, the foowng were common at the mcroscae eve n a of the examned

bogenc carbonates: (1) arge compostona heterogenety and (2) eement/Ca

ratos, skeeta/she mcrostructure, sufur dstrbuton, and nsoube skeeta

organc characters a correated (nonnear) wth one another.

Fg. 30.1 Eementa dstrbuton and skeeta mcrostructure of branchng cora Acropora nobs.

(a) EPMA Mg map. (b) EPMA Sr map. (c) EPMA S map. (d–g) Skeeta mcrostructure of

poshed- etched surface by SEM. (Fgures moded from Shra eta. 2008a). Coor scae bar on

rght ndcates gross count per pxe

Fg. 30.2 Eementa dstrbuton and skeeta mcrostructure of deep sea cora Caryophya

ambrosa ambrosa. (a) EPMA Mg map. (b) EPMA Sr map. (c) Skeeta mcrostructure observed

n thn secton under transmtted ght mcroscopy. (Fgures moded from Shra et a. 2005).

Coor scae bar on rght ndcates gross count per pxe

30 An Eementa Fractonaton Mechansm Common toBogenc Cacum Carbonate

286

30.4 Discussion

The correlation with microstructure suggests that microscale heterogeneity is

likely induced by the biomineralization processes, the magnitude of variation

being too great to be explained by ambient environmental changes. Similar corre-

lations between chemical composition and microstructure have been documented

in other coral skeleton studies (e.g., Meibom et al. 2004, 2008), bivalve shell

Fig. 30.3 Elemental distribution and shell microstructure of ocean quahog Arctica islandica. (a)

Sr/Ca and Mg/Ca maps partly overlapped on S/Ca map. (b–d) High-magnication images of ele-

mental maps and shell microstructure along sixth annual growth line from ventral margin (red

rectangles in (a)). From left to right, (1) EPMA Sr/Ca map, (2) EPMA S/Ca map, (3) SEM micro-

structure image, and (4) inset of (3). HOM homogeneous structure, ISP irregular simple prismatic

structure, CA crossed acicular structure, N.C. shell microstructure is not clear. (Figures modied

from Shirai etal. 2014). Color scale bar on bottom right in panel (a) indicates gross count ratio (ct/

ct count/count) per pixel

Fig. 30.4 Elemental distribution and shell microstructure of hydrothermal mussel Bathymodiolus

platifrons. (a) EPMA Mg map with NanoSIMS Mg/Ca prole overlaid (right axis for scale). (b)

EPMA Sr map with NanoSIMS Sr/Ca prole overlaid. (c) EPMA S map. (d) Shell microstructure

of polished-etched surface by SEM with NanoSIMS Mn/Ca prole overlaid. (Figures modied

from Shirai etal. 2008b)

K. Shirai

287

(Dauphin etal. 2005, 2013; Füllenbach etal. 2017), foraminifera (Sadekov etal.

2005; Kunioka etal. 2006), brachiopod (Pérez-Huerta etal. 2011), and Ostracoda

(Morishita et al. 2007), being lines of evidence pointing to an almost universal

relationship between elemental distribution and microstructure in any biogenic cal-

cium carbonate, as well as suggesting the existence of a common physiochemical

mechanism governing elemental fractionation.

Recent studies have reported that organic molecules present in the calcifying

solution enhance Mg incorporation in inorganically precipitated calcite (Stephenson

et al. 2008) and amorphous calcium carbonate (Wang et al. 2009). In the latter

model, aqueous carboxylated molecules, which have greater selectivity for binding

to Ca than Mg, increase the relative activity of Mg against Ca in the solution, pro-

ducing high Mg/Ca amorphous calcium carbonate, considered a precursor phase of

crystalline carbonate (e.g., Pouget etal. 2009). Thus, organically mediated fraction-

ation processes may occur in the early stages of crystallization (Wang etal. 2009),

with the mechanism that determines the composition of amorphous calcium car-

bonate also likely controlling biogenic calcite composition. Since the organic mem-

brane has a characteristic electrostatic structure regulating biomineralization (e.g.,

Marin etal. 2008; Ren etal. 2011), it is also possible that such a structure of insol-

uble organics inuences elemental incorporation into carbonate. Since biomineral-

ization occurs in calcifying uid (or calcifying space), the organic (and inorganic)

composition of which is highly regulated by the organism, I suggest that both solu-

ble and insoluble organic composition and concentration in calcifying uids control

the microscale elemental distribution commonly found in biogenic calcium

Fig. 30.5 Elemental distribution and shell microstructure of planktonic foraminifera Globorotalia

menardii. (a) Shell microstructure under transmitted light microscopy after EPMA analysis (b)

From left to right, EPMA Mg/Ca map, EPMA S/Ca map, shell microstructure of polished-etched

surface by SEM and transmitted light microscope image. (c) SEM microstructure image, enlarge-

ments of inset of (a). (d) Enlargements of inset of (b). From left to right, (d1) EPMA Mg/Ca map,

(d2) EPMA S/Ca map, (d3) EPMA Ca map, (d4) EPMA backscattered electron image, and (d5)

transmitted light microscope image. Pink bars represent organic membrane. White dashed lines in

(d) act as a guide for comparing each gure on the same position

30 An Elemental Fractionation Mechanism Common toBiogenic Calcium Carbonate

288

carbonate. Changes in the microstructure and sulfur distribution can be considered

as signatures of change in the organic composition and sulfur-containing organic

matrix in the calcifying medium, respectively. These processes are likely common

in any biomineralization. Such an organically mediated fractionation hypothesis

can also explain why elemental composition is not correlated with other elements

in a quantitative manner, since the function of macromolecules in calcium carbon-

ate precipitation depends not only on their structure and composition but also on

their supramolecular assemblage (e.g., Marin etal. 2008). A detailed discussion of

this elemental fractionation mechanism is reported in Shirai et al. (2014) and

Füllenbach etal. (2017).

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adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

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30 An Elemental Fractionation Mechanism Common toBiogenic Calcium Carbonate

291

Chapter 31

Biomineralization ofMetallic Tellurium

byBacteria Isolated FromMarine

Sediment Off Niigata Japan

MadisonPascualMunar, TadaakiMatsuo, HiromiKimura,

HirokazuTakahashi, andYoshikoOkamura

Abstract Three facultative anaerobe mesophilic bacteria were isolated from

marine sediment collected off Niigata, Japan. Sequencing of complete 16S ribo-

somal DNA revealed 99% homology with Shewanella algae, Pseudomonas pseudo-

alcaligenes, and P. stutzeri. Phylogenetic analyses suggest novel strain status thus

new strains were designated as S. algae strain Hiro-1, P. pseudoalcaligenes strain

Hiro-2, and P. stutzeri strain Hiro-3. Minimum inhibitory concentration assays

using increasing concentrations of Na2TeO 3 revealed resistance of S. algae strain

Hiro-1 at 15 mM, and P. pseudoalcaligenes strain Hiro-2 and P. stutzeri strain

Hiro-3 both showed resistance at 4mM.Transmission electron microscopy revealed

intracellular aggregation of metallic tellurium nanorods with a minimum unit size

of 60-nm nanoparticle.

Keywords Marine sediment · Pseudomonas pseudoalcaligenes · Pseudomonas

stutzeri · Shewanella algae · Tellurite reduction · Tellurium nanorods

31.1 Introduction

Tellurite is a strong oxidizing agent that is highly toxic to most microorganisms

(Fleming 1932; Taylor 1999; Chasteen etal. 2009; Arenas-Salinas etal. 2016). The

compound’s toxicity was reported to induce oxidative stress, which eventually leads

to cell death. Some microorganisms can resist this toxicity, either by enzymatic

reduction with the aid of nitrate reductase or by overexpression of glutathione

(GSH) to maintain homeostasis inside the cell (Avazeri et al. 1997; Sabaty etal.

2001; Turner 2001; Turner etal. 2012; Pugin et al. 2014). Tellurate (TeO42−) and

tellurite (TeO32−) oxyanions can also serve as electron acceptors in anaerobic

M. P. Munar · T. Matsuo · H. Kimura · H. Takahashi · Y. Okamura (*)

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,

Hiroshima University, Higashihiroshima, Japan

e-mail: ziphiro@hiroshima-u.ac.jp; okamuray@hiroshima-u.ac.jp

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_31

292

respiration by purple sulfur bacteria (Csotonyi etal. 2006; Baesman et al. 2007).

Reduction of tellurite into pure metallic elemental tellurium (Te0) can be observed

as formation of black tellurium precipitate (Tucker etal. 1962).

Production and recovery of this scarce metalloid is required for the sustainability

of green technologies such as solar cells in the future. However, the exact mecha-

nism of tellurium reduction is still unknown. Most studies conducted on tellurite

reduction (TR) employed bacterial species with very low resistance to the element,

and the poor survival of cells in the presence of tellurite is a major impediment to

fully elucidating the enigmatic reduction mechanism (Arenas-Salinas etal. 2016).

Biomineralization of tellurium nanoparticles (TeNPs) has been documented in sev-

eral bacterial genera including Rhodobacter, Escherichia, Shewanella, Geobacter,

Sulfurospirillum, Bacillus, Pseudomonas, Erwinia, Agrobacter, Staphylococcus,

and Selenihalanaerobacter (Moore and Kaplan 1994; Avazeri etal. 1997; Trutko

etal. 2000; Sabaty etal. 2001; Di Tomaso etal. 2002; Borsetti etal. 2003; Oremland

etal. 2004; Csotonyi etal. 2006; Baesman etal. 2007, 2009; Turner etal. 2012;

Borghese etal. 2014). Some species within these bacterial genera possess innate

resistance to the toxic metalloid and are thereby potential candidates for microbio-

logical reduction and recovery of the valued rare Earth element. Here, we report the

isolation and identication of tellurite-resistant and tellurite-reducing bacterial

strains that can be used for the development of efcient metal recovery strategies.

Further analyses of these strains may add additional insights into factors prerequi-

site for efcient bioremediation.

31.2 Materials andMethods

31.2.1 Isolation andCultivation

A marine sediment sample collected at a depth of 100m off Niigata, Japan (38°05′N,

139°04′E) was generously provided by Dr. Takeshi Terahara of the Tokyo University

of Marine Science and Technology. The sediment was inoculated and incubated at a

nal volume of 50mL with RCVBN medium (Burgess etal. 1991) in completely

lled 50-mL centrifuge tubes (Falcon) under continuous illumination for 1month.

The culture showed prominent growth of a biolm-forming purple bacteria as

observed by the deep purple pigment production in the media. A 1-mL sample of the

resulting culture was transferred into 7-mL fresh RCVBN medium supplemented

with 1-mM sodium tellurite and cultured in completely lled 8-mL screw-capped

tubes at 24°C under continuous illumination (38μmol/m2/s). TR activity was visu-

ally recorded by the formation of black tellurium precipitate. The successive pour

plate method and streak plate method were used to isolate tellurite-resistant and

tellurite-reducing bacteria on RCVBN agar media (pH 7.6, 37°C). Puried colonies

were re-streaked in RCVBN agar plates with 1mM Na2TeO3 to observe TR activity.

Colony characteristics were observed after 72h of growth. To determine the TR

activity and minimum inhibitory concentration (MIC), the cultures were incubated

with varying concentrations of Na2TeO 3 (1, 2, 4, 6, 8, 10, 12, and 15mM). Inoculum

M. P. Munar et al.

293

was standardized by using culture with similar OD values for each strain. Bacterial

growth in pH and temperature optimum experiments was measured using spectrom-

eter at OD550 nm (WPA CO-7500 Colorimeter, Biochrom Ltd., UK).

31.2.2 16S rDNA Amplication, Cloning, andSequencing

The 16S rDNA was amplied using PCR mix including 10X KOD Plus-Neo Buffer,

2mM dNTPs, 25mM MgSO4 (TOYOBO Co., Ltd., Osaka, Japan), 10μM each

forward and reverse primer 16S 27F (F-5′-AGAGTTTGATCNTGGCTCAG-3′)

and 16S ipRSR2 (R-5′-AAGGAGGTGATCCANCCGC-3′) (Eurons Genomics,

Tokyo, Japan), 0.05U/μL KOD Neo POL (TOYOBO Co., Ltd., Osaka, Japan), and

2μL genomic DNA.Thermal cycling conditions were as follows: pre-denaturation

2mins at 94°C for 1 cycle, followed by 35 cycles of denaturation at 95°C for 10s,

annealing at 60°C for 10s, and extension at 68°C for 60s, and nal extension at

68°C for 60s (T100™ Thermal Cycler, Bio-Rad, CA, USA).

After PCR, amplied fragments were excised from the gel. Puried 16S rDNA

fragments were cloned into the ZERO-Blunt TOPO vector following the kit proto-

col (Invitrogen, USA). Clones able to grow on LB/Kanamycin plates were used for

colony PCR to verify insertion using M13 primers (F-5′-

TGTAAAACGACGGCCAGT-3′; R-5′-CAGGAAACAGCTATGACC-3′)

(Eurons Genomics, Tokyo Japan). Thermal cycling conditions were as follows:

pre-denaturation 5mins at 94°C for 1 cycle, followed by 28 cycles of denaturation

at 94°C for 10s, annealing at 70°C for 20s, and extension at 72°C for 45s. Positive

clones with veried inserts were sub-cultured and plasmids were extracted using a

Fastgene plasmid mini kit (Nippon Genetics Co., Ltd., Tokyo, Japan) following the

manufacturer’s protocol. The sequences of the resulting clones were analyzed by

Eurons Genomics Co., Ltd. (Tokyo, Japan). SnapGene (SnapGene Software,

www.snapgene.com) was used to check chromatogram quality and to guide quality

trimming. High quality reads were assembled using CodonCode Aligner

(CodonCode Corporation, www.codoncode.com) and assembled sequences were

used for BLAST analysis (NCBI).

31.2.3 Phylogenetic Analysis

With MEGA6 software, Neighbor-Joining, Maximum-Likelihood, Minimum

Evolution and Maximum Parsimony with Kimura-2-parameter distance correction

and 1000 bootstrap value were used to identify the three pure cultures (Kimura

1980; Felsenstein 1985; Saitou and Nei 1987; Rzhetsky and Nei 1992; Nei and

Kumar 2000; Tamura et al. 2013). NCBI GenBank database was surveyed for

closely related strains with partial and complete 16S ribosomal DNA sequences

included in the phylogenetic analyses. The 16S rDNA sequences of all species iden-

tied were aligned with the muscle DNA alignment method in MEGA 6 software.

31 Biomineralization ofMetallic Tellurium byBacteria Isolated FromMarine…

294

Aligned sequences were visually evaluated to ensure that the subsequent cladogram

would result from polymorphisms rather than sequence errors, gaps, and branch

pulling due to differences in sequence directions or sizes. A total of 26 Shewanella

and 24 Pseudomonas species and strains were included in the phylogenetic analyses

with equal length of nal quality trimmed sequences of 1360 bp and 1391 bp,

respectively.

31.2.4 Benchmarking ofNew Isolates withType Strains

Type strains Shewanella algae (NBRC 103173T), Pseudomonas pseudoalcaligenes

(NBRC 14167T), and Pseudomonas stutzeri (NBRC 14165T) were obtained from

the National Institute of Technology and Evaluation Biological Resource Center

(NBRC) in Japan. The type cultures were initially revived using the prescribed

growth medium for each culture, then were grown in RCVBN media to compare

colony morphology with our new isolates. Cultures were streaked onto plates to

observe single pure colonies of the type strains and new isolates. RCVBN agar

spread with 200μL 1mM Na2TeO 3 was used to evaluate the TR activity of the type

strains. Plates were incubated at 37°C for 6days were used for observation of mor-

phology and TR activity under stereomicroscope (TW-360, WRAYMER, Japan).

31.2.5 Electron Microscopy ofNew Isolates

Transmission electron microscopy (TEM) was performed on the three new isolates

to observe their crystal morphologies and localizations. Enrichment cultures of each

strain exposed at 1mM Na2TeO3 incubated for 101days were used for TEM.The

long incubation period would allow for the observation of all possible crystal mor-

phologies. A 1-mL culture of each strain was washed twice with milliQ water by

centrifugation at 10,000 × g for 5min. The harvested cells were resuspended in mil-

liQ water and mounted on 150-mesh copper grids coated with collodion (Nisshin

EM Co., Ltd., Tokyo, Japan).

31.3 Results

31.3.1 Three Marine Mesophiles withTellurite Resistance

andReduction Activity

The mesophiles isolated into pure culture included two Pseudomonas species and one

Shewanella species. BLAST search using the full-length 16S rDNA sequences of the

three isolates revealed 99% homology to P. pseudoalcaligenes (Query length: 1529bp),

M. P. Munar et al.

295

P. stutzeri (Query length: 1529bp), and S. algae (Query length: 1538bp) with E-value

of 0.00. Based on phylogenetic analyses using four different building methods, the

three isolates were found to be unique strains. Representative trees are shown in

Fig.31.1. Thus, we designate new strains on S. algae, P. pseudoalcaligenes, and P.

stutzeri with strain Hiro-1 (DDBJ Accession no.: LC339942), Hiro-2 (DDBJ Accession

no.: LC339940), and Hiro-3 (DDBJ Accession no.: LC339941), respectively. The two

Pseudomonas isolates showed TR activity up to 4mM tellurite with a MIC of 6mM

tellurite.

The S. algae isolate showed TR activity up to 10mM tellurite with a MIC of

15 mM tellurite (Fig. 31.2). Negative control samples containing only RCVBN

(Fig. 31.2, tube C1) or RCVBN spiked with 1 mM Na2TeO 3 (data not shown)

showed no visible TR or bacterial growth indicating no spontaneous reduction or

contamination, respectively.

31.3.2 Colony Characteristics ofNew Isolates

S. algae strain Hiro-1 formed colonies with round form, entire margin, convex ele-

vation, mucoid consistency, and orange-brown color. P. pseudoalcaligenes strain

Hiro-2 formed colonies with irregular form, undulate margin, raised elevation, vis-

cid consistency, and translucent color. P. stutzeri strain Hiro-3 formed colonies with

wrinkled form, undulate margin, raised elevation, rugose consistency, and yellow

color. The morphology and TR activity were also compared to the type strains, and

the new isolates showed similar colony morphology and TR activity to the type

cultures (Fig.31.3). All three new strains grew optimally at pH 7.0. Growth at pH

7.0 was signicantly higher than growth at all other pH levels tested based on

unpaired t-test with 95% condence level (p-value <0.05). Optimum growth tem-

perature coincided with mesophilic cardinal temperatures. For all three strains,

there was no signicant difference between the growth at 25°C, 37°C, and 45°C

based on unpaired t-test with 95% condence level (p-value >0.05). Little to no

growth was observed at 4°C.

31.3.3 Intracellularly Deposited Tellurium Particles

TEM revealed that tellurium particles were deposited within the cell for all three

strains (Fig.31.4). In S. algae strain Hiro-1, 60-nm minimum units of nanorod par-

ticles were conjugated along the crystallographic axis, forming long rod crystals

that nally assembled into a bundle within the cell (Fig.31.4a). However, this cul-

ture was too old, and the cells were easily lysed. External, inorganically growing

crystals were also found (data not shown). Strain Hiro-2, interestingly, formed

needle- shaped particles 60 nm in size (Fig. 31.4b), whereas strain Hiro-3 formed

rod-shaped crystals resembling the nanorods from the strain Hiro-1 (Fig.31.4c).

31 Biomineralization ofMetallic Tellurium byBacteria Isolated FromMarine…

296

Fig 31.1 Unrooted neighbor-joining tree. (a) S. algae strain Hiro-1 (a). (b) P. pseudoalcaligenes

strain Hiro-2 (b). P. stutzeri strain Hiro-3 (c). Accession numbers are in parentheses. Numbers at

nodes represent bootstrap values from 1000 resampled datasets. Scale bar indicates 0.5% sequence

divergence. Kimura-2-parameter was used for distance correction

M. P. Munar et al.

297

Both Pseudomonas spp. maintained cell integrity, and both showed smaller and

scattered nanoparticles within the cell. Long crystals were observed in ripped cells,

suggesting that the length of tellurium crystal must be regulated within the cellular

environment.

31.4 Discussion

Deep marine sediments support the growth of anaerobic microorganisms that may

use various metals as nal electron acceptors in the respiratory chain, such as purple

sulfur bacteria. S. algae has bioremediation potential against uranium, plutonium,

tellurite ions, nitrite, and halogenated organic compounds (Almagro etal. 2005;

Klonowska etal. 2005). Mucoid, round colonies are common colony morphologies

for Shewanella species (Simidu etal. 1990; Nozue etal. 1992; Venkateswaran etal.

1998; Holt etal. 2005; Gao etal. 2006; Kim etal. 2007) which is also evident in our

isolate. P. pseudoalcaligenes and P. stutzeri have bioremediation potential against

cyanide and tellurite (Romero et al. 1998). P. pseudoalcaligenes subsp. citrulli,

Fig. 31.2 Tellurite reduction activity (TR activity) and minimum inhibitory concentration (MIC)

of tellurite. Control tube C1 is RCVBN media alone and control tube C2 is RCVBN with culture

inoculum. The other tubes are labeled with the concentration (mM) of Na2TeO 3 added to the

RCVBN media inoculated with the culture. Image shown is representative of two replicate reac-

tions taken after 3 weeks of incubation. (a) S. algae, (b) P. pseudoalcaligenes, and (c) P. stutzeri

31 Biomineralization ofMetallic Tellurium byBacteria Isolated FromMar ine…

298

isolated from diseased watermelon, and P. alcaligenes, a human opportunistic

pathogen, are both reported to have translucent consistency (Monias 1928; Ralston-

Barrett et al. 1976; Schaad et al. 1978) which is consistent with our isolate. P.

stutzeri isolated as an opportunistic human pathogen has wrinkled and hard/dry

characteristics (Lalucat etal. 2006) which is also observed in our isolate.

Our new isolates from deep marine sediment have also shown extreme resistance

and reduction activity in very high concentrations (above 1mM) of tellurite ions.

This extreme resistance could be attributed to the species-specic reduction mecha-

nisms, differential cell physiology, or genetic adaptation mechanisms in extremely

toxic environments.

Fig. 31.3 Colony morphology and TR activity of the three new isolates and their corresponding

type cultures, S. algae (a), P. pseudoalcaligenes (b), and P. stutzeri (c)

Fig. 31.4 TEM observation of intracellular tellurium particles in S. algae strain Hiro-1 (a), P.

pseudoalcaligenes strain Hiro-2 (b), and P. stutzeri strain Hiro-3 (c)

M. P. Munar et al.

299

Species-specic biomineralization has been observed in several bacterial strains.

Common tellurium morphologies vary from spheres to nanospheres and from rods

to nanorods (Turner etal. 2012). Spatial distribution of nanoparticles also shows

species-specic variation. The most common localization of metal biodeposition is

at the cell surface or in the cytoplasm (Turner etal. 2012). The new isolates showed

intracellular mineralization of metallic tellurium nanorods. This was also evident

from the blackening of bacterial colonies, while surrounding tellurite ions present in

the agar media were unaffected. Intracellular crystal formation denotes inux of

metal ions into the cell cytoplasm, and thus unknown ion transporters or channels

might be involved in this phenomenon. Once inside the cell, enzyme-catalyzed

reduction of tellurite ions into elemental tellurium precedes crystal nucleation and

growth. Tellurium crystals showed minimum unit size of 60nm which falls under

the category of nanoparticles (<100nm) and therefore has a potential use for nano-

technology applications (Arenas-Salinas etal. 2016).

Together, these results show that the three new isolates identied in this study

effectively reduce tellurite even at high concentrations. This reduction occurs within

the cell, but determining the exact mechanisms will require further study. These

strains may be useful for bioremediation, as well as for recovery of this valuable

metalloid for use in manufacturing.

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31 Biomineralization ofMetallic Tellurium byBacteria Isolated FromMarine…

303

Chapter 32

Calcium Oxalate Crystals inPlant

Communities oftheSoutheast

ofthePampean Plain, Argentina

StellaMarisAltamirano, NataliaBorrelli, MaríaLauraBenvenuto,

MarianaFernándezHonaine, andMargaritaOsterrieth

Abstract Calcium oxalate crystals (COC) are one of the most prevalent and widely

distributed biomineralizations in plants. The aim of this work is to analyze and com-

pare the data previously reported about the presence and production of COC in

leaves of plant species from forests, wetlands, and agroecosystems of the southeast

of the Pampean Plain. Diaphanization, clearing of tissues with 50% sodium hypo-

chlorite, and cross sectioning of the leaves were realized. The material was mounted

with gelatin–glycerin, and COC were identied and described with optical, polar-

ization, and scanning electron microscopes. Crystal size and density were calcu-

lated. Calcication mainly occurred in leaf mesophyll. In terrestrial species, crystals

were closely associated with vascular bundles, while in aquatic species, they were

associated with aerenchyma. Druses, prisms, and raphides were observed in the

leaves of all species analyzed. Average crystal size was smaller in terrestrial species

than aquatic ones (12 and 80 μm, respectively), but average crystal density was

higher (246 and 23 crystals/mm2, respectively). These different patterns in COC

production and distribution may be related to taxonomical characteristics, the types

of cells where crystals precipitate, their function, and the differential transpiration

rates, among other factors.

Keywords Biomineralizations · Phytoliths · Calcium oxalate crystals · Terrestrial

and aquatic ecosystems · Forest · Agroecosystem · Pampean plain

S. M. Altamirano · N. Borrelli (*) · M. L. Benvenuto · M. F. Honaine · M. Osterrieth

Instituto de Geología de Costas y del Cuaternario (IGCyC), FCEyN, UNMdP– CIC,

Mar del Plata, Argentina

Instituto de Investigaciones Marinas y Costeras (IIMyC), CONICET– UNMdP,

Mar del Plata, Argentina

e-mail: nlborrel@mdp.edu.ar; fhonaine@mdp.edu.ar; mosterri@mdp.edu.ar

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_32

304

32.1 Introduction

Biomineralizations in plants are called phytoliths (Coe etal. 2014), and calcium

oxalate crystals (COC) are one of the most common (Metcalfe 1985). The calcium

absorbed from the environment through the roots is combined with oxalate (one of

the products of plant metabolism) to produce crystals of different morphologies

such as prisms, styloids, raphides, druses, and crystal sand that precipitate within

the vacuole of specic cells called idioblasts (Franceschi and Horner 1980;

Franceschi and Nakata 2005; Bauer etal. 2011).

COCs play essential structural and physiological functions (Franceschi and

Horner 1980; Franceschi and Nakata 2005). COCs serve as a Ca+2 sink and source,

preventing Ca accumulation and ensuring the normal cells functions, protect against

herbivory and chewing insects, and play structural functions (Ilarslan etal. 1997;

Prychid and Rudall 1999; Molano-Flores 2001; Braissant etal. 2004; Franceschi

and Nakata 2005; Korth etal. 2006; Bauer etal. 2011). They also have taxonomic

importance since their morphology and distribution in plant tissues and organs are

characteristic of taxa (Franceschi and Horner 1980; Franceschi and Nakata 2005;

Lersten and Horner 2008). Moreover, once in the soil, oxalotrophic bacteria pro-

mote COC oxidation because they function as a carbon, energy, and electron source,

inuencing the carbon and calcium cycle in soils (Braissant etal. 2004; Verrecchia

etal. 2006).

In spite of the biological, ecological and biogeochemical importance of COC in

plants, there are few researches about the COC description and quantication in

relation to the type of community and/or environment, especially in our country.

The aim of this work is to analyze and compare the data previously reported about

the presence and production of COC in leaves of plant species from forests, wet-

lands, and agroecosystems of the southeast of the Pampean Plain, Argentina.

32.2 Materials andMethods

32.2.1 Study Sites

The southeast of Buenos Aires province (38°12′ S, 57°48′ W) belongs to the geo-

morphological unit known as “Perinange aeolian hills,” which comprises a relief of

morphologically complex hills, with relative heights of up to 30m and concave–

convex proles with intermediate straight patches and slopes between 6% and 8%

(Osterrieth etal. 1998) (Fig.32.1). The hills originated from processes of primary

aeolian accumulation, modied later by supercial wash (Osterrieth and Martínez

1993). In this region, the climate is mesothermic and subhumid, with little or no

water deciency, including an annual precipitation of 809mm (Burgos and Vidal

1951). Los Padres Basin (37° 55′ 14.47″ and 38° 1′ 50.56″ S, 57° 52′ 21.96″ and

S. M. Altamirano et al.

305

57° 43′ 0.95″ W) is also located in the southeast of the Pampean region. Los Padres

wetland is a shallow permanent lake (maximum depth 2.4m) with an area of 216ha

bounded by the Tandilia Range, a block mountain system (Cionchi etal. 1982). The

main processes that produced the wetland were tectonic events and wind erosion

(Cionchi etal. 1982). The waterbody receives input from rainwater and groundwa-

ter and constitutes open systems recharge–discharge (Cionchi etal. 1982).

Grasslands were the pristine vegetation predominant in the study area across the

Quaternary (Cabrera 1976). Approximately 150years ago, because of the intense

agricultural and horticultural activity in the Pampean Plain, these native plant com-

munities had been replaced by crops, where the soybean is one of the most repre-

sentative (Aizen etal. 2009). Also, at about 50years ago, a process of articial

forestation with the aim of creating recreation areas generated the introduction of

forest species like Eucalyptus sp., Pinus sp., Cupressus sp., and Acacia sp., among

others.

Fig. 32.1 (a) Location of the study area. (b) Topographic prole of the study sites (Line A–B in a)

32 Calcium Oxalate Crystals inPlant Communities oftheSoutheast ofthePampean…

306

32.2.2 Sample Units

Terrestrial and aquatic vegetation was collected from these environments:

(a) Forest mainly composed of Acacia melanoxylon R. Brown (Fabaceae:

Mimosoideae) and Eucalyptus globulus Labill (Myrtaceae), both associated

with Celtis ehrenbergiana (Klotzsch) Liebm. (Celtidaceae), a native species.

(b) Agroecosystem with soybean (Glycine max L., Fabaceae: Faboideae) crop.

roides (Mart.) Griseb. (Amaranthaceae), Ludwigia peploides (Kunth)

P.H.Raven (Onagraceae), Polygonum hydropiperoides Michx. (Polygonaceae),

Rumex crispus L. (Polygonaceae), Hydrocotyle bonariensis Lam. (Apiaceae),

Typha latifolia L. (Typhaceae).

32.2.3 Description andQuantication ofCalcium Oxalate

Crystals

For each species, leaves from at least two plants at owering or fruiting stage were

sampled, washed with distilled water, and cleaned with an ultrasound bath (Test-

Lab, TBC 10 model) in order to remove any adhered material. Afterward, tissue

clarication (Dizeo de Strittmater 1973) and cross-sectioning were applied. The

material was mounted with gelatine–glycerine, and calcium oxalate crystals were

identied and described with a petrographic (Olimpus BX 51P) and optical micro-

scope (Leitz Wetzlar D35780) at 400× magnication. Photographs were taken with

a Kodak EasyShare CX7530 digital camera.

Average crystals size was calculated from the measuring of about 10–90 crystals

per species, depending on the COC production of the species. Crystal density (n°

crystals/mm2) was determined within an area of 0.196mm2 in the claried leaf sam-

ples. Between ve and ten areas per leaf were analyzed according to the size of the

leaves.

32.3 Results andDiscussion

COC were mainly located in parenchyma tissue and randomly distributed in the

mesophyll (Fig. 32.2a, f–j). Moreover, in terrestrial species (A. melanoxylon, E.

globulus, C. ehrenbergiana, G. max, H. bonariensis), COC were associated to vas-

cular bundles (Fig.32.2a–e), whereas in some aquatic species (T. latifolia, R. cris-

pus) COC were also distributed in the cells around the air spaces of aerenchyma

tissue (Fig.32.2k) (Graciotto Silva-Brambilla and Moscheta 2001; Jáuregui-Zúñiga

etal. 2003; Cervantes-Martínez etal. 2005; Torres Boeger etal. 2007; Borrelli etal.

2009, 2011, 2016). These COC distribution patterns could be related with their

S. M. Altamirano et al.

307

Fig. 32.2 Calcium oxalate crystals in leaves of the terrestrial and aquatic species analyzed. Druses

in the mesophyll and prisms associated to vascular bundles in E. globulus (a). Prisms associated

with vascular bundles in E. globulus (b), A. melanoxylon (c), and G. max (d–e). Druses and raph-

ides in the mesophyll of L. peploides (f–g). Druses in the mesophyll of P. hydropiperoides (h–i)

and R. crispus (j). Druses in the aerenchyma of R. crispus (k). Raphides in T. latifolia (l). vb:

vascular bundles. Arrows: COC.Scale bars: 10μm (a–e, g, i), 100μm (f, h, j–k)

32 Calcium Oxalate Crystals inPlant Communities oftheSoutheast ofthePampean…

308

different functions. The sequestration of Ca+2 in the COCs lets the normal function-

ing of chlorenchyma cells and could be also involved in the diffraction of light

improving the photosynthesis process (Franceschi and Nakata 2005; Horner 2012).

Moreover, the presence of COCs closely associated with vascular bundles in ter-

restrial species could be explained by their higher transpiration rate. As Ca+2 is dis-

tributed along the entire plant via xylem, it is possible that the precipitation of

biomineralizations in these areas prevents the mobilization of calcium excess

through cells (Prychid and Rudall 1999; Franceschi and Nakata 2005; Lersten and

Horner 2008; Gilliham etal. 2011). On the other hand, the differential COC distri-

bution in leaf tissues between terrestrial and aquatic species could be explained by

the reduced xylem system and the important aerenchyma tissue characteristic of

aquatic plants (Fahn 1990). In addition to the structural function of COCs in the

aerenchyma (Kuo-Huang etal. 1994; Prychid and Rudall 1999), the presence of

calcium could increase cell wall plasticity around air spaces (Kausch and Horner

1983).

As it was previously reported, there are differences about COC morphologies

between species (Franceschi and Horner 1980; Franceschi and Nakata 2005;

Lersten and Horner 2008). Terrestrial species produced druses (E. globulus,

C. ehrenbergiana, H. bonariensis) and prisms (E. globulus, A. melanoxylon,

G. max) (Fig. 32.2a–e, Table 32.1) (O’Connell et al. 1983; Ilarslan et al. 1997;

Borrelli et al. 2009, 2016; He et al. 2012, 2013), while aquatic plants produced

druses (R. crispus, P. hydropiperoides, L. peploides, A. philoxeroides) and raphides

(T. latifolia, L. peploides) (Fig.32.2f–l, Table32.1) (Kausch and Horner 1983; Kuo-

Huang et al. 1994; Prychid and Rudall 1999; Graciotto Silva-Brambilla and

Moscheta 2001; Lytle 2003; Duarte and Debur 2004; Borrelli etal. 2011). This dif-

ferential production of morphologies is related to taxonomical characteristics, the

types of cells where crystals precipitate and their function (Franceschi and Nakata

2005; Borchert 1984).

Table 32.1 Size and density of COC in the analyzed species

Plant species

Average COC size (μm)

Average COC density (N°

crystals/mm2)

Druses Prisms Raphides Druses Prisms Raphides

Terrestrial

vegetation

Acacia melanoxylon 12 283

Eucalyptus globulus 15 120

Celtis

ehrenbergiana

9 243

Glycine max 14 340

Hydrocotyle

bonariensis

9 247

Aquatic

vegetation

Ludwigia peploides 45 210 32 7

Polygonum

hydropiperoides

58 38

Rumex crispus 37 14

Typha latifolia 76 20

S. M. Altamirano et al.

309

Differences were also observed in relation with crystals size and density

(Table32.1). Generally, in terrestrial species, average crystal size wassmaller than

aquatic species (12 and 80μm, respectively), but average crystal density washigher

(246 and 23 crystals/mm2, respectively) (Table 32.1). This trend could be related

with the differential transpiration rates between aquatic and terrestrial species,

among others.

In summary, calcium is an important macronutrient necessary for the normal

development of plants. COC production let the different species to regulate Ca+2

concentration in cells along with many other physiological and structural functions

that enhance the normal cell functioning. Differences in terrestrial and aquatic envi-

ronments inuence the COC production and distribution among leaf tissues.

Acknowledgments This work was supported by the Agencia Nacional de Promoción Cientíca

y Tecnológica (PICT 1583/2013), the Universidad Nacional de Mar del Plata (EXA 741/15), and

CONICET PIP (11220130100145CO).

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

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the copyright holder.

32 Calcium Oxalate Crystals inPlant Communities oftheSoutheast ofthePampean…

313

Chapter 33

Iron andCalcium Biomineralizations

inthePampean Coastal Plains, Argentina:

Their Role intheEnvironmental

Reconstruction oftheHolocene

MargaritaOsterrieth, CeliaFrayssinet, andLucreciaFrayssinet

Abstract Biomineralizations are biogenic composites, crystalline or amorphous,

produced by the metabolic activity of organisms distributed all over the world. The

aim of this work was to evaluate the presence of iron and calcium biomineraliza-

tions and their inuence in the physicochemical and mineralochemical variations in

paleo and actual pedosedimentary sequences of the coastal plains in Mar Chiquita.

The complex interaction of calcium with iron biomineralizations, as framboidal and

poliframboidal pyrites associated with gypsum, barite, calcite, halite, and iron oxy-

hydroxides, have demonstrated the active and complex biogeochemistry that occurs

in the temperate–wet paleoesturaries and estuaries of the coastal Pampean Plains.

Particularly the consequences that different human activities could have, such as the

possible acidication processes as result of the iron sulde oxidation.

Keywords Framboidal pyrite · Poliframboidal pyrite · Calcium oxalate · Calcite ·

Biogeochemistry · Soil acidication

33.1 Introduction

Biominerals are deposited in intra- or extracellular spaces as the consequence of

metabolic activity. Biomineralizations processes are genetically controlled and are

also a widespread phenomenon in nature that can take place on both marine and

terrestrial systems, acting as a global source and sink of soluble ions (Lowenstam

1981; Osterrieth 2004). Among the most common biomineralizations can be men-

tioned those ones composed by calcium, iron, and amorphous silica (Mann 2001),

M. Osterrieth (*) · C. Frayssinet · L. Frayssinet

Instituto de Geología de Costas y del Cuaternario (IGCyC), FCEyN, UNMdP– CIC,

Mar del Plata, Argentina

Instituto de Investigaciones Marinas y Costeras (IIMyC), CONICET– UNMdP,

Mar del Plata, Argentina

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_33

314

particularly in coastal environments, where the interaction between marine and ter-

restrial processes is very intense, as well as the biotic and anthropic activity.

The geomorphological and biotic factors in concordance with the pedological

processeshave created a complex soilsystem, where the active iron and calcium

biogeochemistry might allow the acidication processes, as result of the iron sulde

oxidation, when biogenic calcium is available (Osterrieth etal. 2017).

In Argentina, the rst record about framboidal pyrites biomineralizations associ-

ated with paleo-salt marshes environments dates from 1992 (Osterrieth 1992), with-

out further information, while in other regions, there is a large number of reports

about the pyrites formation and its biogeochemical implications (Stribling 1997;

Wilkin etal. 1996; Roychoudhury etal. 2003).

The research of calcium biomineralizations at pedesodimentary sequences asso-

ciated to bioclastic levels and bioerosion processes is very scarce in Argentina

(Osterrieth etal. 2000, 2016, 2017).

In the Pampean plains, coastal morphodynamics throughout the Holocene

(10,000 BP until now) have been very active and have produced variations linked to

the installation of coastal barriers, conditioning the evolution of dunes, marshes,

and the unique coastal lagoon of Argentina: Mar Chiquita lagoon (Osterrieth 2005).

The aim of this work was to evaluate the presence of iron and calcium biominer-

alizations and their inuence in the physicochemical and mineralochemical varia-

tions in paleo and actual pedosedimentary sequences of the coastal plains in Mar

Chiquita, Argentina.

33.2 Materials andMethods

33.2.1 Study Site

Study site is located in the coastal area of Mar Chiquita, Buenos Aires province

(Fig.33.1).

The regional weather is mesothermic and subhumid. The vegetation is character-

ized by communities of psammophytic, halophytic, and freshwater plants, as well as

by woodlands. The main soils at the study area are Udolls and Aquents (Osterrieth

2005).

33.2.2 Methods

The soils and exhumed soils were analyzed across a coastal section of 10km length

(Fig.33.1); description of the modal proles was reported according Soil Survey

Staff (1996). Different pedosedimentary sequences of the Holocene present in geo-

morphological units (UG): paleoestuarine (UG1) and estuarine-salt marsh (UG2)

were analyzed (Fig.33.1a, b).

M. Osterrieth et al.

315

Dating by 14C acceleration mass spectrometry (AMS) was performed (Beta

Analytic, USA; LATIR-UNLP) on soil organic matter and bioclastic material.

Organic matter content, pH, calcium carbonate, and particle size distribution

were done by routine methods (Walkley and Black 1965; Dawis 1970; Galehouse

1971, respectively).

Disturbed and undisturbed samples at different scales of resolution were ana-

lyzed using polarization microscope (Olympus BX 51P) and scanning electron

microscope (SEM: JEOL JSM6460LV). Mineralochemical studies were performed

using an energy dispersive X-ray spectrometer (EDXS, between 15 and 25kV).

33.3 Results andDiscussion

Mar Chiquita region is mostly included in the Biosphere Reserve (Man and

Biosphere Reserve Program, UNESCO) called “Parque Atlántico Mar Chiquita”

(UNESCO 2016). This reserve is a coastal ecosystem with specic organism diver-

sity and, affected by complex biogeochemical processes, is a shallow body of brack-

ish water affected by low amplitude tides and constitutes an estuarine environment

with a very particular behavior (Marcovecchio etal. 2006). The morphodynamics of

this area is unique as marine, estuarine, and eolian deposits interdigitate and inter-

calate between them (Osterrieth 2005).

33.3.1 Paleoestuarine: Exhumed Soils (UG1)

33.3.1.1 Morphological andPhysicochemical Characteristics

The exhumed soils were affected by an active erosion and showed diagenetic and

biogeochemical specic characteristics associated with ancient coastal deposits,

linked to the last Holocene transgressive–regressive cycle (Fig.33.2a, b, d-1). Soils

Fig. 33.1 Study area. Toposequence, geopedological units (UG1, UG2), and proles studies

(P1–P2)

33 Iron andCalcium Biomineralizations inthePampean Coastal Plains, Argentina…

316

were characterized as Hapludolls (2A-2Cb horizons), Sulfaquents (3Ab and

4ACkgb-4Cgkb horizons), and Fluvaquents (5Cgkb horizons), all of them with low

to moderated pedological development (Fig.33.2d-1).

At the subsupercial horizons, pH values were alkaline (8–9), as consequence of

the saline and/or brackish water inuence, with high exchangeable sodium contents

and with abundant bioclastic materials that were affected by processes of bioerosion

and calcium carbonates and oxalates reprecipitation (Fig.33.2k) (Osterrieth 2005).

The supercial horizon was slightly alkaline to neutral (7–8) due to the high organic

matter content. The grain size distribution showed a sandy texture in 2A level,

changing to silt loam towards the bottomof the prole. In all the exhumed soils, the

clay content was very scarce (Fig.33.2d).

Fig. 33.2 (a) Panoramic view of the exhumed soils (UG 1). (b) Photo of del modal prole. (c)

Micromorphological photo (3Ab). (d) Geological prole, grain size analysis, and biomineraliza-

tions present in the prole. (e) Framboidal pyrites and gypsum in the soil matrix. (f) Framboidal

pyrites, gypsum, and barite on vegetal debris, (g) associated with bioclast. (h) Detailed view of

framboidal pyrites. (i) Tubes calcium biomineralizations (CB) in matrix soil. (j) EDXS result of

soil matrix, in the prole, (k) bioclast bioeroded. (l) CB on clast and matrix exhumed soil. BC:

bioclasts. Yellow arrow: framboidal pyrites (FP). e–l: SEM photos

M. Osterrieth et al.

317

The organic matter content was medium (between 6% and 10%) and has

decreased with depth.

Carbonates were present in all the prole, as whole or fragmented shells, scat-

tered in the soil mass. The calcium carbonate content was variable and increased

toward the lower horizons, with an average between 6% in 2A and 15% in 2Cb, 3Ab

horizons, but rising to more than 35% in paleosols (level 4 and 5), associated with

bioclastic materials.

33.3.1.2 Iron andCalcium Biomineralizations

Previous research on exhumed paleo marshes have determined that the iron content

was between 56 and 95μmol Fe/g and have reected that the largest iron proportion

was in the form of crystalline iron oxides (28–76%) and lepidocrocite (6–16%),

while the proportion associated with ferrihydrite and pyrite was lower (0–9% and

1–17%, respectively), especially at the upper levels of the exhumed soils (2A and

3Ab in Fig.33.2d-1). According with the chemical fractionation data on these hori-

zons, well dened framboidal pyrites were found associated with the plant debris

cover (Fig.33.2d-3, j). These pyrites increased in quantity and morphological vari-

ety toward the bottom of the prole, where the anoxic conditions were more resis-

tant (Osterrieth etal. 2016).

The presence of framboidal and poliframboidal pyrites associated with gypsum,

barite, calcite, halite (Fig. 33.2c, e–h), and iron oxyhydroxides was frequent all

along the prole. The sequences of pyrite formation (suldation) as well as their

metastable forms (mackinawite and greigite) were observed. Framboidal pyrites

showed diverse morphologies: combined microcrystals, octahedral–dodecahedral

subedrals, and cubic and irregular octahedral microcrystals subedrals to anedrals

with sizes between 0.2 and 5μm, closely associated with plant debris, bioclasts, and

pores and channels of the soil matrix (Fig.33.2g, h). Degradation processes (sulfu-

rization) were inferred by geochemical and EDXS results, as presence of crystalline

iron oxides, lepidocrocite, and ferrihydrite were observed immersed in the matrix of

the peds. This sulfurization was related to the complex redox processes of these

environments, such as the aeration that is generated in the rhizosphere and also by

the intense bioturbation of invertebrates (Osterrieth etal. 2016). Once the sequence

was exposed to oxic conditions and eroded by storm episodes, the oxidation of sul-

des and the intense dissolution and transformation of framboidal pyrite into iron

crystalline phases could have been the responsible for the acidic conditions, mainly

in the surface horizons (Roychoudhury etal. 2003)

The association of framboidal pyrites have originated elongated and subspherical

poliframboidal pyrites, with sizes of 50–80μm wide and 190–140μm length. They

were immersed and cemented by thin lms of amorphous silica and iron (Fig.33.2c).

These secondary framboidal pyrites were authigenic, generated “in situ” under anoxic

conditions (Osterrieth 1992). The framboidal pyrite genesis in these coastal soils of

the Pampean Plain was bacterial, through biogeochemical reduction processes that

33 Iron andCalcium Biomineralizations inthePampean Coastal Plains, Argentina…

318

have produced a certain type of microcrystals, very different from those generated by

purely chemical action (Osterrieth etal. 2016; Wilkin etal. 1996).

A lot of isolated octahedral and dodecahedral pyrite’s crystals associated with

framboids and with different types of biolms, mainly composed by silica were

observed. These biolms might be related to the high amounts of diatoms and sili-

cophytoliths found that could have been altered by the extreme pH values conse-

quence of the framboidal pyrites generation and degradation cycle.

These paleoestuarine deposits, with abundant wholly and partially fragmented

bioclasts, included mollusk shells such as Heleobia sp., dated 1,670 ± 70year BP

by 14C, and Tagelus plebeius dated between 1,710 ± 60 and 2,880 ± 90year BP

(Osterrieth, 2005). Also, soil organic carbon was dated, ranging from <200year BP

to486 ± 60year BP.

This bioclastic material constituted by calcite/aragonite biomineralizations was

affected by dissolution, bioerosion (Fig.33.2k), and reprecipitation of biogenic cal-

cium carbonates and oxalates (bacteria, fungi, and algae). The reprecipitation pro-

cesses were relevant on the bioclasts (Fig.33.2i, l) and in the matrix between grains

and bioclasts (Fig. 33.2l). The matrix of the peds had dense calcied laments,

raphides, styloids, tubes, rods, and short and long needles with sharp or straight

ends, composed by whewellite (monohydrate calcium oxalate) or weddellite (poly-

hydrated calcium oxalate) (Fig.33.2i, l) (Verrecchia and Verrecchia 1994, Verecchia

etal. 1995; Osterrieth etal. 2000, 2017). In general, all the morphologies found here

could be associated with microbial activity at the matrix, coating peds, pores, and

the bioclastic-matrix interface (Fig.33.2i, l). Moreover, it was common to see fram-

boidal pyrite associated to bioclasts, being part of a complex biolm composition

dominated by calcium (41%), chlorine (29%), silica (13%), carbon (5%), iron (5%),

sodium (4%), and aluminum (3%) (Fig.33.2j).

33.3.2 Estuarine Actual Soils (UG2)

33.3.2.1 Morphological andPhysicochemical Characteristics

The soils (Prole 2) were located within the marginal plain and the tidal channels of

the Mar Chiquita lagoon (Fig.33.1b) affected by the lagoon’s level variation and

also by the episodic sea advances that had increased the presence of iron suldes in

the surface. These soils classied as oxyaquic Udiuvents (Fig.33.3b) were affected

by the presence of mollusk shells and intensely bioturbated by Uca uruguayensis

and Neohelice granulate (Fig.33.3).

The vegetation community was composed of Cortaderia selloana, Panicum rac-

emosum, Sacocornia sp., Juncus sp., etc. (Fig.33.3a, b).

M. Osterrieth et al.

319

Soil reaction ranged from moderately alkaline in the surface to alkaline at the

base of the sequence (pH:7.5–9). The organic matter content was moderate in the

AC horizon (6.5%) and has descended sharply toward the bottom of the prole

(1.3%). The calcium carbonate content was scarce (6%) in the supercial horizon

(AC) and increased at the lower horizons (3Cgk and 4Cgkb horizon) associated with

the presence of bioclastic materials (33%).

The sequence was texturally homogeneous with ne and very ne sand fractions

being dominant, while the AC horizon showed a slight increase in clays and silts

(Fig.33.3d-2).

Fig. 33.3 (a) Panoramic view of the UG2. (b) Photo of del modal prole (P2). (c)

Micromorphological slide (AC). (d) Geological prole, grain size analysis, and biomineralizations

in the prole. (e) Framboidal pyrites in and isolates pyrite crystals in the marsh soil matrix. (f)

Detailed view of framboidal pyrites. (g) Heleobia sp. bioeroded; (h) SEM, soil matrix with CB; (i)

matrix with laments, rods CB; (j) detailed view of CB: rods, raphides, and styloids associated

with the bacterium; (k) detailed view of rosettes CB associated with fungi. Yellow arrow: framboi-

dal pyrites (FP). (e–k): SEM photos

33 Iron andCalcium Biomineralizations inthePampean Coastal Plains, Argentina…

320

33.3.2.2 Iron andCalcium Biomineralizations

In these soils the presence of iron and calcium biomineralizations was common too,

but the framboidal and poliframboidal pyrites were less abundant when compared

to the paleoesturine soils. The quantity and morphologies of the calcium biominer-

alizations observed in this prole were similar to those registered in UG1.

The low degree of pyritization registered here, which is below the typical values

reported for modern estuarine sediments of tropical environments (Wilkin et al.

1996), could be explained by the lower proportion of iron obtained here (36–75μmol

Fe/g.) (Osterrieth etal. 2016). The sequences of pyrite formation (suldation) were

observed with diverse morphologies, for example, octahedral–dodecahedral micro-

crystals and irregular octahedral microcrystals subedrals to anedrals (Fig.33.3c, f).

The presence of gypsum or barite associated to framboidal pyrites was very scarce,

although the oxic conditions were more common in this soils than in the

paleomarisms.

In these soils, the aeration generated by the high density of crab burrows and S.

densiora has promoted the oxic conditions that leaded to a lower pyrite framboidal

production (Osterrieth etal. 2016).

Bioclasts were affected by bioerosion through the action of microorganisms with

a subsequently calcium reprecipitation as secondary oxalates and carbonates

biomineralizations (Fig. 33.3g–k). These biomineralizations were also added or

weakly bound to the skeletal components, which allowed them to be incorporated

into the matrix of soils and sediments. Mineralochemical studies conrmed the

presence of calcium and variable carbon contents, subject to the crystal’s develop-

ment stage. Thus, it was possible to dene the genetic sequence of calcite via cal-

cium oxalate (weddellite and whewellite) associated with hyphae, algae, soil

bacteria, and actinomycetes. A remarkable variety of dichotomous tubes was found

as clear evidence of fungal genesis, added to elongated tubes of multiple sizes and

diameters, and complex interdigitated crystalline textures of oxalate and calcium

carbonates, that also indicated biological production (Fig.33.3g–k). Type and diver-

sity of calcium biomineralizations increased directly in relation with the bioclastic

parental material and biogeochemical conditions (Fig.33.3d-3).

33.4 Final Remarks

The important role of the iron and calcium biomineralizations lies on the possibility

to further understand the biogeochemical processes and to deepen the knowledge

about the complex interaction of the pedological processes on the paleo and recent

coastal environments. They also allow us to deduce that many environmental pro-

cesses do not correlate with chronological evolution, while biogeochemical aspects

related to biomineralizations do.

The presence of iron biominerals allowed us to dene the redoxymorphic condi-

tions in paleoestuarine and estuarine pedosedimentary sequences.

M. Osterrieth et al.

321

Calcium biomineralizations were found and associated with processes of disso-

lution and reprecipitation of calcium carbonates and oxalates associated to fungus,

algae, and bacteria in actual and exhumed soils.

Calcium and iron biomineralizations with other cations and organic components

allowed the formation of organomineral complex which plays an important role in

the availability of macro- and micronutrients for the biota development and in the

persistence of the aggregates and the resistance to erosion processes in these coastal

soils.

Iron and calcium biomineralizations found in paleo marshes allow us to make

inferences related to the management of actual salt marshes, warning us about the

possible acidication processesgenerated by the iron sulde oxidation associated to

different human activities in the temperate–wet coastal environments of the Pampean

Plains.

Acknowledgments This work was supported by the AGENCIA-PICT 1583/2013, UNMDP-

EXA 741/15, and CONICET- PIP-11220130100145CO.

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coastal areas of the Pampean plain, Argentina. Environ Earth Sci 75:671–672

Osterrieth M, et al. (2017) Calcium Biomineralizations associated with bioclastic deposits in

coastal pedostratigraphic sequences of the Southeastern Pampean Plain, Argentina. In: Rabassa

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

M. Osterrieth et al.

Part VIII

Mollusk Shell Formation

325

Chapter 34

Skeletal Organic Matrices inMolluscs:

Origin, Evolution, Diagenesis

FrédéricMarin, AurélienChmiel, TakeshiTakeuchi, IrinaBundeleva,

ChristopheDurlet, EliasSamankassou, andDavorinMedakovic

Abstract The mollusc shell comprises a small amount of organic macromolecules,

mostly proteins and polysaccharides, which, all together, constitute the skeletal

organic matrix (SOM). In the recent years, the study of the SOM of about two doz-

ens of mollusc species via transcriptomics and/or proteomics has led to the identi-

cation of hundreds of shell-associated proteins. This rapidly growing set of data

allows several comparisons, shedding light on similarities and differences at the

primary structure level and on some peculiar evolutionary mechanisms that may

have affected SOM proteins. In addition, it constitutes a prerequisite for investigat-

ing the SOM repertoires of sub-fossils or fossil specimens, closely related to known

extant species, in order to revisit diagenetic processes, i.e. how SOM proteins

degrade during fossilization. These two aspects are briey exemplied here: on the

one hand, Aplysia californica, the sea hare, exhibits a vestigial internal shell that has

kept a proteomic signature similar to that found in fully functional external shells.

On the other hand, subfossil specimens of the giant clam Tridacna, collected in

French Polynesia, precisely dated and analysed by proteomics for their SOM con-

tent, comprise several preserved proteins that can still be identied by their peptide

signature, in spite of information losses likely due to diagenetic transformations.

F. Marin (*) · A. Chmiel · I. Bundeleva · C. Durlet

UMR CNRS 6282 Biogéosciences, Bâtiment des Sciences Gabriel,

Université de Bourgogne Franche-Comté, Dijon, France

e-mail: frederic.marin@u-bourgogne.fr; irina.bundeleva@u-bourgogne.fr;

christophe.durlet@u-bourgogne.fr

T. Takeuchi

Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University,

Okinawa, Japan

E. Samankassou

Department of Earth Sciences, University of Geneva, Geneva, Switzerland

e-mail: Elias.Samankassou@unige.ch

D. Medakovic

Center for Marine Research Rovinj, Rudjer Boskovic Institute, Rovinj, Croatia

e-mail: medakovic@cim.irb.hr

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_34

326

Keywords Mollusc · Shell · Proteomics · Protein · Sequences · Functional

domains · Diagenesis

34.1 Introduction

The mollusc shell is a remarkable composite material made of calcium carbonate at

99% and of a minor organic fraction, the skeletal organic matrix (SOM). During the

mineral deposition process, the SOM is secreted by the shell-forming organ, the

mantle, and remains occluded. It is considered to be the main regulator of crystal-

lization. In addition, it exhibits an interesting potential for preservation in fossil or

subfossil samples (Hare etal. 1980). Classical biochemical characterizations indi-

cate that the SOM consists in a mixture of proteins and polysaccharides (Marin

etal. 2012). For decades, the SOM was considered as a ‘black box’ and analysed

biochemically in bulk. Nowadays, high-throughput screening of SOMs, via the

combined use of proteomics and transcriptomics, has allowed the identication of

hundreds of shell proteins that are putatively involved in shell biosynthesis, in about

two dozens of model mollusc genera comprising mostly bivalves, gastropods, and,

in a lesser extent, cephalopods (Marin etal. 2016). This wealth of molecular data

has considerably blurred the outlines of the SOM. However, taken individually,

each of these ‘shell repertoires’ represents a key component of the calcifying

machinery for making a shell, and shell repertoires can be compared to each other,

shedding light on the macroevolution of calcifying matrices (Kocot et al. 2016;

Marie etal. 2017). In addition to giving information on the calcication process and

its macroevolution, molecular data collected from shells of extant molluscs is a

prerequisite for obtaining– whenever possible– similar proteins in archaeological

or fossil shell samples, an emerging eld dened as paleoproteomics (Demarchi

etal. 2016; Wallace and Schiffbauer 2016).

In the present paper, we briey describetwo unpublished examples on the use of

proteomics to identify shell proteins: the rst example relates to the Californian sea

hare, Aplysia californica, a heterobranch gastropod that belong to a family, the

Aplysiidae, characterized by an internal atrophied shell which is weakly calcied.

The second example relates to subfossil specimens of the giant clam, Tridacna sp.,

collected in French Polynesia and precisely dated. In both cases, proteomics was

performed from their extracted SOM.

34.2 Materials andMethods

34.2.1 Materials

Fresh shells of the Californian sea hare Aplysia californica were obtained from

RSMAS at the University of Miami (Ph. Gillette) after sacrice of the living ani-

mals according to ethical rules. Six shells of the giant clam Tridacna sp., including

fresh, recent and subfossil specimens, were collected by one of us (E.S) on two

F. Marin et al.

327

sites of French Polynesia: Motu Piti Aau, Motu Mute, Bora Bora (Society Islands)

and Motu Tepapuri, Mangareva (Gambier archipelago). The fresh Tridacna speci-

mens were used as reference material.

34.2.2 Structural andGeochemical Characterizations

Series of structural characterizations of the shells were performed by SEM observa-

tions on polished sections or on freshly broken shell pieces that were slightly etched

(EDTA 1% wt/vol, 3–5min.). Minute fragments were sampled and powdered and

the powder analysed by FT-IR spectroscopy, in order to check the mineralogy (ara-

gonite). For Tridacna samples, thick sections were made for cathodoluminescence,

epiuorescence and XRD analyses. In addition, the subfossil samples were dated

via 14C measurements (Beta Analytic, Miami, FL, USA).

34.2.3 Extraction oftheAplysia andTridacna SOMs

All Tridacna shells were scrupulously abraded and cleaned (two or three extended

bleaching treatments with sodium hypochlorite) in order to remove putative con-

taminants. The clean powders were decalcied overnight with acetic acid, and the

soluble and insoluble fractions were fractionated by centrifugation. All the subse-

quent steps leading to freeze-dried matrices were performed as previously described

(Ramos-Silva etal. 2014). For Aplysia californica, the thinness of shells required

adapting the cleaning/extraction protocol, which consisted rst in protein desorp-

tion in successive bathes of TBS buffer containing Tween 20 (0.1%), NaN3 (0.001%),

pH 9.2 for 1week. After drying and reduction into powder, the samples were decal-

cied and centrifuged, leading to the fractionation of the soluble and insoluble

matrices. The soluble fraction was desalted by several centrifugations/resuspension

in water, in Vivaspin 20 cells (3kDa cutoff), while the insoluble was rinsed with

water. Both fractions were freeze-dried.

34.2.4 Proteomics onSOMs

All lyophilized samples were submitted to proteomic analysis (3P5 platform,

Institut Cochin, Université Paris Descartes, Paris), after trypsic digestion, as previ-

ously described (Immel etal. 2016). For Aplysia californica, assigning identied

peptides to known proteins was performed by using Mascot program (version 2.5,

MatrixScience, London, UK) against the nonredundant NCBInr database. The

search was restricted to ‘Other Metazoa’ dataset, which comprises a large collection

of transcriptomic and genomic sequences from Aplysia californica, publicly

34 Skeletal Organic Matrices inMolluscs: Origin, Evolution, Diagenesis

328

accessible at NCBI (www.ncbi.nlm.nih.gov/). For Tridacna sp., an unpublished

EST database provided by one of us (Dr. T.Takeuchi) from mantle tissues of the

coral reef-associated crocus giant clam Tridacna crocea was used for protein

identication.

34.3 Results

34.3.1 Proteomics onAplysia californica Shell Matrices

The internal shell of Aplysia californica is lightly calcied, chitinous, made of ara-

gonite of the crossed-lamellar type (see Fig.34.1a, b). It was submitted to a com-

plete structural, chemical, biochemical and proteomic characterization that will be

the subject of an extended publication in preparation. In the present paper, we sim-

ply summarize few of the outcomes obtained by proteomics. Our investigations

generated several hits with proteins– known or unknown– from Aplysia califor-

nica. In total, we obtained 40 hits with proteins identied by more than two peptides

and several additional hits with proteins identied with one peptide. We classied

the protein hits according to the similarity of each of their primary structure to

known functional domains or domains with a peculiar signature in terms of amino

acid composition. As shown in Fig. 34.1c, we obtained six categories: enzymes,

protease inhibitors, ECM/ECM-like (extracellular matrix), cation-interacting pro-

teins, proteins containing LCDs/RLCDs (repetitive low complexity domains). The

last category (others) comprises proteins that cannot be included in the ve others.

It is to note that the heterogeneous class of proteins containing LCDs/RLCDs rep-

resents the biggest group of shell proteins. It contains hydrophobic proteins in addi-

tion to P-rich, D-rich and S-rich proteins.

34.3.2 Proteomics onSubfossil Tridacna Samples

fromFrench Polynesia

The subfossil samples of the giant clam Tridacna sp. were carefully checked for

their preservation state, taking the fresh shells as reference. In particular, micro-

samplings made across the thickness of the shell to identify the mineralogy via

FT-IR spectroscopy showed that all shells were fully aragonitic and not recrystal-

lized (data not shown). All of them exhibited the classical crossed-lamellar micro-

structure. In the subfossil shells, we however saw important alterations and

perforations in their outermost and innermost layers (which were subsequently dis-

carded) while the core layer was intact.

Proteomic investigations performed on the fresh shells generated up to 134 pro-

tein hits, 46 of which corresponding to proteins identied by at least two peptides.

F. Marin et al.

329

For subfossil shells, these numbers decreased drastically. For example, the GAM-14

sample, the age of which was precisely determined at 2880 ± 30 BP (before pres-

ent), exhibited a total of 40 hits, but only 4 of them correspond to proteins identied

by at least 2 peptides. Figure 34.2 shows an example of a new LCD-containing

protein (P-rich), unambiguously identied in the fresh Tridacna sample, owing to

100µm

Organic

Aragonite

10 mm

FUNCTIONAL DOMAINSProtein name MW (kDa)p

ENZYMES Tyrosinase-3-lik

7.95.5 N

Hybrid signal transduction histidinekinaseA-like40.49.7 N

PROTEASE INHIBITORS CD109antigen-lik

34.7 7.

BPTI/Kunitz-domain containing protein4-like25.08.9 Y

ECM/ECM-like Collagen alpha-VI /BMPSM. edulis (partial)158.2 6.

CATION-INTERACTING Seductin(ependymin-related):C

0.25.1 N

Hephaestin-like: Fe/C

8.75.2 N

LCDs /RLCDs containing Proline-rich proteinHaeIIIsubfamily1-like19.412.

proteins Ser/Argrepetitivematrixprotein 1-like 88.1 9.

Fibroinheavy chain-like 71.0 10.9 N

Basicproline-richprotein-lik

3.74.8 N

Several uncharacterized proteins (>10)

(hydrophobic, D-rich,S-rich)

OTHERS Microtubule-associated futsch-like198.6 4.

Cytoplasmicintermediatefilamentprotein A65.15.5 N

Vacuolar protein-sortingassociatedprotein 36-like35.69.6 Y

Fig. 34.1 (a) The internal shell of Aplysia californica; the shell is lightly calcied. Top, apex

(posterior); bottom, anterior part, which corresponds to the non-calcied growing shell margin. (b)

Microstructure of the shell, observed in cross section; the dorsal side is on the right, the ventral, on

the left. (c) Abridged list of proteins identied in the SOM of A. californica by proteomics. LCDs/

RLCDs stands for low complexity domains/repetitive low complexity domains, ECM for extracel-

lular matrix. The columns on the right indicate their theoretical molecular weight (MW) in kDal-

tons, their calculated isoelectric point (pI) and the presence (Y) or absence (N) of signal peptides.

Signal peptides were identied by SignalP 4.1, while molecular weights and isoelectric points

were calculated by ProtParam (after removal of the signal peptides when present). Both tools are

accessible at http://www.expasy.ch/tools/. The fact that several secreted proteins do not exhibit a

signal peptide could indicate that some of the sequences are not complete

34 Skeletal Organic Matrices inMolluscs: Origin, Evolution, Diagenesis

330

ETMNKVILIVFSGLLAVQLVSAQSHTTWAAAQVPGLGRMTPPTTDYPEYMLHMAVG

EIMRAPTENKAAYAAAKVYNPVMDMSDKVQQALEDRVLQLRHPPGTPYYRRKLDFD

VMQLVIGAYYKTLNISAPQQLGSFYGPPPANHWAGASQPVGPPARQPGPLPPAGPP

AGPAMGPPTSIRRGFRPRAQGIYSPFEPTPWELDRAVQDIHMARTEKQAVKAAAGVH

RIGLDLADIVVNALEEKIARLRRPNWTGFRPPPIPRGLNVHGLVRHAFYEIQRIAQAKAA

ADAAAAAAAAAKAKKTPPPPTPRAGSKIPTTLPPTPPPKPYKKPRQPSKPNPPPSPKKT

KPPKRDFMTDFIQNRRKQRQPPPAPKLFKQAQITRPPFVQPVRRQTLNPFPTQPSVPP

YFEPTKITRPPYVQPQRRDPVDPFFSQPSYRPKPQVEPFRPPPNVPRPPKINWKKKAAA

KPVPTSVSLTEKGPTSISAASSNSNKSPVSYETIPSQNSAKPAFMKIPKPNVPPAPFRSEP

PKPKSLFPKGNSGRPSDIPKAVLSSNKGKGKRPSSKTVEPLFQSTETTPSPEEQQLFNRY

PGLFENKARMRVANLAQHRLETVGPSAEISKPPLKGPNPQPPKVSNKKMPVSKPPQQ

AAVKVAPKIIKPSKVWSPLGNLGSNINEILKFSIDGPSESVPIPTAAPLTTKAPTTTTKKPT

TTTPRKQEKVKPIRKTKVKRRRKVVSKAKSKSFAIKLAKKKPEKPKKPQGADKLTQLHKLL

EGISPSQLQTLVDLIKAKANEGKPKPLPKPEPLPKPKPIFAPPPPPGSEHKGPPREFRSQ

MQSSRSGPYRPNSDYGPPPDNRGPPPDWARGPGGRPRGPPGPPGPGGPGGPGMR

GGPDLSNPQIARLIRVMKQGGHPKNNFLSGRTPGSSAAAAGGEAPEAGEGPTGLLGN

PLMMSMLMNRGGQGGGGGIASLLGGGAAGGANPLAALMPGGAGGAGGGEGGM

NPAMLAAIMGGGQGGGGGGMGALGGGYGGMLAGLGL

ETMNKVILIVFSGLLAVQLVSAQSHTTWAAAQVPGLGRMTPPTTDYPEYMLHMAVG

EIMRAPTENKAAYAAAKVYNPVMDMSDKVQQALEDRVLQLRHPPGTPYYRRKLDFD

VMQLVIGAYYKTLNISAPQQLGSFYGPPPANHWAGASQPVGPPARQPGPLPPAGPP

AGPAMGPPTSIRRGFRPRAQGIYSPFEPTPWELDRAVQDIHMARTEKQAVKAAAGVH

RIGLDLADIVVNALEEKIARLRRPNWTGFRPPPIPRGLNVHGLVRHAFYEIQRIAQAKAA

ADAAAAAAAAAKAKKTPPPPTPRAGSKIPTTLPPTPPPKPYKKPRQPSKPNPPPSPKKT

KPPKRDFMTDFIQNRRKQRQPPPAPKLFKQAQITRPPFVQPVRRQTLNPFPTQPSVPP

YFEPTKITRPPYVQPQRRDPVDPFFSQPSYRPKPQVEPFRPPPNVPRPPKINWKKKAAA

KPVPTSVSLTEKGPTSISAASSNSNKSPVSYETIPSQNSAKPAFMKIPKPNVPPAPFRSEP

PKPKSLFPKGNSGRPSDIPKAVLSSNKGKGKRPSSKTVEPLFQSTETTPSPEEQQLFNRY

PGLFENKARMRVANLAQHRLETVGPSAEISKPPLKGPNPQPPKVSNKKMPVSKPPQQ

AAVKVAPKIIKPSKVWSPLGNLGSNINEILKFSIDGPSESVPIPTAAPLTTKAPTTTTKKPT

TTTPRKQEKVKPIRKTKVKRRRKVVSKAKSKSFAIKLAKKKPEKPKKPQGADKLTQLHKLL

EGISPSQLQTLVDLIKAKANEGKPKPLPKPEPLPKPKPIFAPPPPPGSEHKGPPREFRSQ

MQSSRSGPYRPNSDYGPPPDNRGPPPDWARGPGGRPRGPPGPPGPGGPGGPGMR

GGPDLSNPQIARLIRVMKQGGHPKNNFLSGRTPGSSAAAAGGEAPEAGEGPTGLLGN

PLMMSMLMNRGGQGGGGGIASLLGGGAAGGANPLAALMPGGAGGAGGGEGGM

NPAMLAAIMGGGQGGGGGGMGALGGGYGGMLAGLGL

TRINITY_DN253411_c2_g2_i3|m.459507

Proteinsequencecoverage: 38%

Proteinsequencecoverage: 4%

Fig. 34.2 Example of a novel protein identied in the fresh (a) and in the subfossil (b) Tridacna

sample. This protein is the translation of the sequenced transcript TRINITY_DN253411_c2_g2_

i3|m.459507. This protein is rich in proline (17.2%), glycine (10.1) and alanine (9.6%) residues

and its theoretical calculated pI is basic (10.63). Its function in biomineralization is unknown. In

grey italic, signal peptide. The peptides identied by proteomics are underlined. Note that the full

protein sequence is well covered (38%) in the case of the fresh Tridacna shell while the coverage

is poor (4%) for the subfossil shell. This drop may be explained by information losses at the pep-

tide level due to diagenetic transformations (hydrolysis, modication of chemical groups on amino

acids)

F. Marin et al.

331

good protein sequence coverage by peptides (38%, 17 peptides) all along the

sequence. In the subfossil sample, the percentage of coverage of this protein

sequence by peptides drops to 4% only (5 peptides), which suggests that the other

non-covered parts of the sequence may be submitted to diagenetic transformation

and/or hydrolysis. A complete view of all the results will be resumed in a publica-

tion in preparation.

34.4 Discussion

This paper illustrates how proteomics contributes to answer questions related to the

functions, evolution and diagenesis of SOMs in mollusc shell. In the rst case, we

explored the protein composition of the internal shell of the Californian sea hare,

Aplysia californica. Aplysiidae are usually considered as a very derived gastropod

family that has emerged only 25million years ago (Klussmann-Kolb 2004) during

the Oligocene epoch. This family is characterized by the presence of an atrophied

and weakly calcied internal shell, which has completely lost its primary function,

the protection of the soft tissues. In spite of this regressive evolution, it is remark-

able to observe that the shell of A. californica has conserved a protein repertoire that

exhibits a similar signature as the ones from fully functional external shells, in terms

of diversity of protein families present in the matrix.

The second example deals with the diagenetic processes that affect organic

matrices associated to calcium carbonate biominerals. In a previous paper, we

showed that articial diagenesis experiments performed on fresh nacre powder sam-

ples resulted in two phenomena recorded by proteomic analyses: a decrease of the

number of identied proteins correlated to harsh diagenetic conditions and, in paral-

lel, a decrease of the number of peptides identied per protein (Parker etal. 2015).

Our analyses performed on the SOMs of subfossil Tridacna tend to correlate this

nding. This example gives interesting perspectives for the coming time, i.e. the

possibility to track the diagenetic pathway of each shell protein, taken individually,

in sub-fossil/fossil of increasing age.

Acknowledgements This work was supported by NEWFELPROprogramme (Dr.D.Medakovic),

by funds from OIST (T.Takeuchi), by EC2CO project (I.Bundeleva, F.Marin) and by annualfunds

from UMR Biogeosciences (F.Marin).

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

F. Marin et al.

333

Chapter 35

Functional Analysis onShelk2 ofPacic

Oyster

JunTakahashi, ChiekoYamashita, KenjiKanasaki, andHaruhikoToyohara

Abstract Shelk2, a novel shell matrix protein from the Pacic oyster, Crassostrea

gigas, is reported to be involved in shell biosynthesis of the prismatic layer. Results

of RNAi experiment on shelk2 showed that Shelk2 has a key role in shell regenera-

tion. When dsRNA of shelk2 was injected into the adductor muscle of Pacic oyster,

the prismatic layer did not grow normally during shell regeneration. Observation of

regenerated shell using scanning electron microscopy (SEM) revealed that the size

of each column in the prismatic layer was reduced, and the edge of the column top

looked rounder. From these results, it was deduced that the columns were less

tightly bound with each other than in normally regenerated shells. Furthermore, the

surface of the column appeared to be rough. Unexpectedly, the expression level of

shelk2 mRNA was not reduced but remarkably enhanced by the knockdown experi-

ment. Further experiments including gene and protein expression will be necessary

for a better understanding of its function and role in oyster shell regeneration.

Keywords Biomineralization · Knockdown · Mollusk · Pacic oyster · Shelk2 ·

Shell · Silk-like protein

35.1 Introduction

Mollusk is the second largest metazoan taxon with many members possessing min-

eralized hard tissues formed as a result of biomineralization. The molluscanshell is

synthesized and maintained by the epithelial cells of the mantle, which is a specic

tissue present only in mollusks. Generally, the molluscan shell is composed of

>90% inorganic materials that mainly consist of CaCO3 and <10% organic matrices,

including polysaccharides and proteins. Various organic matrices play an important

J. Takahashi · H. Toyohara (*)

Graduate School of Agriculture, Kyoto University, Kyoto, Japan

e-mail: juntaka@kais.kyoto-u.ac.jp; toyohara@kais.kyoto-u.ac.jp

C. Yamashita · K. Kanasaki

Progress Co., Ltd, Osaka, Japan

e-mail: c-yamashita@progress-water.com; k-kanasaki@progress-water.com

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_35

334

role in the crystallization and/or framework formation of the shell, while most of

them reported so far do not share identity in their amino acid sequences among spe-

cies, with the exception of acidic proteins (Takahashi etal. 2013).

The identication of most organic matrix substances, including proteins, so far

has been accomplished by the decalcication of shells and subsequent extraction

with specic solutions (Marin etal. 2000). This conventional method is suitable for

the identication of relatively abundant proteins, but certain vital proteins cannot be

obtained because of their low solubility and/or instability in solution.

Instead of the shell itself, we focused on the mantle where the genes involved in

shell regeneration are expressed to identify essential proteins involved in shell bio-

synthesis. We have successfully cloned mantle edge-specic genes from Pacic

oyster, Crassostrea gigas, by means of a subtractive hybridization method, then

found two novel genes, shelk1 and shelk2 (Takahashi et al. 2012). The mRNA of

shelk2 was specically expressed in the outer fold of the mantle edge, suggesting

that it is possibly involved in the synthesis of the prismatic structure. In situ hybrid-

ization revealed gradual increase in shelk2 mRNA expression during shell regenera-

tion, suggesting the possible involvement of Shelk2in shell formation (Takahashi

etal. 2012).

Deduced amino acid sequences of both proteins were highly homologous to

those of arthropod silk broins (Hayashi and Lewis 1998; Hinman and Lewis 1992).

Interestingly, tandem repeats of poly-alanine (poly-Ala) motifs were identied in

the amino acid sequence of Shelk2 of C. gigas. Poly-Ala motifs have also been

reported in silk broins of arthropods (Guerette etal. 1996) and two shell matrix

proteins of mollusks, including the MSI60 of Japanese pearl oyster (Sudo et al.

1997) and Shelk2 of Crassostrea nippona (Takahashi et al. 2012). However, the

function of Shelk2 still remains unknown. Therefore, in this study, we made an

attempt to elucidate their function via knockdown experiment.

35.2 Materials andMethods

Adult Pacic oysters (shell length, 5–7cm; shell height, 7–11cm) were purchased

from the market and maintained in articial seawater for a day before using them for

the RNAi experiments.

For the synthesis of shelk2 dsRNA, we used T7 RiboMAX Express RNAi System

(Promega, Madison, WI, USA) following the manufacturer’s instructions. The

dsDNA templates of shelk2 and EF-1α for both RNA syntheses were cloned into

pTAC-2 plasmid (BioDynamics Laboratory, Tokyo, Japan), prepared using TaKaRa

Ex Taq DNA polymerase (TaKaRa, Shiga, Japan) or PrimeSTAR GXL DNA poly-

merase (TaKaRa) with PCR primers shown in Table 35.1. These primers were

designed on the basis of C. gigas shelk2 sequence (GenBank ID: AB474183) and

EGFP sequence. Thermal cycler T-Gradient Thermoblock (Biometra, Goettingen,

Germany) was used for the amplication according to the conventional reaction

program.

J. Takahashi et al.

335

The Pacic oyster shells were cut on the ventral side near the adductor muscle

into approximately 3-cm wide portions using a pair of nippers. To knock down the

shelk2, the designed shelk2 dsRNA (10μg or 30 μg in 200 μL PBS) or EGFP

dsRNA (30μg in 200μL PBS, for control) was injected into the adductor muscle of

each oyster (Suzuki etal. 2009; Funabara etal. 2014; see Fig.35.1a). The oysters

were then kept in articial seawater for 7days without feeding. Then their mantles

and the newly regenerated prismatic layers (Fig.35.1b) were collected for qPCR

experiments and SEM observation, respectively.

For SEM observation of the regenerated shell, Miniscope TM3000 (Hitachi

High-Technologies, Tokyo, Japan) was used at two magnications (×500 and

×2000).

For qPCR analyses, total RNA was extracted from the collected mantles using

Sepasol-RNA I Super G (Nacalai tesque, Kyoto, Japan), while using Handy Sonic

UR-20P (Tomy Seiko, Tokyo, Japan) for mantle homogenization. We used

Sequence (5' -> 3')

S2-101 Fw ATGCTGAAGCTTGTCTCCATCGTTTGCCTT

S2-102 Rv TTAATAGGTCTTTTTATGTCTGATGCCACC

T7 S2-117 Fw GGATCCTAATACGACTCACTATAGGATGCTGAAGCTTGTCTCC

T7 S2-121 Rv GGATCCTAATACGACTCACTATAGGTTAATAGGTCTTTTTATGTCTGATGCC

EGFP-903 Fw ATGGTGAGCAAGGGCGAGGAGCTG

EGFP-904 Rv TTACTTGTACAGCTCGTCCATGCC

T7 EGFP-901 Fw GGATCCTAATACGACTCACTATAGGATGGTGAGCAAGGGCGAG

T7 EGFP-902 Rv GGATCCTAATACGACTCACTATAGGTTACTTGTACAGCTCGTC

Cg_EF1a-802 Fw AAGTCTTGGAAGAGGCACCA

Cg_EF1a-803 Rv CAGCCTTCTCAACCTCCTTG

S2-126 Fw CTCCATCGTTTGCCTTTTTG

S2-127 Rv AGTCCTCCAATGACACCACC

dsRNA synthesis

Name

qPCR analysis

Table 35.1 Primers for dsRNA syntheses and qPCR analyses

Fig. 35.1 Knockdown experiment and shell regeneration. (a) Shell surrounding the adductor

muscle was excised by a pair of nippers within 3cm, and dsRNA was injected into the adductor

muscle. A constant volume (200μl) of PBS solution containing 30μg of EGFP dsRNA and 10μg

or 30μg of shelk2 dsRNA was injected into each group (n=5). (b) Plastic-like structure of new

shell was regenerated after a day of injection, and it was more clearly observed after the next

2days (arrowheads). We collected the structure and the mantle edges after 7days of injection

35 Functional Analysis onShelk2 ofPacic Oyster

336

PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa) for RT-PCR and rst

strand cDNA synthesis. Primers for qPCR were also designed on the basis of C.

gigas shelk2 sequence and C. gigas EF-1α sequence (GenBank ID: AB122066). For

the qPCR reaction, KOD SYBR qPCR Mix (TOYOBO, Osaka, Japan) was used in

StepOnePlus Real-Time PCR System (Life Technologies Japan, Tokyo, Japan)

employing the comparative CT (ΔΔCT) method.

35.3 Results andDiscussion

35.3.1 Regeneration ofShell Prismatic Layer Observed

bySEM

Figure 35.2 shows the SEM results (top view) of the newly generated plastic-like

structure in the prismatic layer. In general, the prismatic layer gradually grew from

the lower left to the upper right direction during the natural regeneration of a cracked

shell, as shown in Fig.35.2a. During this process, it is assumed that gap among the

columns is lled densely. When dsRNA of EGFP was injected as the control experi-

ment, the prismatic layer grew in a similar manner (Fig.35.2c, d).

In contrast, when dsRNA of shelk2 was injected, the prismatic layer did not grow

normally (Figs.35.2e–h). In particular, the size of each column was reduced, and

the reduction was more remarkable by the 30-μg injection than by the 10-μg injec-

tion (Fig.35.2g, h). In addition, the edge of the column top looked rounder; resul-

tantly the columns were not tightly bound to each other compared with the control

experiment as well as the natural regeneration. Furthermore, the surface of the col-

umn top looked rough, whereas those of the control experiment and the natural

regeneration were smooth.

35.3.2 Real-Time PCR

To determine the effect of shelk2 knockdown by RNAi, the expression of shelk2

mRNA was evaluated (Fig. 35.3). Unexpectedly, the expression level of shelk2

mRNA was considerably higher than that in the control experiment, in which EGFP

dsRNA (Fig.35.3) or PBS (data not shown) was injected. Generally, target gene

expression is reduced in the knockdown experiments. Actually in the experiments of

shells, the expression of Pinctada fucata genes including Pif and Nacrein were

reduced by the previous knockdown experiment (Suzuki etal. 2009; Funabara etal.

2014), although their expressions were examined 7 or 8days after injection similar

to our experiments. In fact, reduction was observed in the knockdown experiment of

another oyster silk-like gene, shelk1, in our experiment (data not shown).

J. Takahashi et al.

337

As a result of shelk2 knockdown, shelk2 mRNA was expressed remarkably dur-

ing shell regeneration, suggesting that Shelk2 would increase. Then the increase in

the amount of the protein would induce the reduction in the column size of the

prismatic layer. However, detailed studies on the change in expression levels of

shelk2 mRNA after injection are required for the full understanding of its remark-

able expression.

Fig. 35.2 SEM observation of the regenerated prismatic layers at two magnications (×500 and

×2000). The bar indicates 30μm. (a, b) Shell was excised, but no operation was performed. (c, d)

dsRNA of EGFP was injected (control). (e, f) Shelk2 dsRNA (10μg) was injected. (g, h) Shelk2

dsRNA (30μg) was injected

35 Functional Analysis onShelk2 ofPacic Oyster

338

35.3.3 Plan forSubsequent Studies

We have unexpectedly detected the remarkable expression of shelk2 mRNA by real-

time PCR analysis, but no information was available on the expression level of

Shelk2. We are now trying to raise an antibody against Shelk2 for the detection of

its expression and subsequent observation using SEM and western blotting during

regeneration following knockdown experiments.

Since shelk2 has multiple copies (Takahashi etal. 2012), the reactionary excess

expression of the genes at multiple sites would be due to the temporal shelk2 mRNA

suppression caused by the RNAi. To validate the speculation, we attempt to identify

the overexpressed gene after RNAi experiment. Further studies on the molecular

mechanism of oyster shell synthesis, especially on the remarkably rapid regenerat-

ing process, would lead to the application in medical and cosmetic elds.

References

Funabara D, Ohmori F, Kinoshita S, Koyama H, Mizutani S, Ota A, Osakabe Y, Nagai K, Maeyama

K, Okamoto K, Kanoh S, Asakawa S, Watabe S (2014) Novel genes participating in the forma-

tion of prismatic and nacreous layers in the pearl oyster as revealed by their tissue distribution

and RNA interference knockdown. PLoS One 9:e84706

Fig. 35.3 Knockdown of shelk2 by means of RNAi. The expression levels of shelk2 mRNA in the

mantle, which are normalized to those of EF-1α, were determined with real-time quantitative

PCR.Five oysters were used in each experiment group. The graph bar shows the shelk2 mRNA

expression level of 7days after injection with dsRNAs against 10μg (bar number: 1–5) or 30μg

(6–10) of shelk2, and those of the EGFP group (average of 5 oysters) is attributed a relative value

of 1.0. Unexpectedly, the shelk2 expression levels increased more than ten times

J. Takahashi et al.

339

Guerette PA, Ginzinger DG, Weber BHF, Gosline JM (1996) Silk properties determined by gland-

specic expression of a spider broin gene family. Science 272:112–115

Hayashi CY, Lewis RV (1998) Evidence from agelliform silk cDNA for the structural basis of

elasticity and modular nature of spider silks. JMol Biol 275:773–784

Hinman MB, Lewis RV (1992) Isolation of a clone encoding a second dragline silk broin. JBiol

Chem 267:19320–19324

Marin F, Corstjens P, Gaulejac B, Jong EVD, Westbroek P (2000) Mucins and molluscan cal-

cication: molecular characterization of mucoperlin, a novel mucin-like protein from the

nacreous shell layer of the fan mussel Pinna nobilis (Bivalvia, Pteriomorphia). JBiol Chem

275:20667–20675

Sudo S, Fujikawa T, Nagakura T, Ohkubo T, Sakaguchi K, Tanaka M, Nakashima K, Takahashi T

(1997) Structures of mollusk shell framework proteins. Nature 387:563–564

Suzuki M, Saruwatari K, Kogure T, Yamamoto Y, Nishimura Y, Kato Y, Nagasawa H (2009) An

acidic matrix protein, Pif, is a key macromolecule for nacre formation. Science 325:1388–1390

Takahashi J, Takagi M, Okihana Y, Takeo K, Ueda T, Touhata K, Maegawa S, Toyohara H (2012)

A novel silk-like shell matrix gene is expressed in the mantle edge of the Pacic oyster prior to

shell regeneration. Gene 499:130–134

Takahashi J, Kishida T, Toyohara H (2013) Poly-alanine protein Shelk2 from Crassostrea species

of oysters. In: Watabe S, Meyama K, Nagasawa H (eds) Recent advances in pearl research.

Terrapub, Tokyo, pp167–181

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

35 Functional Analysis onShelk2 ofPacic Oyster

341

Chapter 36

Mollusk Shells: Does theNacro-prismatic

“Model” Exist?

YannickeDauphin andJean-PierreCuif

Abstract The “nacro-prismatic” shells are the most studied mollusks, and they are

often said to be “the” model to unravel the biomineralization mechanisms.

Nevertheless, the nacro-prismatic structure is not unique, despite most data are pro-

vided by only three genera. The aragonitic nacre is taxon dependent: in cephalopods

and gastropods, nacre is columnar, whereas bivalves have a spiral or sheet nacre.

The inner structure of gastropod and cephalopod columnar nacre differs. The shape

of the tablets is specic of the taxa. Calcitic and aragonitic prisms exist. The com-

position of the organic matrices extracted from calcitic prisms with a similar shape

and mineralogy strongly differs. The inner structure of aragonite prisms is complex,

with a central zone and divergent elongated crystallites at the periphery. Additionally,

the relationships between nacre and prisms are also taxonomically related. From

these data, whatever the scale at which they are studied, every component of the

“nacro-prismatic” model– nacre, prisms, and prism–nacre topographic relations–

is highly variable, so that this “model” does not exist; it is a structure.

Keywords Mollusks · Nacre · Prismatic layer · Model

36.1 Introduction

The most common structure in mollusk shells is the aragonite crossed-lamellar

layer, but the most studied is the “nacro-prismatic” arrangement. Almost all data

about mollusks are from three bivalve genera with at large shells: Atrina, Pinna,

and Pinctada. These genera are taxonomically related (Pteriomorphia), with

Y. Dauphin (*)

Institut de systématique, Evolution, Biodiversité, ISYEB– UMR 7205, Muséum national

d’Histoire naturelle, Paris, France

e-mail: yannicke.dauphin@sorbonne-universite.fr

J.-P. Cuif

Centre de recherche sur la paléodiversité et les paléoenvironnements, CR2P– UMR 7207,

Muséum national d’Histoire naturelle, Paris, France

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_36

342

large polygonal prismatic units and an inner nacreous layer, so that separating

the two layers for detailed analyses is not difcult. They are often used as “the”

model to understand the biomineralization processes. Sometimes, Unio and

Mytilus, both with a nacro-prismatic structure, are also used as a model. The

concept of model to describe this structure suggests that all these nacro-pris-

matic shells are identical in terms of structure and composition. The examination

of the structure and composition of the layers and of the prismatic and nacreous

units, as well as their relationships, demonstrates that the nacro-prismatic

arrangement is not unique. It is impossible to enter into the detailed description

of all mollusk shells. So, the present article will concentrate largely on the dif-

ferences between the nacro-prismatic shells.

36.2 Materials andMethods

Details about the origin of the samples and preparative process and setup of the

diverse used techniques are given in the relevant publications listed in the References.

36.2.1 Materials

Bivalves (Pinctada, Pinna, Nucula, Neotrigonia, Unio), gastropods (Haliotis,

Trochus, Turbo), and cephalopods (Nautilus, Sepia, Spirula) were used. Depending

on the genera, several species were studied (Pinna, Sepia, Haliotis, among others).

36.2.2 Methods

Micro- and nanostructures were studied using thin sections, fractures, and polished

etched surfaces for the scanning electron microscope (secondary and backscattered

electron modes, Philips SEM XL30, FEI Phenom) and atomic force microscope

(Veeco Nanoscope Dimension 3100). Electron microprobes (energy- and

wavelength- dispersive spectrometry) (Link AN10000, CAMECA SX50, SX100)

were used for quantitative elemental chemical composition and distribution maps.

Chemical distribution maps were also performed using NanoSIMS (CAMECA

N50), TOF-SIMS (TOF-SIMS IV Ion-Tof GmbH), and XANES (ID21, ESRF).

Thermogravimetric analyses allow to quantify the organic matrix content. Infrared

and Raman spectrometry were used on both bulk samples and extracted organic

matrices. Liquid chromatography and electrophoresis were used for molecular

weights and acidity of the soluble matrices. Lipid content was known using thin-

layer chromatography. Amino acid analyses were done on both soluble and insolu-

ble matrices.

Y. Dauphin and J.-P. Cuif

343

36.3 Results

36.3.1 Microstructures

Prisms are aragonite (Neotrigonia, Unionidae, Cephalopoda) or calcite

(Pteriomorphia) (Fig.36.1a–c) (Boggild 1930; Taylor etal. 1969, 1973; Ben Mlih

1983; Checa etal. 2014; Cuif etal. 2011). In some species, calcitic and aragonitic

prisms coexist (Dauphin etal. 1989). The inner structure of the prisms is also vari-

able, but the morphological and microstructural diversity is not related to the min-

eralogy (Sepia, Fig.36.1b; Haliotis, Fig.36.1c) but to the taxa. The inner structure

of aragonite prisms is complex, with a central zone and divergent elongated crystal-

lites at the periphery. Calcitic prisms are mono- or polycrystalline. The nacre is

aragonite, but the tablets are deposited in vertical columns in gastropods and cepha-

lopods, whereas they are in lenses in bivalves (Wise 1970). The inner structure of

gastropod and cephalopod columnar nacre differs. Moreover, the shape and the

inner structure of the tablets are species dependent (Nautilus, Fig.36.1d; Pinna,

Fig. 36.1e; Sepia, Fig. 36.1f) (Mutvei 1978, 1979). In coleoid cephalopods, the

nacreous layer has no tablets (Mutvei 1963) (Fig.36.1f).

Not only the shape, mineralogy, and inner structure of the prisms or tablets differ,

but the transition between the two layers is also taxonomically dependent. When the

prisms are calcite, there is no direct contact between the calcite and the nacre (Cuif

etal. 2011). Both layers are separated by a thick organic membrane and an irregular

layer of brous aragonite (Pinctada, Fig.36.1g). In shells with aragonitic prisms,

the transition is smooth, without an organic membrane (Neotrigonia, Fig.36.1h)

(Dauphin et al. 2014). Chemical differences also exist in the transition zone.

Backscattered electron image of the calcitic–aragonitic transition demonstrates that

the rst aragonitic deposits are not nacre (Pinctada, Fig. 36.1i) (Dauphin et al.

2008). XANES map shows that the chemical composition of the end of the calcitic

prisms is modied (Pinctada, Fig.36.1j) (Dauphin etal. 2003), so that it cannot be

said that the termination of prisms is “abrupt” (Hovden etal. 2015). No organic

membrane exists between aragonitic prisms and nacre (Neotrigonia, Fig. 36.1k)

(Checa and Rodriguez-Navarro 2001; Dauphin etal. 2008, 2014). The amino acid

content of the brous aragonite differs from that of the nacreous layer, as shown by

TOF-SIMS maps (Pinctada, Fig.36.1l, m) (Farre etal. 2011), and the N map con-

rms the difference between the nacre and the brous aragonite in calcitic–arago-

nitic shells (Pinctada, Fig.36.1n) (Dauphin etal. 2008).

It must be added that the elemental chemical composition of a given structure

(nacre, calcitic or aragonitic prisms) is species dependent (Fig. 36.1o) (Dauphin

etal. 1989).

36 Mollusk Shells: Does theNacro-prismatic “Model” Exist?

Fig. 36.1 (a) Unetched fracture showing the complex aragonitic prisms of Neotrigonia. (b)

Aragonitic prismatic layer of the dorsal shield of Sepia– unetched fracture. (c) Calcitic prisms of

Haliotis rufescens, polished and etched fracture. Formic acid 5%, 7s, 20°C. (d) Columnar nacre-

ous layer of Nautilus– unetched fracture. (e) Rectangular nacreous tablets of Pinna – unetched

sample. (f) Type 2 nacre: layered structure without tablets in a lamella of the ventral part of Sepia

– unetched sample. (g) Unetched fracture showing the calcitic prismatic– aragonitic nacreous

345

36.3.2 Organic Components

It is now well-known that mollusk shells are organo-mineral biocomposites. For a

given structure, the quantity and nature of the organic matrices differ and depend on

the taxa as shown by TGA data of the nacre in Nautilus (Cephalopoda), Trochus

(Gastropoda), and Pinctada (Bivalvia) (Fig.36.2a, b). Insoluble matrices comprise

proteins and lipids. It must be noted that the results differ following the sample

preparation (decalcication or lipid extraction using organic solvents, Farre and

Dauphin 2009). Using the same preparative process, the lipidic composition of the

calcitic prisms of Pinna and Pinctada differs (Fig.36.2c). The molecular weights of

the soluble matrices of these prisms also differ (Fig.36.1d) (Dauphin 2003). As for

the insoluble matrices, most analyses are dedicated to the protein contents, mainly

amino acid analyses (Pinctada, Nautilus, Fig. 36.2e). Nevertheless, infrared spec-

trometry of the insoluble matrices shows the presence of lipids and sugars in the

insoluble matrices of nacreous layers (Nautilus, Pinctada, Fig.36.2f). Despite the

similarity of shape and mineralogy of the prisms of Pinna and Pinctada, the acidity

(pI) and aliphatic index (indicative of the thermal stability for globular proteins) of

the insoluble matrices differ (Fig.36.2g).

36.4 Discussion andConclusion

There is a strong contrast between the small number of studied taxa with a nacro-

prismatic structure and the diversity of their shells. The examination of the shape,

inner structure, mineralogy, and composition of both mineral and organic compo-

nents of these shells show the large diversity of these characteristics (Samata 1990),

despite some supercial similarities. The relationships between the two layers are

also variable and controlled by the organism. However, the diversity is not hazard-

ous: every characteristic is taxonomically dependent, usually at a specic level.

Most often, the presence and role of acidic proteins in the biomineralization process

is emphasized, but the role of sugars and lipids is neglected (Kocot etal. 2016). Up

to now, proteomics and genomics data have not permitted to select the possible

mechanisms of the secretion (Suzuki and Nagasawa 2013; Simkiss 2016).

Thus, not only the structure and composition of the nacre and prisms are hetero-

geneous, they are also dependent on the species, so that this heterogeneity does not

Fig. 36.1 (continued) transition in Pinctada, with a thick organic membrane and an irregular layer

of brous aragonite. (h) Aragonitic prism– nacre transition in Neotrigonia – BSE image of a

polished and etched section (HCl 1%10s). (i) BSE map showing the organic membrane and the

brous aragonite between the calcitic prisms and the nacre in Pinctada. (j) XANES map of S in

amino acids in the shell of Pinctada. (k) XANES map of sulfate in Neotrigonia. (l) TOF-SIMS

map of alanine in the shell of Pinctada. (m) TOF-SIMS map of glycine of the same section. (n)

NanoSIMS map of N in Pinctada. (o): Mg and Sr contents of the prismatic calcitic layers in some

species of Haliotis

36 Mollusk Shells: Does theNacro-prismatic “Model” Exist?

Fig. 36.2 (a, b) Thermogravimetric proles showing the differences in the quantity and composi-

tion of the organic matrices in three nacreous layers. (c) Thin-layer chromatography showing the

lipidic composition of the calcitic prisms in two bivalve shells. (d) Liquid chromatography of the

soluble organic matrices of calcitic prisms. (e) Amino acid composition of the insoluble matrix of

nacreous layers. (f) Infrared spectrometry of the insoluble organic matrix of nacreous layers. (g)

Isoelectric point (pI) and aliphatic index (alip) of the insoluble organic matrix of calcitic prisms

347

suit the usual characteristics of a model. They are neither simple nor unique, so that

the nacro-prismatic model concept cannot be sustained.

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vidensk og mathem 9:231–326

Checa AG, Rodriguez-Navarro A (2001) Geometrical and crystallographic constraints determine

the self-organization of shell microstructures in Unionidae (Bivalvia: Mollusca). Proc R Soc

Lond B 268:771–778

Checa AG, Salas C, Harper EM, De Dios Bueno-Perez J (2014) Early stage biomineralization

in the periostracum of the “living fossil” bivalve Neotrigonia. PLOS ONE 9:2. https://doi.

org/10.1371/journal.pone.0090033

Cuif JP, Dauphin Y, Sorauf JE (2011) Biominerals and fossils through time. Cambridge Univ.

Press, Cambridge, 490 p

Dauphin Y (2003) Soluble organic matrices of the calcitic prismatic shell layers of two pteriomor-

phid bivalves: Pinna nobilis and Pinctada margaritifera. JBiol Chem 278(17):15168–15177

Dauphin Y, Cuif JP, Mutvei H, Denis A (1989) Mineralogy, chemistry and ultrastructure of the

external shell layer in ten species of Haliotis with reference to Haliotis tuberculata (Mollusca:

Archaeogastropoda). Bull Geol Inst Univ Uppsala, NS 15:7–38

Dauphin Y, Cuif JP, Doucet J, Salomé M, Susini J, Williams CT (2003) In situ chemical specia-

tion of sulfur in calcitic biominerals and the simple prism concept. JStruct Biol 142:272–280

Dauphin Y, Ball AD, Cotte M, Cuif JP, Meibom A, Salomé M, Susini J, Williams CT (2008)

Structure and composition of the nacre– prism transition in the shell of Pinctada margaritifera

(Mollusca, Bivalvia). Anal Bioanal Chem 390:1659–1169

Dauphin Y, Cuif JP, Salomé M (2014) Structure and composition of the aragonitic shell of a living

fossil: Neotrigonia (Mollusca, Bivalvia). Eur JMineral 26:485–494

Farre B, Dauphin Y (2009) Lipids from the nacreous and prismatic layers of two Pteriomorpha

mollusc shells. Comp Biochem Physiol B152:103–109

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1: introduction and molecular biology. In: Saleuddin S, Mukai S (eds) Physiology of molluscs:

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348

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

Y. Dauphin and J.-P. Cuif

349

Chapter 37

The Marsh’s Membrane: AKey-Role

foraForgotten Structure

Jean-PierreCuif andYannickeDauphin

Abstract Recent imaging methods applied to the growing edge of the Pinctada

margaritifera shell allow for a better appreciation of ancient structural data. Growth

of the Pinctada shell (both lateral extension and thickness increase) is a coordinated

mechanism involving a series of clearly identied steps in contrast to the prevailing

concept of a direct “self-assembly” process.

Keywords Periostracal transit · Flexible shell · Layered growth mode · Marsh

membrane

37.1 Introduction

In contrast to Wada (1961) or Wilbur (1964) who recognized the importance of a

specic structural phase predating the prismatic layer of mollusk shells (Fig.37.1a),

most modern investigators propose microstructural schemes in which the calcite

prisms and their organic envelopes are directly and simultaneously produced at the

growing edges of the shells (Saleuddin and Petit 1983; Volkmer 2007; Soldati etal.

2008). These models share the surprising ability of the prisms to continue to grow

after having been covered by the nacreous layer. This is also the case in the scheme

initiated by Petit (1978) and repeated up to Calvo-Iglesias etal. (2016, g. 10) who

summarized the concept of a remotely controlled formation of the prismatic layer:

“Molecules secreted into the extrapallial cavity would be self-assembled and they

reach the shell growing area without the participation of any cell.”

J.-P. Cuif (*)

Centre de recherche sur la paléodiversité et les paléoenvironnements, CR2P – UMR 7207,

Museum National d’Histoire Naturelle, Paris, France

Y. Dauphin

Institut de systématique, Evolution, Biodiversité, ISYEB – UMR 7205, Muséum national

d’Histoire naturelle, Paris, France

e-mail: Yannicke.dauphin@upmc.fr

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_37

350

In such models, no place exists for the “innermost shell lamella” described by

Marsh and Sass (1983) as “a single continuous layer which forms the inner surface

of the shell … rmly attached to the mineral in the underlying calcied layer”.

Here, through a microstructural approach of the growing edge of a prismatic

shell layer, an attempt is made to establish a junction between the several decade-

old observations clearly neglected in current literature.

37.2 Material andMethods

Adult pearl oysters (Pinctada margaritifera) were collected alive in Tuamotu archi-

pelago. Young samples come from the hatchery of the Direction des Ressources

Marines et Minières (DRMM, the Polynesian governmental ofce for pearl

cultivation).

Observations were carried out with optical microscopy (natural and polarized

light), scanning electron microscopy in both secondary and backscattered electron

modes, and atomic force microscopy in tapping mode. X-ray diffraction measure-

ments were performed at the ID13 beam line of the ESRF (Grenoble).

37.3 Results

37.3.1 Structure oftheFlexible Shell Initiated andDeveloped

inthePeriostracal Grove

On the internal side of the periostracal lm, numerous nonadjacent mineral disks

are visible and regularly distributed (Fig.37.1b, c). Every disk is growing around a

non-mineralized center (Fig.37.1d, arrows). The concentric mode of growth of the

disks is visible of the outer side (Fig.37.1e), whereas their inner side reveals a con-

tinuous granular structure (Fig.37.1f). Using transmitted polarized light, the disks

appear homogenous, each of them with a specic nuance always in the gray levels

of the Newton scale (Fig.37.1g). The single-crystal behavior of the disks is well

Fig. 37.1 (continued) disks are clearly visible (d: arrows); Bar 30μm (c), 15μm (d). (e) Central

nodule surrounded by concentric growth layers. Bar 8μm. (f) Granular appearance of the inner

surface of a disk. Bar 6μm. (g) Optical view of disks (transmitted polarized light). Note the homo-

geneity of the gray levels for every disk. Bar 15μm. (h–i) Three series of nine points where X-ray

diffraction was carried out on a single disk. Note the superposition of the diffraction spots (i). (j–l)

Series of growing disks. Thickness does not increase during diametral growth; periostracum

almost completely discarded excepted in left part of the picture. Bar 20μm. (m) The exible shell

at the end of the periostracal phase, just before passage to the rigid shell status. Note the 3μm

thickness of the disks. Bar 50μm. (n) Scheme of a section of the shell growing edge cut perpen-

dicularly to shell surface. Focus is made on the transit of the disks carried on the internal side of

the periostracum, from deposition of the disk organic centers (oc) up to the upside-down move-

ment (ud, arrow) by which the disks become the initial substrates for prism growth

J.-P. Cuif and Y. Dauphin

351

Fig. 37.1 (a) Wada (1961) reprinted in Wilbur (1964). Growing edge surface of a Pinctada fucata

shell viewed in transmitted polarized light. Bar 50μm. (b) SEM view of the outer surface of the

periostracal membrane. Through the membrane the growing disks are visible. Note their centers.

Bar 30μm. (c–d) Optical view of an equivalent area (episcopic polarized light). Centers of the

37 The Marsh’s Membrane: AKey-Role foraForgotten Structure

352

established by the perfect superposition of the spots in a series of 27 X-ray diffrac-

tions in a single disk (Fig.37.1h, i). Through their concentric growth mode during

their transportation by the periostracum acting as a conveyor belt, the disks reach

their maximal size, becoming in close contact at the distal end of the periostracal

grove (Fig.37.1j–m). Their thickness remains about 3–4μm (Fig.37.1m, n, black

arrow).

37.3.2 Transition fromFlexible toRigid Shell: Occurrence

ofaNew Growth Mode

The distal end of the periostracal transit is a turning point in shell formation.

Through an upside-down movement, the disks are placed in geometrical continuity

with the previously built “rigid shell.” By this movement, the internal sides of the

disks (with non-mineralized centers) become the outer side of the shell growing

edge. The outer surface of the shell growing edge shows the roughly circular crests,

the dimensions of which correspond to the nal stages of the discoid units devel-

oped in the “exible shell” stage (Fig.37.2a).

SEM view of the internal side in the same area reveals the polygonal morphology

of the mineral building units (Fig.37.2b) whose single crystal behavior is obvious

in transmitted polarized light (Fig. 37.2c). Transition from discoid to polygonal

morphology of the mineral units is well illustrated by transmitted polarized light

(Fig.37.2d). The limits of the previously discoid units are still visible (Fig.37.2d:

blue arrows), while the mineral phase has been extended up to become in contact to

the neighboring units (Fig.37.2d: red arrows). This close contact between the newly

formed polygonal units ensures the shell rigidity.

This developmental step shows the rst occurrence of an additional component

of the shell: the internal side of the crystal-like polygonal units is now covered by a

continuous organic membrane (Fig.37.2b: Mm) so that the mineral phase is sand-

wiched between the external periostracum and this internal organic membrane.

Simultaneously, a completely different biomineralization pattern can be observed.

Instead of an individual lateral extension of the disks, mineralization occurs as a

synchronic process insuring a simultaneous increase of shell thickness (Fig.37.2e).

Once more, the organic lm is visible at the internal side of the polygonal units

(Fig.37.2e, arrows). It is still present (Fig.37.2f, arrows) when the repeated layered

growth process leads the thickness of these calcareous units to be superior to their

lateral dimensions, justifying the term “prism.” Closer observations of the basal

surface of the prisms leave no doubt about the presence of this well-individualized

membrane (Fig.37.2g, arrows). This membrane, permanently present at the internal

surface of the prisms, exhibits a granular structure rather similar to the granular

structure of the mineral phase of the prisms (Fig.37.2h–j).

J.-P. Cuif and Y. Dauphin

353

37.4 Discussion

37.4.1

Inadequacy ofthe“Direct Crystallization” Model

toAccount forFormation ofthePrismatic Layer

inthePinctada Shell

The “molecular self-assembly process” underlying recent schemes and explicitly

formulated by Calvo-Iglesias etal. (2016) cannot be applied to the outer shell layer

of the Pinctada margaritifera. The two-phase mechanism briey described in the

present report was suggested by Wada (1961, Figure8) and Wilbur (1964, Figure10),

who observed circular crystalline units growing “in oolitic aggregation” becoming

progressively polygonal by mutual contact. The crystalline properties of the mineral

units forming the “exible shell” were established by Suzuki etal. (2013), but the

presence of a non-mineralized center in each of these calcareous units basically

modies the interpretation of their formation and role.

37.4.2 Origin oftheCrystallographic Individuality

oftheCalcite Prisms ofthePinctada Shell

From the deeper parts of the periostracal grove, the calcareous disks transported on

the internal side of the periostracum exhibit a non-mineral center, suggesting that

this organic glomerule was deposited in close vicinity to the group of cells dedi-

cated to the formation of the periostracum itself (see histological sections in Jabbour

etal. 1992). Polarization microscopy and multiple X-ray diffractions show that, in

conformity to the results of Suzuki etal. (2013), the disks are crystal-like units, each

of them with a specic crystalline orientation (assessed by the distinct gray levels

corresponding to a 3–4 μm thickness measured by SEM; Fig. 37.1m). It can be

assumed that these specic crystalline orientations take origin in the slightly diverse

orientations of the organic substrates of the disks.

From this very early origin of crystallographic orientation in the transitional

phase from exible to rigid shells (Fig.37.2d), the crystallographic orientation of

the rst mineral polygons forming the rigid shell relies on the crystallographic ori-

entation of the disks after their upside-down movement. In further growth steps of

the prisms, crystallographic orientation of every newly formed polygon is repeated.

Thus, conclusion arises that the long-recognized crystal-like behavior of the

prisms forming the outer shell layer of the Pinctada is determined in the deeper part

of the periostracal grove and is by no means the result of a “self-assembly

process.”

37 The Marsh’s Membrane: AKey-Role foraForgotten Structure

354

37.4.3 The Crystal-Like Disks asExamples ofNon-Ion-by-Ion

Crystallization

In the search of evidences for a biological control of crystallization, we must note

that all along their growth, the freely and independently crystallizing disks exhibit a

concentric layering as obvious traces of their stepping growth mode. If disk crystal-

lization was based on an ion-by-ion mechanism, the presence of calcite faces ori-

ented in conformity with those of crystals produced by purely chemical precipitation

should appear in these distant and freely growing units. This is not the case: no

exception has been observed to the circular morphology and concentric mode of

growth for the crystal-like units. This provides a contrario evidence for a nonionic

but biochemically controlled mode of crystallization (i.e., non-purely chemical).

37.4.4 The Marsh’s Membrane asCoordinator oftheLayered

Growth Mode ofthePrisms

Occurrence of the organic membrane covering the newly formed polygons

(Fig.37.2e) and its persistency during further development of the prisms introduce

a new factor to be taken into account in the crystallization process. Its granular

structure was noted from the early descriptions (Fig.37.2k) and among the very rare

mentions that have been made of this shell component. Yan etal. (2008) have well

noted the time-based variability in the development of these grains, leading these

authors to qualify the Marsh’s membrane as a “dynamic structure.” This means that

the Marsh’s membrane is involved in the mineralization process, a conclusion here

conrmed (Fig.37.2g–j) and by previous observations (Cuif etal. 2014).

One of the most striking features of the calcite prisms in Pinctada margaritifera

is the microstructural change that regularly occurs after about 150 μm growth

length. After an initial stage with a single crystal structure (Fig.37.2l), the prisms

become polycrystalline (Fig.37.2m) (Cuif etal. 2011, 2014; Checa etal. 2013).

Fig. 37.2 (continued) the front raw and the changing colors revealing the increasing thickness of

the polygonal units. Bar 50μm. (d) Formation of the rigid shell: limits of the disks, still visible by

the round-shaped trace (blue arrows), are overcome by new mineral deposits that bring the neigh-

bor units in close contact (red arrows). Transmitted polarized light; Bar 20μm. (e) The Marsh’s

membrane is visible at the base of the newly formed polygons (arrows). Note the decayed perios-

tracum making visible the outer surface of the initial disks and their centers. Bar 10μm. (f) The

Marsh’s membrane at the inner surface of young prisms. Compare to picture of the compartment

lamella by Bevelander and Nakahara (1980). Bar 25μm. (g) Closer view of the Marsh’s mem-

brane. Note the transversal striation and the granular structure. Bar 5μm. (h–j) Aspect of the

Marsh’s membrane: h, SEM view, Bar, 5μm; i, SEM view, Bar, 2μm; j, AFM view, Bar, 150nm.

(k) Reprint of a Marsh gure (TEM). Note the granular pattern of the Marsh membrane, corre-

sponding to the high-resolution SEM view here reported. Bar: 0.5μm. (l–m) Thin sections (trans-

mitted polarized light) in the upper part of prisms (up to 150μm) and in their lower part. Note the

passage from single crystal- like to polycrystalline organization. Bar: 50μm (l) and 30μm (m)

J.-P. Cuif and Y. Dauphin

Fig. 37.2 (a) SEM view of the outer surface of the rigid shell at its growing edge. Bar 30μm. (b)

Internal side of a polygonal unit at the distal raw of the rigid shell. Mm: organic membrane. SEM

view, Bar 25μm. (c) Polygonal units in transmitted polarized light. Note the various gray levels in

356

Such a coordinated change contradicts the theory of “crystal growth competi-

tion” that postulates a selection of the best oriented crystals leading to a progressive

increase of the mean diameter in a given shell.

37.4.5 The Key Role oftheMarsh’s Membrane

inMicrostructural Evolution ofthePrisms

Pinctada calcite prisms are submitted to microstructural changes during aging (Cuif

etal. 2014), and composition of both the mineral and organic phases is modied

before the occurrence of nacre deposition (Cuif etal. 2011). Microstructural varia-

tions of the prisms during shell growth provide evidence for time-based genetically

programmed secretion process.

What is remarkable is that the repeated back-and-forth movements of the mantle

due to the rhythmic mode of life of the animal leave no trace in prism microstruc-

ture. This contrast epitomizes the key role of the Marsh’s membrane in shell forma-

tion. When the animal withdraws its mantle for shell closure, the Marsh’s membrane

stays in place at the basis of the prismatic layer. Thus, when the mantle returns to an

actively mineralizing position, growth of the microstructural units (each of them

with its specic crystalline orientation) can restart without any apparent

interruption.

Under many respects understanding the Marsh’s membrane as an active interface

between mantle secretions and shell is an important issue for both microstructural

analysis and any attempt to create biomimetic materials.

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Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

37 The Marsh’s Membrane: AKey-Role foraForgotten Structure

359

Chapter 38

Pearl Production by Implantation ofOuter

Epithelial Cells Isolated fromtheMantle

ofPinctada fucata andtheEffects ofBlending

ofEpithelial Cells withDifferent Genetic

Backgrounds onPearl Quality

MasahikoAwaji, TakashiYamamoto, YasunoriIwahashi, KiyohitoNagai,

FumihiroHattori, KaoruMaeyama, MakotoKakinuma,

ShigeharuKinoshita, andShugoWatabe

Abstract In the current method of pearl production, the mantle fragment of a donor

pearl oyster is transplanted into a host pearl oyster together with an inorganic bead

(pearl nucleus). After this surgical procedure, only outer epithelial cells (OEC) in

the transplanted mantle survive in a host pearl oyster and form a pearl sac to begin

pearl formation. Therefore, implantation of only the OEC instead of the mantle

fragment would be a possible alternative to the current procedure. To examine the

potential of pearl production by implanting OEC in Pinctada fucata, we developed

M. Awaji (*)

National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency,

Minami-Ise, Japan

e-mail: awajim@affrc.go.jp

T. Yamamoto · Y. Iwahashi · K. Nagai

Pearl Research Laboratory, K.MIKIMOTO & CO., LTD, Shima, Japan

e-mail: kenkyujo@mikimoto.com; y-iwahashi@mikimoto.com; k-nagai@mikimoto.com

F. Hattori · K. Maeyama

MIKIMOTO COSMETICS, Mie, Japan

e-mail: hattori.468@mikimoto-cosme.com; maeyama.511@mikimoto-cosme.com

M. Kakinuma

Graduate School of Bioresources, Mie University, Tsu, Mie, Japan

e-mail: kakinuma@bio.mie-u.ac.jp

S. Kinoshita

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,

The University of Tokyo, Tokyo, Japan

e-mail: akino@mail.ecc.u-tokyo.ac.jp

S. Watabe

School of Marine Biosciences, Kitasato University, Minami, Sagamihara, Kanagawa, Japan

e-mail: swatabe@kitasato-u.ac.jp

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_38

360

a cell implantation method using the pearl nucleus carrying a small pit inoculated

with OEC.As a result, approximately 70% of the inserted nuclei formed the nacre-

ous layer when the OEC were inoculated at 5 × 104 cells/nucleus. Then, OEC iso-

lated from two genetically different types of pearl oysters that signicantly differed

in shell nacre color (yellowness) were mixed at four different ratios, and the pre-

pared OEC mixtures were transplanted to investigate the effects of the blend on the

yellowness of pearls to be harvested. The yellowness of harvested pearls differed

signicantly in accordance with the mixing ratio. Similarly, OEC isolated from two

types of pearl oysters that showed a signicant difference in the thickness of their

shell nacre aragonite tablets were mixed at four different ratios and transplanted.

Mean thickness of the aragonite tablets of the harvested pearls differed according to

mixing ratio. These results suggest the method to control pearl quality by blending

OEC obtained from pearl oysters genetically improved by selective breeding for

traits related to pearl quality.

Keywords Pearl oyster · Outer epithelial cells · Implantation · Blending ·

Yellowness · Aragonite tablets

38.1 Introduction

Currently, the method of pearl production using pearl oyster Pinctada fucata

involves transplanting a small fragment of the mantle of a donor pearl oyster into a

host pearl oyster together with a small shell bead called a pearl nucleus (Masaoka

etal. 2013). After transplantation, only outer epithelial cells (OEC) in the trans-

planted mantle fragment survive in the host pearl oyster and form a pearl sac sur-

rounding the pearl nucleus to begin pearl formation (Awaji and Suzuki 1995).

Therefore, implantation of only OEC instead of the mantle fragment would be a

possible alternative to the current pearl production procedure.

Quality of a pearl is determined by various traits: size, color, luster, shape, and

blemish (Jerry etal. 2012; Atsumi etal. 2014). Among these traits, color is mainly

developed by the amount of yellow pigments and the interference color of pearl

nacre. The amount of yellow pigment in nacre is known to be largely affected by the

genetic backgrounds of a donor pearl oyster (Wada 1985). The interference color is

determined by thickness of the aragonite tablets of pearl nacre, which is also affected

by the genetic backgrounds of a donor pearl oyster combined with water tempera-

ture and nutritional condition of a host pearl oyster (Linard etal. 2011; Muhammad

etal. 2017; Odawara etal. 2017). Therefore, if we can produce pearls by implanting

OEC, it might be possible to control yellowness or the interference color of pearls

by blending the OEC isolated from donor pearl oysters with different genetic back-

grounds regarding these traits.

To assess the possibility of these technical improvements in pearl production, we

developed a method to produce pearls by implanting OEC and examined the effects

of blending of OEC isolated from donor pearl oysters with different genetic back-

grounds on the yellowness and interference color of pearls.

M. Awaji et al.

361

38.2 Materials andMethods

38.2.1 Implantation ofaPearl Nucleus withOuter Epithelial

Cells intoa Host Pearl Oyster

OEC were separated from the mantle of a pearl oyster by the methods described in

Awaji and Machii (2011). Briey, a pallial zone of the pearl oyster mantle was

excised out and washed in sterile balanced salt solution for marine mollusks (BSS,

Awaji and Machii 2011) containing 1 mg/ml of kanamycin sulfate (11815-024,

Thermo Fisher Scientic K.K., Yokohama, Japan) with several changes of the

medium. Then the mantle strips were digested for 6h at 25 °C with a mixture of

1.25mg/ml of dispase (17105-041, Thermo Fisher Scientic K.K.) and 0.5mg/ml

of collagenase (034-10533, Wako Pure Chemical Industries, Ltd., Osaka, Japan) in

sterile BSS buffered at pH 7.5 with 20 mM HEPES (346-01373, Wako Pure

Chemical Industries, Ltd.). After the digestion, the outer epithelium on an outer side

of the mantle strip was carefully peeled off using forceps under a binocular micro-

scope. The obtained outer epithelium was washed in the buffered BSS containing

0.4mg/ml of hyaluronidase (151272, MP Biomedicals, LLC, Santa Ana, USA) to

prepare suspension of the OEC clusters (Fig.38.1a). Cell density was determined

by staining cell nuclei with 0.1% crystal violet in 0.1M citric acid (Sanford etal.

1951). Briey, precipitated cell clusters were incubated in 0.5ml of the crystal vio-

let solution for 1h at room temperature with agitation, and the number of cell nuclei

stained dark blue with the dye was counted using a hemocyte counter. For pearl

production, 1μl of OEC suspension (1–5 × 104 cells/μl) was inoculated into a small

pit (1mm diameter and 0.5–1.0 mm depth) of a pearl nucleus (4.5 mm diameter,

Fig.38.1b), and the pearl nucleus was inserted into a host pearl oyster, as described

in Awaji etal. (2014).

Fig. 38.1 Pearl formation by implanting outer epithelial cells isolated from a pearl oyster mantle.

(a) Suspension of outer epithelial cell clusters prepared for implantation; (b) a pearl nucleus car-

rying a small pit; (c) pearls formed by the implantation of a pearl nucleus carrying a pit inoculated

with outer epithelial cells. A white arrow indicates a dimple formed on the surface of a pearl at the

site of a pit. A black star denotes a pearl nucleus harvested from a host oyster without pearl layers.

Scale = 100μm (a), 2mm (b), 5mm (c)

38 Pearl Production by Implantation ofOuter Epithelial Cells Isolated…

362

38.2.2 Effects oftheBlending ofOuter Epithelial Cells

onYellowness ofPearls

Several strains of P. fucata genetically different in shell nacre color have been main-

tained at Mikimoto Pearl Research Laboratory. Among these strains, strains Y and

W are signicantly different in yellowness of the shell nacre, strain Y being yellow-

ish, while strain W being whitish. We used these two strains for the experiment.

OEC isolated from these strains were blended at four different mixing ratios (Y:W

= 3:0, 2:1, 1:2, 0:3) and implanted for pearl production at 6.0 × 105 cells/nucleus.

The host pearl oysters were reared in Ago Bay, Mie Prefecture, for 5months until

pearl harvesting. Yellowness of the harvested pearls was measured using a fast spec-

trophotometric color meter (CMS-35SP, Murakami Color Research Laboratory,

Tokyo, Japan) and expressed as yellowness index (YI, YI=(1.250X-1.038Z)/Y),

where X, Y, and Z represent tristimulus values of CIE 1931 XYZ color space, as

described in Awaji etal. (2014).

38.2.3 Effects oftheBlending ofOuter Epithelial Cells

ontheThickness ofthePearls’ Aragonite Tablets

Strains YW and BWW are genetically different in terms of thickness of shell arago-

nite tablets, and the interference color of shell nacre differs between these strains.

OEC were isolated from these strains and blended at the same ratios as in the experi-

ment on yellowness. The blended cells were implanted for pearl production at 5.0 ×

104 cells/nucleus. The host pearl oysters were reared in Ago Bay for 5months until

pearl harvesting. Thickness of the aragonite tablets of harvested pearls was calcu-

lated from the images obtained with a color 3D laser microscope (VK-9700,

Keyence Corporation, Osaka, Japan) and a scanning electron microscope (S-2380N,

Hitachi High Technologies Corporation, Tokyo, Japan) using VK-H1A1 software

(Keyence Corporation) and ImageJ1.50-b, respectively.

38.3 Results

38.3.1 Pearl Production byImplantation ofOuter Epithelial

Cells

Nacreous pearls could be produced by the implantation of OEC, and the inoculation

of 5 × 104 cells/nucleus led to formation of aragonite layers in approximately 70%

of the inserted nuclei (Awaji etal. 2014). An example of the harvested pearls is

shown in Fig.38.1c.

M. Awaji et al.

363

38.3.2 Effects oftheBlending ofOuter Epithelial Cells

onYellowness ofPearls

The harvested pearls were different in terms of yellowness (Fig.38.2a). Blending of

OEC from strains Y and W at the ratio of 2 to 1 (Y2+W1in Fig.38.2a, b) showed

signicantly higher yellowness than the 0 to 3 group (Y0+W3), and the 1 to 2 group

(Y1+W2) was considered intermediate yellowness (Awaji etal. 2014). This result

Fig. 38.2 Effects on the yellowness of harvested pearls of blending the outer epithelial cells

isolated from two pearl oyster strains that signicantly differed in shell nacre yellowness. The

outer epithelial cells from the strains with yellow (Y) or white nacre (W) were mixed at four

different ratios and transplanted into host oysters with the pearl nucleus carrying a pit. (a) A

photograph showing difference in the yellowness of pearls harvested from four experimental

groups. Scale = 10mm; (b) the yellowness of the pearls expressed by the yellowness index (YI).

Numbers in the columns indicate the sample size, and bars indicate 95% condence limits of the

average. An asterisk indicates a signicant difference (Steel-Dwass test, p<0.05). (Cited from

Awaji etal. 2014)

38 Pearl Production by Implantation ofOuter Epithelial Cells Isolated…

364

implied that we could modify the yellowness of pearls by the blending of OEC

isolated from genetically different pearl oyster strains.

38.3.3 Effects oftheBlending ofOuter Epithelial Cells

ontheThickness ofthePearls’ Aragonite Tablets

Blending of OEC from strains YW and BWW caused difference in the thickness of

the aragonite tablets of nacre. In the observations by scanning electron microscopy,

implantation of OEC from strains YW and BWW at the blending ratio of 3 to 0

(YW3+BWW0) or 0 to 3 (YW0+BWW3) resulted in signicant difference in the

tablet thickness, and the groups with the blending ratio of 2 to 1 (YW2+BWW1) or

1 to 2 (YW1+BWW2) showed intermediate thickness (Fig.38.3). The surface struc-

ture of the nacreous pearls could be observed clearly with a color 3D laser micro-

scope. On this surface, a signicant difference in the thickness of the aragonite

tablets was observed in accordance with the mixing ratio, with the blended groups

showing intermediate thickness. These results implied that we could modify the

thickness of the aragonite tablets, namely, the interference color of the pearls, by the

blending of OEC.

Fig. 38.3 Scanning electron microscopy images showing effects on the thickness of aragonite

tablets in the pearl nacre of blending the outer epithelial cells isolated from two pearl oyster strains

that signicantly differed in the thickness of aragonite tablets of shell nacre. The outer epithelial

cells from the strains with thick (YW) or thin (BWW) aragonite tablets were mixed at four differ-

ent ratios and transplanted into host oysters with the pearl nucleus carrying a pit. Pearl surface is

located to the left. Scale = 10μm

M. Awaji et al.

365

38.4 Discussion

The obtained results indicate that we could modify the color of pearls by implanting

blended OEC isolated from pearl oysters with genetically different phenotypes for

pearl color traits. Intermediate phenotypes observed in the blended groups suggest

that OEC isolated from two different strains can survive in a host pearl oyster to

form a chimeric pearl sac after the implantation. Further studies to conrm the chi-

meric formation of a pearl sac by the blended OEC are needed, by using, for exam-

ple, genomic DNA markers that can identify the origin of OEC (Masaoka etal.

2013). The practical use of the OEC implantation for pearl culture, however, is still

challenging since a pit of a pearl nucleus remains as a dimple on the surface of a

harvested pearl. OEC implantation methods without making a pit on a pearl nucleus

are needed.

Pearl formation by the implantation of OEC would also serve to clarify mecha-

nisms underlying shell and pearl formation at a molecular level in combination with

gene transfection technologies. Various genes and molecules have been reported to

be involved in shell formation, but details of their function have remained mostly

unknown. Implantation of OEC transfected with a gene of interest for its overex-

pression would be a novel tool to analyze functions of the target gene. Gene trans-

fection techniques effective for OEC are now under investigation.

For pearl oysters, breeding programs to develop superior donor and host pearl

oyster strains are ongoing (Jerry et al. 2012; Wada 1985). The present studies

became possible because several strains that exhibit different phenotypes for nacre

color traits have been established through breeding programs. Although the use of

pearl oyster strains showing different phenotypes for traits related to shell structures

has been uncommon in shell formation studies, they would serve as novel tools for

clarifying the mechanisms that underlie shell and pearl formation in detail.

Acknowledgment Part of this study was supported by JSPS KAKENHI Grant Numbers

JP23658169, JP26292108, JP17K19282.

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Japanese and hybrid Pinctada fucata in response to the aquaculture temperature condition in

Ago Bay, Japan. Aquaculture 477:35–42

Odawara K, Ozaki R, Takagi M (2017) Inuence of the thickness of the nacreous elemental lam-

ina of the pearl oyster Pinctada fucata used as donor oysters on the pearls. Nippon Suisan

Gakkaishi. https://doi.org/10.2331/suisan.16-00090 (in Japanese with English abstract)

Sanford KK, Earle WR, Evans VJ (1951) The measurement of proliferation in tissue culture by

enumeration of cell nuclei. JNatl Cancer Inst 11:773–795

Wada KT (1985) The pearls produced from the groups of pearl oysters selected for color of nacre

in shell for two generations. Bull Natl Res Inst Aquat 7:1–7 (in Japanese with English abstract)

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

M. Awaji et al.

367

Chapter 39

Functional Analyses ofMMP Genes

intheLigament ofPinctada fucata

KazukiKubota, YasushiTsuchihashi, ToshihiroKogure, KaoruMaeyama,

FumihiroHattori, ShigeharuKinoshita, ShoheiSakuda,

HiromichiNagasawa, EtsuroYoshimura, andMichioSuzuki

Abstract The bivalve hinge ligament is the hard tissue that functions to open and

close shells. The ligament contains brous structures consisting of aragonite crys-

tals surrounded by a dense organic matrix. This organic matrix may contribute to the

formation of brous aragonite crystals, but the mechanism underlying this forma-

tion remains unclear. Recently, we showed that tissue inhibitor of metalloproteinase

(TIMP) and matrix metalloproteinase (MMP) is related to the formation of the liga-

ment in Pinctada fucata. BLAST search of genome database revealed that seven

K. Kubota · S. Sakuda · H. Nagasawa · M. Suzuki (*)

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life

Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

e-mail: asakuda@mail.ecc.u-tokyo.ac.jp; anagahi@mail.ecc.u-tokyo.ac.jp;

amichiwo@mail.ecc.u-tokyo.ac.jp

Y. Tsuchihashi

Mie Prefecture Fisheries Research Institute, Mie, Japan

e-mail: tsuchy00@pref.mie.jp

T. Kogure

Department of Earth and Planetary Science, The University of Tokyo, Tokyo, Japan

e-mail: kogure@eps.s.u-tokyo.ac.jp

K. Maeyama · F. Hattori

MIKIMOTO COSMETICS, Mie, Japan

e-mail: maeyama.511@mikimoto-cosme.com; hattori.468@mikimoto-cosme.com

S. Kinoshita

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,

The University of Tokyo, Tokyo, Japan

e-mail: akino@mail.ecc.u-tokyo.ac.jp

E. Yoshimura

Department of Applied Biological Chemistry, Graduate School of Agricultural

and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Department of Liberal Arts, The Open University of Japan, Chiba, Japan

e-mail: ayoshim@mail.ecc.u-tokyo.ac.jp

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_39

368

MMP genes are encoded in the genome of P. fucata. To identify the specic MMP

that may contribute to ligament formation, the expression level of each MMP was

measured in the mantle isthmus, which secretes the ligament. The expression of

MMP54089 increased after scratching of the ligament, while the expressions of

other MMPs did not increase after doing the same operation. To identify the role of

MMP54089in forming the ligament structure, double-stranded (ds) RNA targeting

MMP54089 was injected into the living P. fucata to suppress the function of

MMP54089. Scanning electron microscopic images showed disordered growing

surfaces of the ligament in individuals injected with MMP54089-specic

dsRNA.These results suggest that PfTIMP and MMP54089 play important roles in

the formation of the brous ligament structure.

Keywords Pinctada fucata · Ligament · Matrix metalloproteinase

39.1 Introduction

The components of the ligament are secreted by the mantle isthmus, which is the

mantle tissue attached to the shell hinge. Bevelander and Nakahara (1969) reported

that small brous aragonite crystals and organic frameworks are secreted from cells

of the mantle isthmus and align to form long aragonite bers. These bers and the

organic matrix are connected and transported to the shell to form the ligament

microstructure. The crystals grow vertically toward the growing surface of the liga-

ment. In a cross section of the ligament, a pseudohexagonal aragonite crystal was

observed (Kahler etal. 1976; Marsh and Sass 1980), which appears to be the euhe-

dral shape of the aragonite crystal. The diameter of the crystal is 50–100nm, and the

c axis is parallel to the long axis. In contrast, the a and b axes are oriented randomly.

Aragonite crystals with the defect of {110} twinning were observed in the ligament

(Kahler etal. 1976; Marsh and Sass 1980).

Suzuki et al. (2015) reported that the aragonite crystals in the ligament of P.

fucata contain ligament intracrystalline peptide (LICP). LICP regulates the growth

of the aragonite crystal and maintains the orientation of the c axis to keep the arago-

nite crystals small. These small aragonite crystals can be aligned in the same direc-

tion and stacked to form aragonite bers. The mechanism by which these small

crystals are gathered and arranged in the organic matrix of the ligament is still

unknown. To reveal the mechanisms underlying the formation of the complicated

aragonite microstructures present in the ligament, structural and functional analyses

of the organic matrix between the aragonite bers are necessary. Recently, we

reported that tissue inhibitor of metalloproteinase (TIMP) was identied from the

insoluble fraction of the ligament in P. fucata (Kubota et al. 2017). This report

shows the function of matrix metalloproteinase (MMP) for the formation of the

ligament.

K. Kubota et al.

369

39.2 Materials andMethods

We used the BLAST search method to identify the MMP genes. We also prepared

three types of pearl oyster samples to synthesize the cDNA for the templates of

RT-PCR: sample 1 received no wound as a control, sample 2 received a wound on

the shell by breaking the edge of the outer side of the shell with a nipper as the non-

specic injured control, and sample 3 received a wound on the ligament from the

outside to the inside with a cutter. After making a wound, each oyster was cultured

in about 10L seawater for 36h. The PCR used the following cycling conditions: 35

cycles of 30s at 94 °C (3min 30s for the rst cycle), 30s at 55 °C, and 30s at

72°C.The expression level of each MMP was checked by agarose gel electropho-

resis of the PCR products. The gel contained 0.004% ethidium bromide so that

amplied DNA could be visualized. After the electrophoresis, the uorescence of

the gel was observed under the exposure of the light at the wavelength of 365nm.

dsRNAs of MMP genes were prepared for the RNAi experiments. 30μg of each

dsRNA was dissolved with 50μL PBS buffer and injected to adductor muscle of

Pinctada fucata. We used four young oysters (7cm in shell size) in this experiment,

because it was necessary to observe the growth surface of the ligament. Four days

after injection, the growth surface of the ligament was observed by scanning elec-

tron microscope (SEM). We observed the ligament growth edges of three individu-

als using SEM independently. These observations showed similar results. Table39.1

shows the list of primers used in these experiments.

Table 39.1 Primer sequences for MMPs

21914-5 AAAGAAAACTACAGACAAAG

21914-3 GTTTTACGGCGAGGTATTAT

07860-5 AGCTACACAAAGGTATGCCT

07860-3 ATAGCATCGAATTTCATGTT

60936-5 TGGTGATGCTGACATTATGA

60936-3 TCCTCCTGGAGTTTCCAGTA

32404-5 AAGTGGTCAGATGTGACGCC

32404-3 ATCTGTCATTAAGTTTGACG

23659-5 CGTAGCGGCCCACGAGTTTG

23659-3 TTGGGGTACCCAGGTGGGGG

54089-5 AAGCTTGTCTTTCGCACCTC

54089-3 TAAAGAGTTTGTATTCCTCT

14973-5 AAAACAAGTAGAAAATGCAA

14973-5 TTAAGTAATCTCCATCGAAT

54089-5(ds) GGGTAATACGACTCACTATAGGGAGCAGTTTAACTTAGGACCA

54089-3(ds) GGGTAATACGACTCACTATAGGGTTAGGATCATATCCAGCGTA

14973-5(ds) GGGTAATACGACTCACTATAGGGTCACCATTTCCAGATGTCAT

14973-3(ds) GGGTAATACGACTCACTATAGGGTCAAAGAATATCAAAAGAGG

39 Functional Analyses ofMMP Genes intheLigament ofPinctada fucata

370

39.3 Results

39.3.1

MMP Genes inP. fucata

Blast search showed seven MMP genes in the P. fucata genomic database (Takeuchi

etal. 2012): pfu_aug1.0_21.1_21914 (21914), pfu_aug1.0_1282.1_07860 (07860),

pfu_aug1.0_11733.1_60936 (60936), pfu_aug1.0_14122.1_32404 (32404), pfu_

aug1.0_4795.1_23659 (23659), pfu_aug1.0_14629.1_54089 (54089), and pfu_

aug1.0_322.1_14973 (14973) (Fig. 39.1). Among these MMPs, some conserved

domains were identied. All MMPs in the database have the MMP superfamily

domain that contains the activity site of MMP. MMP21924, MMP07862,

MMP32404, MMP54089, and MMP14973 have a putative peptidoglycan-binding

domain. No putative peptidoglycan-binding domain is found in human MMPs.

MMP21914, MMP07860, MMP23659, and MMP14973 have hemopexin-like

repeat domain, which is also found in most of human MMPs. Only MMP14973 has

a signal peptide in the N-terminal sequence.

MMP21914

MMP0786

MMP60936

MMP32404

MMP23659

MMP5408

MMP1497

(539 aa

)

(802 aa

)

(382 aa

)

(294 aa

)

(329 aa

)

(453 aa

)

(680 aa

)

100aa

Fig. 39.1 The domain structures of MMPs from P. fucata. MMP21914 consists of a putative

peptidoglycan-binding domain, an MMP superfamily domain, and hemopexin-like repeats.

MMP07860 consists of a putative peptidoglycan-binding domain, an MMP superfamily domain,

and hemopexin-like repeats. MMP60936 consists of only an MMP superfamily domain.

MMP32404 contains of a putative peptidoglycan-binding domain and an MMP superfamily

domain. MMP23659 consists of an MMP superfamily domain and hemopexin-like repeats.

MMP54089 consists of a putative peptidoglycan-binding domain and an MMP superfamily

domain. MMP14973 consists of a putative peptidoglycan-binding domain, an MMP superfamily

domain, and hemopexin-like repeats. Black boxes show the putative peptidoglycan-binding

domain. Dotted boxes show the hemopexin-like repeats. Slashed boxes show the MMP superfam-

ily domain. Gray box shows the signal peptide

K. Kubota et al.

371

39.3.2 MMP Gene Expressions inResponse toWounds

To further investigate the expression level of each MMP in the mantle isthmus, the

wound repair process was investigated, and MMP expression levels during this pro-

cess were compared (Fig.39.2). We assumed that expression of the MMP related to

formation of the ligament would increase during the wound repair response of the

ligament. Living pearl oysters were cultured in an aquarium for 1week at 20 °C

after the shell had been damaged by a knife. We used three treatment conditions: a

control without any wounds, a wound on the shell margin, and a wound on the liga-

ment. Signicant changes in the expression levels of MMPs 32404, 23659, and

54089 were detected under these three conditions. Although the expression of

MMP32404 decreased when the oyster was wounded on its shell margin, the expres-

sion levels in the control was higher than that in the ligament wound treatments,

indicating that MMP32404 did not play a role in regeneration of the ligament. The

expression level of MMP23659 increased after both shell margin and ligament

wounds, indicating that MMP23659 has a non-specic wound-repair function in the

shell. The expression level of MMP54089 increased only in the ligament wound

treatment, indicating that MMP54089 plays a role specically in regeneration of the

ligament.

39.3.3 RNAi Experiment

To investigate the function of MMP54089in the ligament, an RNAi experiment was

performed. dsRNA targeting MMP54089 was injected into four living pearl oysters,

which were then cultured in an aquarium for 4days at 20°C.The hinge ligament is

attached to the nacreous layer of the shell (Fig.39.3a, b). The growing edge of the

ligament was observed by SEM 4days after injection. In the control treatment of

actin

21914

07860

60936

32404

23659

54089

14973

1 2 3

3240

236

1 2 3

Fig. 39.2 RT-PCR

analysis of MMP

expression after wound

treatment. (1) Unwounded

control, (2) shell wounded

with a nipper, (3) ligament

wounded with a cutter

39 Functional Analyses ofMMP Genes intheLigament ofPinctada fucata

372

(EGFP)-specic dsRNA injection, the tips of aragonite crystals were observed as

white dots along the surface of the growing edge (Fig.39.3c). Similar microstruc-

tures were observed after injections with MMP14973 dsRNA (Fig.39.3d). In con-

trast, disordered microstructure was observed after MMP54089-specic dsRNA

injection (Fig.39.3e). The disordered organic matrix exhibited a wavy surface, and

the aragonite crystals were not aligned, suggesting that MMP54089 plays a key role

in the regulated formation of the brous microstructure of the ligament.

39.4 Discussion

Two hypotheses regarding the functions of PfTIMP and MMP54089in the ligament

were formulated. First, MMP54089 may degrade extracellular organic bers to

soften them and increase the space between them. The loosened extracellular

organic bers tend to adjust to the same orientation. A previous report showed that

physical stress increased the activity of human MMP2 as well as its expression and

also increased the toughness of a blood vessel analog in a cell-seeded collagen gel

(Seliktar etal. 2003). A similar phenomenon may occur in the ligament of P. fucata.

MMP54089 of P. fucata degrades extracellular organic bers in the ligament and

Fig. 39.3 (a) The shell of P. fucata. The white rectangle showed the region observed in (b). (b)

SEM observations of the ligament and nacreous layer. (c) SEM observations of the surface in the

ligament’s growing edge in the sample of EGFP-dsRNA injection. (d) The surface in the liga-

ment’s growing edge in the sample of MMP14973-dsRNA injection. (e) The surface in the liga-

ment’s growing edge in the sample of MMP54089-dsRNA injection

K. Kubota et al.

373

arranges them to regulate the direction of their growth. In the second hypothesis,

MMP54089 digests proteins to prepare the peptides that contribute to calcium car-

bonate crystallization. A previous work suggested that amelogenin in the enamel of

human teeth is digested by human MMP20, and the peptide fragments of amelo-

genin promote the crystallization of calcium phosphorus in the teeth (Vuk et al.

2011). MMP54089 may digest some matrix proteins such as LICP to prepare the

released peptides for calcium carbonate crystallization (Suzuki etal. 2015). Further

investigation is required to reveal the relationships among PfTIMP, MMP54089,

extracellular organic bers, and calcium carbonate crystallization.

References

Bevelander G, Nakahara H (1969) An electron microscope study of the formation of the ligament

of Mytilus edulis and Pinctada radiata Calcif. Tiss Res 4:101–112

Kahler G, Sass R, Fisher F (1976) The ne structure and crystallography of the hinge ligament of

Spisula solidissima (Mollusca: Bivalvia: Mactridae). JComp Physiol 109:209–220

Kubota K, Tsuchihashi Y, Kogure T, Maeyama K, Hattori F, Kinoshit S, Sakuda S, Nagasawa H,

Yoshimura E, Suzuki M (2017) Structural and functional analyses of a TIMP and MMP in the

ligament of Pinctada fucata. JStruct Biol 199:216–224

Marsh M, Sass R (1980) Aragonite twinning in the molluscan bivalve hinge ligament. Science

208:1262–1263

Seliktar D, Nerem R, Galis Z (2003) Mechanical strain-stimulated remodeling of tissue-engineered

blood vessel constructs. Tissue Eng 9:657–666

Suzuki M, Kogure T, Sakuda S, Nagasawa H (2015) Identication of ligament intra-crystalline

peptide (LICP) from the hinge ligament of the bivalve, Pinctada Fucata. Mar Biotechnol

17:153–161

Takeuchi T, Kawashima T, Koyanagi R, Gyoja F, Tanaka M, Ikuta T, Shoguchi E, Fujiwara M,

Shinzato C, Hisata K, Fujie M, Usami T, Nagai K, Maeyama K, Okamoto K, Aoki H, Ishikawa

T, Masaoka T, Fujiwara A, Endo K, Endo H, Nagasawa H, Kinoshita S, Asakawa S, Watabe S,

Satoh N (2012) Draft genome of the pearl oyster Pinctada fucata: a platform for understanding

bivalve biology. DNA Res 2:117–130

Vuk U, Feroz K, Haichuan L, Halina E, Li Z, Wu L, Stefan H (2011) Hydrolysis of amelogenin by

matrix metalloproteinase-20 accelerates mineralization invitro. Arch Oral Biol 56:1548–1559

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

39 Functional Analyses ofMMP Genes intheLigament ofPinctada fucata

375

Chapter 40

Chitin Degraded by Chitinolytic Enzymes

Induces Crystal Defects ofCalcites

HiroyukiKintsu, TaigaOkumura, LumiNegishi, ShinsukeIfuku,

ToshihiroKogure, ShoheiSakuda, andMichioSuzuki

Abstract Mollusk shells have unique microstructures and mechanical properties

such as hardness and exibility. Calcite in the prismatic layer of P. fucata is

extremely tough due to small crystal defects and localized organic networks inside

calcites. Electron microscopic observations have suggested that such crystal defects

are caused by the organic networks during calcite formation. Our previous work

reported that the chitin which is the main component of organic networks and chi-

tinolytic enzymes that bind to chitin were identied. In this article, to investigate the

effects of chitin and chitinolytic enzymes on the formation of calcites, calcites were

synthesized in chitin gel after treatment with chitinolytic enzymes. Chitin bers

seemed to become smooth and loosened after degradation. The crystal defects

became larger as the chitin bers became more degraded by chitinolytic enzymes in

a dose-dependent manner. These results suggest that the shape of chitin ber, which

is regulated by the degradation of chitinolytic enzymes, contributes to the formation

of small crystal defects.

Keywords Biomineralization · Prismatic layer · Chitinase · Chitin · Pinctada

fucata

H. Kintsu · S. Sakuda · M. Suzuki (*)

Department of Applied Biological Chemistry, Graduate School of Agricultural

and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

e-mail: asakuda@mail.ecc.u-tokyo.ac.jp; amichiwo@mail.ecc.u-tokyo.ac.jp

T. Okumura · T. Kogure

Department of Earth and Planetary Science, The University of Tokyo, Tokyo, Japan

e-mail: okumura@eps.s.u-tokyo.ac.jp; kogure@eps.s.u-tokyo.ac.jp

L. Negishi

Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan

e-mail: lnegishi@iam.u-tokyo.ac.jp

S. Ifuku

Department of Chemistry and Biotechnology, Graduate School of Engineering,

Tottori University, Tottori, Japan

e-mail: sifuku@chem.tottori-u.ac.jp

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_40

376

40.1 Introduction

Biominerals are biogenic mineralized tissues containing a small amount of organic

matrices which regulate crystal nucleation, orientation, polymorphism, and mor-

phology of inorganic substances (Belcher etal. 1996; Chen etal. 2008; Falni etal.

1996; Weiner and Addadi 1997). The shell of Pinctada fucata, Japanese pearl oys-

ter, has two layers: prismatic and nacreous layer. The prismatic layer focused on in

this study is composed of calcite prisms that are surrounded by thick polygonal

organic frameworks as intercrystalline organic matrices. Each prism in the prismatic

layer of P. fucata consists of several small calcites. Scanning electron microscope

(SEM) and transmission electron microscope (TEM) analyses have revealed that

each calcite contains subgrain units of several hundred nanometers divided by crys-

tal lattice distortions known as small-angle grain boundaries (Okumura etal. 2012,

2013); however, the crystal structures of adjacent grains were continuous. Calcites

that contain such crystal defects are tougher and stiffer than single calcites of

another type of shell that contain no crystal defects because the defects inhibit the

propagation of cracks (Olson etal. 2013). TEM observations have also revealed that

organic matrices inside the small calcite of P. fucata prisms are localized in net-

works (Okumura etal. 2012). The location of the organic network correlates with

the location of the crystal defects, indicating that the organic network affects the

formation of the crystal defects. Our previous report claried chitin and chitinolytic

enzyme as the components of this organic network (Kintsu etal. 2017), in which we

indicated that the chitin treated by chitinolytic enzymes may induce the formation

of crystal defects.

In this article, we performed calcium carbonate crystallization using chitin

hydrogel and chitinolytic enzymes in vitro to assess the effects of such organic

matrices on lattice distortion inside the synthesized crystals.

40.2 Materials andMethods

Calcium carbonate crystallization in the chitin hydrogel. Chitin hydrogel was pre-

pared according to the previous method (Tamura et al. 2006). The prepared chitin

hydrogel was incubated with Yatalase (an enzyme complex containing chitinase and

chitobiase activities from Corynebacterium sp. OZ-21; TaKaRa) as chitinolytic

enzyme for 24h. After incubation, chitin hydrogel was ltered and washed with

10mM calcium chloride to remove Yatalase. The chitin hydrogel was spread on a

plate and put into desiccator lled with the gas of 5 g of ammonium carbonate

(Kanto Chemical) to crystallize calcium carbonate in the chitin hydrogel for 24h.

Chitin hydrogel was dissolved in 50% sodium hypochlorite to collect crystals.

Calcium carbonate crystallization using chitin nanober. A chitin nanober

solution (1.1% (w/v) was prepared according to the previous report (Ifuku etal.

2010). A solution of 10mM calcium chlorite was added to the chitin nanober

H. Kintsu et al.

377

solution, followed by calcium carbonate crystallization in the chitin nanober solu-

tion using the same method described above.

Fixation of chitin gel. Chitin gel was xed by using the method of cell xation

with glutaraldehyde, osmium tetroxide, and potassium permanganate for reference

with some modications (Gunning 1965). Chitin gel was incubated sequentially in

glutaraldehyde, osmium tetroxide, and potassium permanganate solution for 1h.

After xation, the sample was dehydrated by ethanol and embedded in Spurr resin

(Polysciences, Inc., USA). Sections prepared by ultramicrotome using a diamond

knife.

Variance of lattice spacing determined from XRD spectra. The variance of lattice

spacing in calcite crystal was estimated from peak broadening of XRD spectra using

a RINT-Ultima+ diffractometer (Rigaku) with graphite-monochromated Cu Kα

radiation emitted at 40kV and 20mA according to the method reported previously

(Okumura etal. 2012). The variance of lattice spacing (Δd/d) of the samples was

calculated using Williamson-Hall plot (Williamson and Hall 1953) as follows:

094

()

cos/sin.

40.3 Results andDiscussion

40.3.1 Observation ofChitin Fibers by TEM

In an initial phase of prism formation, after the organic frameworks in the prismatic

layer are constructed, the space surrounded by the organic framework is lled with

organic gel solution composed mainly of chitin and matrix proteins; calcium car-

bonate is then crystallized with organic gel solution in the space. The organic gel

solution such as chitin and chitinolytic enzymes we identied can develop into

organic networks during calcite crystallization and affect the crystallization to form

small-angle grain boundaries. Therefore, we performed an invitro calcium carbon-

ate crystallization experiment using chitin hydrogel and chitinolytic enzymes.

Chitin hydrogel was prepared by dissolving chitin powder in methanol saturated

with calcium chloride dehydrate. To compare the differences of chitin bers between

before and after treatment with chitinolytic enzymes at concentration of 1.2mg/mL,

the chitin gels were observed by using TEM.Chitin gel was xed according to a

chemical xation procedure for physiological tissues in order to observe in natural

condition. TEM images of the chitin gels showed that a lot of chitin bers of a few

dozen of nanometer were observed in both conditions and no apparent differences

of thinness and length could be seen (Fig.40.1). However, while chitin bers with-

out treatment of chitinolytic enzymes became entangled with each ber (Fig.40.1a,

b), chitin bers with treatment of chitinolytic enzymes became smooth and loos-

ened, not entangled (Fig.40.1c, d), indicating that the chitinolytic enzymes may

40 Chitin Degraded by Chitinolytic Enzymes Induces Crystal Defects ofCalcites

378

degrade branched molecular chains of chitin which was getting entangled with each

other. We suggest from this result that chitin bers are loosened by chitinolytic

enzymes and become such organic network as observed in the prism of P. fucata.

40.3.2 Synthesis ofCalcium Carbonate Crystals inChitin

Hydrogel Treated withChitinolytic Enzymes

To investigate how chitin and chitinolytic enzymes have effects on the formation of

the small crystal defects in single calcites, calcium carbonate was crystallized in the

chitin hydrogel with or without Yatalase which is a commercially available chitino-

lytic enzyme produced by a Streptomyces strain. After crystallization in the chitin

hydrogel, calcium carbonate crystals were collected and observed by SEM.The

normal shape of calcium carbonate crystals (a typical rhombohedral calcite) was

formed in the chitin hydrogel without Yatalase treatment (Fig.40.2a). In contrast, in

the chitin hydrogel treated with 1.2mg/mL chitinolytic enzyme, the shape of the

crystal was completely changed and appeared to be round (Fig.40.2b). As the con-

centration of chitinolytic enzymes increased, the chitin ber became thinner, and

Fig. 40.1 TEM images of chitin gel. (a) Chitin gel before treatment with chitinolytic enzymes and

(b) its high magnication. (c) Chitin gel after treatment with chitinolytic enzymes at the concentra-

tion of 1.2mg/mL and (d) high magnication

H. Kintsu et al.

379

the surface microstructure of the calcite crystals in the chitin hydrogel changed.

These results enabled us to guess that the thinness of chitin is a key factor to induce

crystal defects.

Although it is indicated that thinness and length of chitin ber are important, it is

difcult to achieve a uniform thickness and length of chitin bers using chitinolytic

enzymes. Chitin nanobers prepared by mechanical cleavage were used because the

chitin nanober has a uniform thickness of several dozens of nanometers. Calcium

carbonate was precipitated in the chitin nanober solution using the same method

as described above without chitinolytic enzymes. SEM image of formed calcium

carbonate crystals showed that chitin nanober can also affect crystallization and

the surface microstructure became polygonal (Fig.40.2c).

40.3.3 X-Ray Diffraction (XRD) Analyses ofCalcite Crystals

To estimate the reason for the crystal defects among synthesized calcite crystals,

XRD spectra of calcite samples were analyzed to calculate the variance of lattice

spacing (Δd/d) as crystal defects. Figure40.3 shows the ratio of Δd/d gained from

Williamson-Hall plots (data not shown) of each crystal synthesized in the presence

of chitin pretreated with 0, 1.2mg/mL chitinolytic enzymes, chitin nanobers, and

P. fucata prisms. At concentrations of 0, the Δd/d value was very low and showed

few crystal defects. In contrast, at a concentration of 1.2mg/mL, the Δd/d value was

high, and the Δd/d value in the chitin nanober was nearly equal to that observed at

a concentration of 1.2mg/mL.However, the Δd/d value in the P. fucata prisms was

approximately 1.7-fold higher than that observed at a concentration of 1.2mg/mL.

These results showed that lattice distortion became larger as the chitin ber

became thinner. Although the data are not shown, TEM observation of the cross

section of the calcite claried that thinner chitin bers were more easily embedded

in calcium carbonate crystals. However, the lattice distortion ratio of synthesized

calcite was still much smaller than that of prism calcite. This is perhaps because

there are many acidic matrix proteins identied from the prismatic layer (Gotliv

etal. 2005; Suzuki etal. 2004). Such proteins may also contribute to the increase in

the lattice distortion ratio in calcite prisms.

Fig. 40.2 The calcite crystals synthesized under different conditions. Calcite crystal formed in the

chitin hydrogel treated with chitinolytic enzymes at concentrations of (a) 0 mg/mL, and (b)

1.2mg/mL, and (c) in the chitin nanober solution

40 Chitin Degraded by Chitinolytic Enzymes Induces Crystal Defects ofCalcites

380

Taken together, chitin degradation plays an important role in the formation of

calcite prisms in the prismatic layer. Crystal defects became larger as the chitin

bers were degraded by chitinolytic enzymes, because the increasing surface area

of chitin bers strengthens the physical and/or chemical interaction between cal-

cium carbonate and the chitin ber. This strong interaction may allow chitin bers

to attach to the crystal growth front in a random manner, which prevents calcium or

carbonate ions from being well-oriented around crystal growth planes, leading to

the crystal lattice distortion. Therefore, in the prismatic layer of P. fucata, the thin-

ness of chitin ber may be precisely regulated by chitinolytic enzyme to induce the

small-angle grain boundaries. This novel mechanism may provide useful insights to

the elds of biomineralization and biomimetic material engineering.

References

Belcher AM, Wu XH, Cristensen RJ, Hansma PK, Stucky GD, Morse DE (1996) Control of crystal

phase switching and orientation by soluble mollusc-shell proteins. Nature 381:56–58

Chen PY, Lin AY, McKittrick J, Meyers MA (2008) Structure and mechanical properties of crab

exoskeletons. Acta Biomater 4:587–596

Falini G, Albeck S, Weiner S, Addadi L (1996) Control of aragonite or calcite polymorphism by

mollusk shell macromolecules. Science 271:67–69

Gotliv BA, Kessler N, Sumerel JL, Morse DE, Tuross N, Addadi L, Weiner S (2005) Asprich: a

novel aspartic acid-rich protein family from the prismatic shell matrix of the bivalve Atrina

rigida. Chem Bio Chem 6:304–314

Gunning BES (1965) The ne structure of chloroplast stroma following aldehyde osmium-

tetroxide xation. JCell Biol 24:79



  

0 mg/ml

1.2 mg/ml

chitin nanofiber

prism of

P. fucata

Fig. 40.3 Variance of lattice spacing calculated from Williamson-Hall plots. The synthesized cal-

cites in the chitin hydrogel treated with chitinolytic enzymes at the concentrations of 0, 1.2mg/

mL, and in the chitin nanober solution, and prisms of the prismatic layer. Error bars indicate the

standard deviation of the measurements (n=3)

H. Kintsu et al.

381

Ifuku S, Nogi M, Yoshioka Y, Morimoto M, Yano H, Saimoto H (2010) Fibrillation of dried chitin

into 10–20nm nanobers by a simple grinding method under acidic conditions. Carbohydr

Polym 81:134–139

Kintsu H, Okumura T, Negishi L, Ifuku S, Kogure T, Sakuda S, Suzuki M (2017) Crystal defects

induced by chitin and chitinolytic enzymes in the prismatic layer of Pinctada fucata. Biochem

Biophys Res Commun 489:89–95

Okumura T, Suzuki M, Nagasawa H, Kogure T (2012) Microstructural variation of biogenic calcite

with intracrystalline organic macromolecules. Cryst Growth Des 12:224–230

Okumura T, Suzuki M, Nagasawa H, Kogure T (2013) Microstructural control of calcite via incor-

poration of intracrystalline organic molecules in shells. JCryst Growth 381:114–120

Olson IC, Metzler RA, Tamura N, Kunz M, Killian CE, Gilbert PU (2013) Crystal lattice tilting in

prismatic calcite. JStruct Biol 183:180–190

Suzuki M, Murayama E, Inoue H, Ozaki N, Tohse H, Kogure T, Nagasawa H (2004) Characterization

of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster

(Pinctada fucata). Biochem J382:205–213

Tamura H, Nagahama H, Tokura S (2006) Preparation of chitin hydrogel under mild conditions.

Cellulose 13:357–364

Weiner S, Addadi L (1997) Design strategies in mineralized biological materials. JMater Chem

7:689–702

Williamson G, Hall W (1953) X-ray line broadening from led aluminium and wolfram. Acta

Metall 1:22–31

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

40 Chitin Degraded by Chitinolytic Enzymes Induces Crystal Defects ofCalcites

383

Chapter 41

Screening forGenes Participating

intheFormation ofPrismatic

andNacreous Layers oftheJapanese Pearl

Oyster Pinctada fucata by RNA

Interference Knockdown

DaisukeFunabara, FumitoOhmori, ShigeharuKinoshita, KiyohitoNagai,

KaoruMaeyama, KikuhikoOkamoto, SatoshiKanoh, ShuichiAsakawa,

andShugoWatabe

Abstract Many genes have been identied to participate in the shell formation so

far. Nevertheless, the whole picture of the molecular mechanisms underlying the

shell formation has remained unknown. In our previous study, we analyzed compre-

hensively genes expressed in the shell-producing tissues and identied 14 genes to

be involved in the shell formation by the RNA interference (RNAi) method. In the

present study, we performed further screening to nd additional novel genes

involved in the formation of the nacreous and prismatic layers. We here selected 80

genes from the EST data as candidates to function in the shell formation, conducted

knockdown experiments by the RNAi method, and observed surface appearances on

the nacreous and prismatic layers. We newly identied 64 genes that could partici-

pate in the shell formation. Taken together with our previous study, 78 genes were

D. Funabara (*) · S. Kanoh

Graduate School of Bioresources, Mie University, Tsu, Mie, Japan

e-mail: funabara@bio.mie-u.ac.jp; kanoh@bio.mie-u.ac.jp

F. Ohmori · K. Maeyama · K. Okamoto

MIKIMOTO COSMETICS, Mie, Japan

e-mail: oomori.353@mikimoto-cosme.com; maeyama.511@mikimoto-cosme.com;

okamoto.632@mikimoto-cosme.com

S. Kinoshita · S. Asakawa

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,

The University of Tokyo, Tokyo, Japan

e-mail: akino@mail.ecc.u-tokyo.ac.jp; asakawa@mail.ecc.u-tokyo.ac.jp

K. Nagai

Pearl Research Laboratory, K.MIKIMOTO & CO., LTD., Hamajima, Shima, Mie, Japan

S. Watabe

School of Marine Biosciences, Kitasato University, Minami, Sagamihara, Kanagawa, Japan

e-mail: swatabe@kitasato-u.ac.jp

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_41

384

supposed to function in the shell formation. These ndings indicate that the

combination of transcriptome and knockdown analyses is a powerful tool to screen

novel genes involved in the shell formation.

Keywords EST · Knockdown · Nacreous layer · Pearl oyster · Prismatic layer ·

RNAi · Shell

41.1 Introduction

Many genes have been identied to participate in the shell formation so far. In clas-

sical ways, proteins were puried from shells after decalcication and their proper-

ties were analyzed. Nacrein, for instance, was puried from shells of the Japanese

pearl oyster Pinctada fucata and characterized in detail (Miyamoto etal. 1996).

Suzuki etal. (2009) employed the RNA interference (RNAi) method to elucidate

possible functions of Pif discovered as an aragonite-binding protein in the shell of

P. fucata. Knockdown of the Pif gene by the RNAi method induced an abnormal

crystal structure of aragonite. This nding conrmed that Pif is really involved in

the nacreous layer formation and proved that the RNAi method is useful to study

genes involved in shell formation. We obtained the EST data of nacreous and pris-

matic layer-producing tissues of P. fucata, which contained 29,682 genes, and found

novel 29,550 genes (Kinoshita etal. 2011). Genes involved in the shell formation

must be contained in these genes. Thus, we compared gene expression patterns

among mantle pallium, edge, and pearl sac tissues using the EST data to nd genes

expressed in a tissue-specic manner. We selected ve genes specically expressed

in the mantle pallium, three highly expressed in the mantle pallium and pearl sac,

and six specically expressed in the mantle edge as candidates to function in shell

formation. Knockdown experiments for these candidate genes induced abnormal

appearances on the inner surface of the shells in the oysters (Funabara etal. 2014).

These ndings demonstrated that a combination of transcriptome analyses and

RNAi knockdown is a powerful tool to screen genes involved in the shell formation.

In the present study, we conducted further screening for genes involved in the shell

formation of P. fucata using the above method.

41.2 Materials andMethods

We selected 195 genes having more than 200 reads from the EST data (Kinoshita

etal. 2011) of the shell-forming tissues, along with 9 genes expressed similarly to

those known to be involved in the shell formation from genes having less than 200

reads in the EST data. We conducted cDNA cloning of the selected genes with prim-

ers designed using the nucleotide sequences of respective genes. dsRNAs of the

selected genes were synthesized using the cDNA clones as templates with a

ScriptMAX™ Thermo T7 Transcription Kit (Toyobo, Osaka, Japan). About 40μg

of dsRNA/100 μl H2O were injected into adductor muscles of 2-year-old pearl

D. Funabara et al.

385

oysters (n=3), followed by rearing them in articial seawater at 23°C for 8days

with feeding plankton once a day. The green uorescence protein (GFP) and Pif

genes were used as negative and positive references, respectively, to verify the

RNAi experiments. Surface appearances of the prismatic and nacreous layers on the

shells of the knockdown oysters were observed with a scanning electron microscope

(SEM), S-4000 (Hitachi, Tokyo, Japan).

41.3 Results

41.3.1 Selection ofCandidate Genes Functioning inShell

Formation

We selected candidate genes having more than 200 reads in the EST data (Kinoshita

etal. 2011) to be possibly involved in the shell formation, except for 14 genes which

we analyzed in our previous study (Funabara etal. 2014) (Table41.1). cDNAs of 71

genes out of the selected 181 genes above were successfully cloned and used for

synthesizing dsRNAs as templates. We selected additionally 9 genes showing

expression patterns similarly to those of known shell formation-related genes such

as PFMG1, KRMP1, N19, and N16 series from those having less than 200 reads

(Table41.1). cDNAs of all the nine genes were cloned and used for the synthesis of

dsRNAs. A total of 80 genes were subjected to the knockdown experiments.

41.3.2 Observation oftheAppearances ontheInner Surface

oftheKnockdown Oyster Shells

Knockdown of 64 out of 80 genes induced abnormal appearances on the inner sur-

face of the shells (Table41.2). Among them, 18 knockdown oysters had abnormal

appearances on both the prismatic and nacreous layers, 45 only on the nacreous lay-

ers, and 1 only on the prismatic layers. The data combined with our previous study

are shown in Fig.41.1. Ninety-four genes, 80in the present and 14in our previous

studies, contained 78 genes that are suggested to be involved in the shell formation

processes. Only one gene changed the surface appearance on the prismatic layer.

41.4 Discussion

We have obtained the data of gene expression patterns and genes possibly involved

in shell formation (Tables 41.1 and 41.2). It is not easy to discuss how genes play

roles in shell formation based on expression patterns in the EST and knockdown

data. We have only short sequences of the respective genes in the EST data. Full-

length sequences or at least open reading frame (ORF) regions of the interest genes

41 Screening forGenes Participating intheFormation ofPrismatic andNacreous…

386

Table 41.1 Gene expression patterns in shell- and pearl-forming tissues

Genea

TPM Total

reads Gene

TPM Total

reads Gene

TPM Total

reads Gene

TPM Total

readsME MP PS ME MP PS ME MP PS ME MP PS

1b6042 6381 7207 1728 52b1456 1386 93 226 104 1922 908 148 224 179 1456 1028 1129 308

2b12,142 17,827 1240 24,620 53b1922 1529 3099 595 105 1267 1063 805 263 187 1834 2282 0 317

3b4833 4672 1351 869 54b1485 1159 3238 549 106 1150 920 1933 365 188 772 1123 2396 406

4 2737 1410 5301 879 55b1470 1326 2923 528 107 2329 2342 2156 589 190 903 836 805 219

5b4062 4146 28 629 56b1776 1529 0 250 108b815 729 2535 391 191 466 430 1480 228

6b3479 2449 5458 1034 57b2538 2951 0 409 109 961 478 1628 282 193 1267 1195 1018 297

7b3130 3644 4200 974 58c2635 275 0 204 110 1616 1482 1286 374 194c2751 1028 0 275

8b1994 2892 185 399 59b2140 2426 629 418 111b1601 1864 1008 375 196 597 347 3349 432

9b3261 2366 19 424 60b2519 1937 1415 463 112b975 1135 601 227 197 1092 1267 722 259

10b2737 2892 111 442 61b1529 1338 3654 612 113c1019 2653 56 298 200c2227 789 0 219

11b3494 2844 65 485 62b2373 1852 2563 595 114b2504 1470 2082 520 209 189 72 1878 222

12b2227 3597 315 488 63b3712 2438 0 459 115b1529 1302 1758 404 215 1631 1338 0 224

13b3217 1972 130 400 64b3217 2461 1878 630 116 2009 1517 0 265 216 932 1111 490 210

14b2125 1816 3173 641 65b3072 1972 0 376 118c641 1924 102 216 218 670 789 860 205

15b5008 3023 1739 785 66c2853 48 0 200 121 830 657 1813 308 228 1325 1350 0 204

16b2009 2593 5107 907 67b2053 1625 3312 635 122 1077 1171 786 257 237 1194 1446 28 206

17b2504 2665 4431 874 68b2737 1470 2285 558 123 1077 753 1147 261 243 1529 1075 361 234

18 1732 1995 3007 611 69b3523 3190 4496 995 124 728 693 1452 265 248 1441 1099 333 227

19 2533 1613 5097 860 70b757 1051 833 230 125 1689 1147 2720 506 250 1296 1338 1332 345

20b2038 1804 3626 683 71b1878 1972 1480 454 127 1791 2246 2017 529 252 1252 1506 0 212

21 1470 1995 5190 829 72b3028 143 0 220 128 1820 1852 1092 398 268 903 1016 1425 301

22b2475 1697 3423 682 73 1354 2031 481 315 129 2038 1482 2044 485 272 1936 1386 37 253

23b2358 2210 3830 761 74b2795 2043 111 375 130 1034 1816 648 293 274 684 633 1471 259

24b2198 1888 4330 777 75b2562 1936 2868 648 132 1194 1565 259 241 292 903 789 1018 238

25b2737 3405 0 473 76b2082 1912 2646 589 133c903 1804 46 218 300 611 203 1369 207

26b2286 2306 4459 832 77b2373 2414 65 372 134 888 930 962 243 301 1616 1290 0 219

D. Funabara et al.

387

27c641 1744 3432 561 78 1441 1410 1721 403 136 1383 944 2054 396 323 1485 1147 83 207

28b3669 3967 0 584 79b2795 2497 2812 705 137 1237 2031 1600 428 336 1092 442 1221 244

29b2417 2115 3867 761 80 2679 2139 1915 570 138 1558 1434 56 233 344 1310 1876 916 346

30b2868 2270 5005 928 81c364 1625 463 211 139 1893 1517 1767 448 384 859 1040 1203 276

31c4586 1267 0 421 82 2053 2605 583 422 141 1252 1577 0 218 395 58 36 2618 290

32b2519 3202 2877 752 83 1776 1972 851 379 143 1558 2031 65 284 399 1441 1673 333 275

33b2446 1613 3719 705 84 1339 2210 453 326 145c4047 167 0 292 407 1325 1517 296 250

34b2067 2151 786 407 85 1689 2402 1295 457 147 1150 1398 361 235 411c131 211 259 214

35b4367 2784 6642 1251 86 1412 1840 546 310 148 1601 1601 0 244 3840 0 0 2812 304

36b2868 2258 2655 673 87 1645 693 1878 374 150 1747 1374 2461 501 3969 0 0 1896 205

37b4906 4756 0 735 88 1150 36 1147 206 152 670 442 3275 437 4121 0 0 1896 205

38b1951 1685 1970 488 89 1456 2031 1610 444 154 670 609 1129 219 4600 0 0 2109 228

39b2140 1995 2997 638 90 1005 621 1018 231 155 1063 741 814 223 5656 0 0 2017 218

40 3188 2485 0 427 91 1208 801 1110 270 157 1398 908 1591 344 7101 0 0 2054 222

41 3596 3465 2711 830 92b1631 2342 0 308 161 1077 1804 0 225 7147 0 0 1952 211

42b2795 2772 2396 683 93 2198 2449 1795 550 162 1063 645 1480 287 11,232 0 0 1961 212

43 1922 1458 3867 672 94 1514 1398 1425 375 164 1776 1792 1230 405 390b1267 896 0 162

44b1907 2629 194 372 95 2140 1756 2738 590 165 2024 1458 1304 402 493b422 574 259 105

45b1645 1649 3210 598 96c437 1446 2701 443 166 2693 2019 0 354 496b87 585 0 55

46b2955 1900 2600 643 97 2096 1792 851 396 167 1019 1243 1489 335 1362b335 36 204 48

47b2636 2342 3034 705 98c1194 2760 194 334 168 320 454 2785 361 3968b0 550 0 4

48b2198 2856 0 390 99 1893 2175 315 346 170 742 442 1175 215 4254b0 0 1138 123

49 2446 2222 157 371 101 1718 2306 2211 550 171 742 382 1499 245 6605b0 574 0 48

50b2941 2330 3451 770 102 1310 1350 1563 372 172 495 693 1674 273 14278b0 48 0 4

51b2417 2103 3608 732 103 2315 2067 1832 530 176 1048 1040 749 240 16419b0 0 28 55

TPM templates per million, ME mantle edge, MP mantle pallium, PS pearl sac

aData and gene numbers from Kinoshita etal. (2011)

bGenes subjected to RNAi experiments in the present study

cGenes analyzed in our previous study Funabara etal. (2014)

41 Screening forGenes Participating intheFormation ofPrismatic andNacreous…

388

are required to discuss their function. To determine the full-length sequences, it is

reasonable that we choose genes in descending order of the numbers of their reads

in the EST data. We can also search the genome database for their gene models by

BLAST searching using the EST sequence data (Takeuchi etal. 2012).

Many studies on shell formation-related proteins have focused on those secreted

from mantle tissues into shells. This way is incapable of analyzing regulatory

Table 41.2 Appearances of the inner surface of shells injected with dsRNAs of the subject genes

GeneaPrismatic Nacreous Gene Prismatic Nacreous Gene Prismatic Nacreous

1 n a 39 a a 79 a a

2 a a 42 n a 92 a a

3 a a 44 n a 108 n a

5 n a 45 n a 111 n a

6 a a 46 a a 112 n a

7 a a 47 n a 114 a n

8 a a 48 n a 115 n n

9 n a 50 n a 390 n n

10 a a 51 n a 493 n n

11 a a 52 n a 496 n n

12 n a 53 n a 1362 n n

13 a a 54 n n 3968 n n

14 a a 55 n a 4254 n n

15 a a 56 n a 6605 n a

16 n a 57 n a 14,278 n n

17 n a 59 a a 16,419 n n

20 n a 60 n a 27ba a

22 n a 61 n n 31ba a

23 a a 62 n a 58bn a

24 n a 63 n a 66bn a

25 n a 64 n n 81ba a

26 n a 65 n a 96ba a

28 n a 67 n n 98ba a

29 a a 68 n a 113bn a

30 n a 69 n a 118bn a

32 n a 70 n a 133bn a

33 n a 71 n n 145bn a

34 n a 72 n n 194ba a

35 n a 74 n a 200bn a

36 a a 75 n a 411bn a

37 n a 76 n n

38 n a 77 n a

n normal appearance, a abnormal appearance

aGene numbers from Kinoshita etal. (2011)

bData from Funabara etal. (2014)

D. Funabara et al.

389

pathways to form shells. We found in our previous study that some shell formation-

related genes encoded proteins lacking a signal peptide, suggesting that such cyto-

plasmic proteins function in shell formation together with secretory ones (Funabara

etal. 2014). We have not determined the full-length sequences for the newly iden-

tied 64 genes to be involved in shell formation yet. They may contain cytoplas-

mic proteins which function in shell formation. The combination of transcriptome

and knockdown analyses would give us some useful information on the shell for-

mation processes from genes to shells.

References

Funabara D, Ohmori F, Kinoshita S, Koyama H, Mizutani S, Ota A, Osakabe Y, Nagai K, Maeyama

K, Okamoto K, Kanoh S, Asakawa S, Watabe S (2014) Novel genes participating in the forma-

tion of prismatic and nacreous layers in the pearl oyster as revealed by their tissue distribution

and RNA interference knockdown. PLoS One 9:e84706

Kinoshita S, Wang N, Inoue H, Maeyama K, Okamoto K, Nagai K, Kondo H, Hirono I, Asakawa

S, Watabe S (2011) Deep sequencing of ESTs from nacreous and prismatic layer producing

tissues and a screen for novel shell formation-related genes in the pearl oyster. PLoS One

6:e21238

Prismatic &

nacreous layers

Nacreous layers

Prismatic la

yers

Normal

appearance

Fig. 41.1 The numbers of individuals having normal and abnormal appearances on the inner sur-

face of the shells of the Japanese pearl oysters Pinctada fucata subjected to the RNAi experiments

as observed by SEM. “Prismatic and nacreous layers,” “nacreous layers,” “prismatic layers,” and

“normal appearances” indicate individuals having abnormal appearances on “both the prismatic

and nacreous layers,” “only on the nacreous layers,” “only on the prismatic layers,” and “normal

appearances” on the shell inner surface, respectively. Numerals indicate the numbers of the genes

41 Screening forGenes Par ticipating intheFormation ofPrismatic andNacreous…

390

Miyamoto H, Miyashita T, Okushima M, Nakano S, Morita T, Matsushiro A (1996) A carbonic

anhydrase from the nacreous layer in oyster pearls. Proc Natl Acad Sci U S A 93:9657–9660

Suzuki M, Saruwatari K, Kogure T, Yamamoto Y, Nishimura T, Kato T, Nagasawa H (2009) An

acidic matrix protein, Pif, is a key macromolecule for nacre formation. Science 325:1388–1390

Takeuchi T, Kawashima T, Koyanagi R, Gyoja F, Tanaka M, Ikuta T, Shoguchi E, Fujiwara M,

Shinzato C, Hisata K, Fujie M, Usami T, Nagai K, Maeyama K, Okamoto K, Aoki H, Ishikawa

T, Masaoka T, Fujiwara A, Endo K, Endo H, Nagasawa H, Kinoshita S, Asakawa S, Watabe S,

Satoh N (2012) Draft genome of the pearl oyster Pinctada fucata: a platform for understanding

bivalve biology. DNA Res 19:117–130

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

D. Funabara et al.

391

Chapter 42

Gene Expression Patterns intheMantle

andPearl Sac Tissues ofthePearl Oyster

Pinctada fucata

ShigeharuKinoshita, KaoruMaeyama, KiyohitoNagai, ShuichiAsakawa,

andShugoWatabe

Abstract The shell of pearl oysters consists of two distinct layers, nacre and pris-

matic. Mantle is the tissue involved in the shell formation, and its ventral part (man-

tle edge) forms the prismatic layers, whereas the dorsal part (pallium) forms the

nacre. In pearl culture, mantle grafts from the pallium of donor are transplanted into

the recipient. Then pearl sac is formed by proliferation of epithelial cells from the

grafted mantle to form pearls. It has been reported that gene expression patterns are

different between mantle edge and pallium in accordance with their distinct func-

tions in the shell formation. However, it is not well addressed whether gene expres-

sion is identical or not between two nacre-forming tissues, pallium and pearl sac.

Here, we examined expression patterns of known genes related to nacre and pris-

matic layer formation in mantle edge, pallium, and pearl sac of Pinctada fucata.

Although the pallium and pearl sac have the same function in terms of nacre forma-

tion, various genes were not expressed identically to the respective tissues, suggest-

ing that shell matrix proteins differently function in the formation of shell nacre and

pearls.

Keywords Pinctada fucata · Pearl · Shell nacre · Prismatic layer · Gene expres-

sion · RNA-seq

S. Kinoshita (*) · S. Asakawa

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,

The University of Tokyo, Tokyo, Japan

e-mail: akino@mail.ecc.u-tokyo.ac.jp; asakawa@mail.ecc.u-tokyo.ac.jp

K. Maeyama

Mikimoto Pharmaceutical CO., LTD, Ise, Mie, Japan

e-mail: maeyama.511@mikimoto-cosme.com2

K. Nagai

Pearl Research Laboratory, K.MIKIMOTO & CO., LTD., Hamajima, Shima, Mie, Japan

e-mail: k-nagai@mikimoto.com

S. Watabe

School of Marine Biosciences, Kitasato University, Minami, Sagamihara, Kanagawa, Japan

e-mail: swatabe@kitasato-u.ac.jp

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_42

392

42.1 Introduction

The shell of pearl oysters consists of two distinct layers: inner nacre and outer pris-

matic layers composed of aragonite and calcite crystals, respectively. The forming

processes of these different shell layers are thought to be regulated by proteins

secreted from epithelial cells in mantle tissues. The ventral part of the mantle (man-

tle edge) forms the prismatic layers, whereas the dorsal part (pallium) forms the

nacreous layers. These two regions secrete different repertoire of shell matrix pro-

teins. In pearl culture, mantle grafts from the pallium region of donor oyster are

transplanted with spherical nuclei into the recipient oysters. Pearl sac is formed by

proliferation of mantle epithelial cells originating from the mantle graft from which

various proteins are secreted to form the nacre surrounding nuclei.

Pearl consists of nacre or “mother of pearl” and is formed inside the body of

pearl oysters; thus the nature of shell nacre and pearls is considered to be identical.

On the other hand, our previous study clearly showed differences in the expression

levels of several shell matrix protein genes between pallium and pearl sac (Wang

etal. 2009). Inoue etal. (2010) investigated the expression of six shell matrix pro-

tein genes and showed that some of them were not expressed identically in pearl sac

and mantle center (nacre-forming region), although a signicant correlation was

observed in their expression patterns between the two tissues. To analyze molecular

mechanisms underlying the shell and pearl formation, it is important to examine

whether gene expression patterns are different or not between pearl sac and mantle.

Here, we compared expression pattern of known genes related to nacre (nacreous

genes) and prismatic layer formation (prismatic genes) in mantle edge, pallium, and

pearl sac of pearl oyster Pinctada fucata by using our previous RNA-seq data

(Kinoshita etal. 2011).

42.2 Materials andMethods

42.2.1 Sample Preparation

Mantle and pearl sac tissues were collected from four individuals of P. fucata main-

tained at the Mikimoto Pearl Research Laboratory, Mie, Japan. Mantle pieces were

grafted to all individuals for pearling 5months before sampling. The mantle edge

and pallium regions were separated from the mantle. Pearl sacs were collected from

gonad of pearl oysters, and contaminated recipient tissues were carefully trimmed.

All tissues, pallium, mantle edge, and pearl sacs used in this study were collected at

the same time.

S. Kinoshita et al.

393

42.2.2 RNA-Seq Analysis

mRNAs were puried from tissues. 3′-fragment sequencing was performed using

the GS FLX 454 system (Kinoshita et al. 2011). After quality trimming of raw

reads, a de novo assembly using MIRA assembler ver. 2.9.45x1 and the BLAST

Clust program from NCBI was used to assemble the reads. Known nacreous and

prismatic gene sequences were searched from assembled contig data set using the

local blastn and tblastn algorithms. Expression levels of gene sequences were

expressed by transcripts per million (TPM). Hierarchical cluster analysis was per-

formed by CLUSTER3.0 using Euclidean distance.

42.3 Results andDiscussion

42.3.1 Clustering ofExpression Patterns inThree Shell-

Formation Tissues fortheKnown Nacreous

andPrismatic Genes

Although many studies have identied genes and proteins that are related to nacre

and prismatic layer formation, the information on their functions has been limited.

Among the genes previously reported, we selected 10 nacreous and 14 prismatic

genes (Table42.1). Based on the expression patterns of these nacreous and pris-

matic genes, hierarchical clustering analysis among three tissues, mantle edge, pal-

lium, and pearl sac, was performed. As shown in Fig.42.1, pallium and pearl sac

were clustered in the same node and separated from the mantle edge, well in accor-

dance with their functions in the nacre formation or prismatic layer formation.

42.3.2 Expression ofKnown Nacreous andPrismatic Genes

Most nacreous genes examined in this study were expressed predominantly in the

pallium (Table42.1). Among them, MSI60, MSI25, and Pif177 showed the highest

expression in the pallium, suggesting their importance in the nacre formation. In

contrast, two nacreous genes, ACCBP and CaLP, were expressed in the mantle edge

more than in the pallium, suggesting their additional roles in the prismatic layer

formation. On the other hand, N66, PFMG1, and N19 family members were detected

only in pallium or pearl sac.

42 Gene Expression Patter ns intheMantle andPearl Sac Tissues ofthePearl Oyster…

394

Meanwhile, the expression levels of most known prismatic genes did not differ

between the pallium and mantle edge (Table42.1). Shematrin5 was expressed in the

pallium much greater than in the mantle edge. These prismatic genes may also play

roles in the nacreous layer formation. This is consistent with our previous report that

knockdown of prismatic genes affects the shell nacre formation in P. fucata

(Funabara etal. 2014). Jackson etal. (2010) also showed that prismatic genes such

as shematrins and KRMPs are important in the formation of the shell nacre in P.

maxima. These prismatic genes except for shematrin2 were marginally detected in

pearl sacs (Table42.1).

Table 42.1 The expression levels of known nacreous and prismatic genes

TPM

ME P PS

Nacreous genes

MSI60 131 2115 259

MSI25 29 406 120

Pif177 44 872 37

Nacrein 1019 2623 56

N66 0 36 0

PFMG1 0 36 0

ACCBP 146 96 0

CalP 553 466 56

N19-1 0 48 0

N19-2 0 0 28

Prismatic genes

Aspein 4062 4146 28

Prismalin-14 1252 1577 0

KRMP1 3217 1972 13

KRMP3 3494 2844 65

KRMP4 1907 2629 194

Prismin 3669 3967 0

Prisilkin-39 641 442 0

Shematrin1 1456 1386 93

Shematrin2 4833 4672 1351

Shematrin3 379 36 0

Shematrin4 29 0 0

Shematrin5 408 1517 0

Shematrin6 4906 4756 0

Shematrin7 903 370 0

TPM transcripts per million, ME mantle edge, P pallium, PS pearl sac

S. Kinoshita et al.

395

Although the pallium and pearl sac have the same function in terms of nacre

formation, our data indicate that the expression patterns of various nacreous genes

are not identical, though similar to each other, between the two tissues. In addition,

most prismatic genes analyzed in this study showed high expression levels in both

mantle edge and pallium but only marginally in pearl sac. One possibility is that

contaminating tissues surrounding the pearl sac decreased the expression of shell

formation-related genes in our pearl sac preparation. Nevertheless our data suggest

different composition of shell matrix proteins between the shell nacre and pearls,

and the importance of the gene expression analysis in the pearl sac to address

molecular mechanisms underlying the pearl formation.

MSI60

MSI25

N19-2

Pif177

PFMG1

N66

ACCBP

N19-1

Shematrin3

Shematrin6

Shematrin7

Prisilkin-39

Prismalin-14

Shematrin4

Shematrin5

Prismin

Nacrein

KRMP1

CalP

KRMP3

Aspein

Shematrin1

Shematrin2

KRMP4

TPM

high

low

TPM=0

Fig. 42.1 Hierarchical

clustering of expression

patterns in three shell-

formation tissues for

known nacreous and

prismatic genes. ME

mantle edge, P pallium, PS

pearl sac. Explanation

about color is needed

42 Gene Expression Patterns intheMantle andPearl Sac Tissues ofthePearl Oyster…

396

References

Funabara D, Ohmori F, Kinoshita S, Koyama H, Mizutani S, Ota A, Osakabe Y, Nagai K, Maeyama

K, Okamoto K, Kanoh S, Asakawa S, Watabe S (2014) Novel genes participating in the forma-

tion of prismatic and nacreous layers in the pearl oyster as revealed by their tissue distribution

and RNA interference knockdown. PLoS One 9:e84706

Inoue N, Ishibashi R, Ishikawa T, Atsumi T, Akoki H, Komaru A (2010) Gene expression pat-

terns and pearl formation in the Japanese pearl oyster (Pinctada fucata): a comparison of gene

expression patterns between the pearl sac and mantle tissue. Aquaculture 308:68–74

Jackson DJ, McDougall C, Woodcroft B, Moase P, Rose RA, Kube M, Reinhardt R, Rokhsar DS,

Montagnani C, Joubert C, Piquemal D, Degnan BM (2010) Parallel evolution of nacre building

gene sites in molluscs. Mol Biol Evol 27:591–608

Kinoshita S, Wang N, Inoue H, Maeyama K, Okamoto K, Nagai K, Kondo H, Hirono I, Asakawa

S, Watabe S (2011) Deep sequencing of ESTs from nacreous and prismatic layer producing

tissues and a screen for novel shell formation-related genes in the pearl oyster. PLoS One

6:e21238

Wang N, Kinoshita S, Riho C, Maeyama K, Nagai K, Watabe S (2009) Quantitative expression

analysis of nacreous shell matrix protein genes in the process of pearl biogenesis. Comp

Biochem Physiol B Biochem Mol Biol 154:346–350

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

S. Kinoshita et al.

Part IX

Appendix

399

Chapter 43

Selected SEM andTEM Images by Late

Dr. Hiroshi Nakahara

MitsuoKakei

Abstract The following SEM and TEM images were taken by late Dr. Hiroshi

Nakahara many years ago, left unpublished, and shown on the screen during lunch-

times in the symposium. He graduated from the course of zoology of the Faculty of

Sciences, University of Hokkaido, and studied abroad in the University of NewYork

and School of Dentistry, University of Texas. After returning home, he taught oral

anatomy at Meikai University, School of Dentistry (former Josai Dental University).

Using electron microscopes both SEM and TEM, he studied the mineralization pro-

cesses of a variety of shellsh as well as vertebral hard tissues such as tooth enamel,

dentin, and bone.

Keywords Bone · Ligament · Monodonta confusa · Nacreous layer · Otolith ·

Pearl · Pinctada fucata · Prismatic layer · Tooth enamel

M. Kakei (*)

Tokyo Nishinomori Dental Hygienist College, Tokyo, Japan

e-mail: mkakei@jcom.home.ne.jp

Dr. Hiroshi Nakahara (1928–2001)

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_43

400

Fig. 43.1 SEM of growing surface of cultured pearl in Pinctada fucata. Spiral patterns are distrib-

uted across the surface (a). Aragonite tablets are arranged (b) (bars: a = 20μm, b = 10μm)

Fig. 43.2 TEM of nacre formation of Pinctada fucata. Growing surface of bivalve nacre is pro-

tected from being exposed to seawater by periostracum (bars: a, b = 2μm, double staining)

M. Kakei

401

Fig. 43.3 SEM of the growing surface of nacreous layer of Pinctada fucata. The growing surface

shows stepwise structure (bar: 5μm)

Fig. 43.4 TEM of a crystal in the nacreous layer of Pinctada fucata. Flat cut sections show poly-

synthetic twin (arrows) (bars: a, b = 500nm)

43 Selected SEM andTEM Images by Late Dr. Hiroshi Nakahara

402

Fig. 43.5 SEM of growing surface of nacreous layer of Monodonta confusa. After treatment with

sodium hypochlorite solution, columnar arrangement of tablets shows pyramid-shaped stacks

(bars: a = 10μm, b = 5μm)

Fig. 43.6 TEM of growing surface of nacreous layer of Monodonta confusa. Tablets of crystals

are created between the interlamellar matrix of sheets. (a) No staining, (b) double staining (bars:

a, b = 5μm)

M. Kakei

403

Fig. 43.7 TEM of growing

surface of nacreous of

Sulculus diversicolor

supertexta. Crystals of

snail nacre are arranged in

the brick wall type,

reinforcing the structural

strength (bar = 5μm)

Fig. 43.8 SEM (a) and TEM (b) of the interlamellar matrix sheets of nacre of Batillus cornutus.

Surface sheets cover the top of aragonite stacks (bars: a = 5μm, b = 4μm)

43 Selected SEM andTEM Images by Late Dr. Hiroshi Nakahara

404

Fig. 43.9 TEM of nacre formation of Calliostoma unicum. (a) Central portion of the pyramid-

shaped stacks shows empty space. (b–e) Sections were nearly parallel to the surface. Each tablet is

divided into sectors (b and c). Organic cores remained at the center of stacks in the stained sections

(d and e) (bars: a = 2.2μm, b = 5μm, c = 10μm, d = 10μm, e = 1μm)

Fig. 43.10 TEM of nacre

formation of Batillus

cornutus. Thick surface

sheet (arrow) is only

formed in the gastropods

and protects the developing

nacre surface from

seawater (double staining,

bar = 10μm)

M. Kakei

405

Fig. 43.11 TEM of

transverse plane of

nacreous layer of Haliotis.

Brick wall-type structure

enhances the structural

strength (bar = 1μm)

Fig. 43.12 TEM of nacre of Lithophaga. Flat-cut crystals are observed without staining (bars: a =

2μm, b = 1μm)

43 Selected SEM andTEM Images by Late Dr. Hiroshi Nakahara

406

Fig. 43.13 TEM of nacre of Sulculus diversicolor supertexta. Thin sections show holes in the

interlamellar matrix of organic sheets. (a) No staining, (b) double staining (bars: a, b = 1.0μm)

Fig. 43.14 TEM of the

organic sheet of nacre of

Batillus cornutus. Holes in

the sheets are clearly

observed in section cut to

nearly parallel to the

interlamellar matrix of

sheets (bar = 250nm)

M. Kakei

407

Fig. 43.15 SEM of the prismatic and nacreous layers of Atrina pectinata. Crystals of bivalve

prismatic layer are arranged in a columnar fashion. (a) Crystals of the prismatic layer show rectan-

gular shape. (b) Crystals of the nacreous layer (bars: a = 10μm, b = 5μm)

Fig. 43.16 SEM of the prismatic layer of Pinctada fucata. After acid treatment of (a), interpris-

matic layers remain as shown in Figs. (b, c). (a) Before decalcication, (b, c) after decalcication

(bars: a, b, c = 10μm)

43 Selected SEM andTEM Images by Late Dr. Hiroshi Nakahara

408

Fig. 43.17 SEM of the

prismatic layer of Cellana

toreuma. Imbricated

pattern of mineral plates is

observed (bar: 2μm)

Fig. 43.18 TEM of aragonite crystals in the ligament of Meretrix lusoria. (a) Longitudinal section

of crystals runs parallel with each other. (b) Cross section of the crystals shows hexagonal structure

(bars: a = 2μm, b = 1μm)

M. Kakei

409

Fig. 43.19 Cross section

of aragonite crystals in the

ligament of Neotrigonia

sp. Arrows indicate the

polysynthetic twin

structure (bar: 200nm)

Fig. 43.20 SEM (a) and TEM (b) show the crossed lamellar structure of Strombus gigas (b double

staining) (bars: a = 10μm, b = 1μm)

43 Selected SEM andTEM Images by Late Dr. Hiroshi Nakahara

410

Fig. 43.21 SEM of the fracture surface of Patelloida saccharina. Long and thin aragonite crystals

form a cross-lamellar structure (bars: a = 100μm, b = 10μm)

Fig. 43.22 TEM of aragonite crystals of Patelloida saccharina. (a) The cross section shows rect-

angular shape. (b) Crystals show twin pattern (bars: a = 1μm, b = 110nm)

M. Kakei

411

Fig. 43.23 TEM of shell of Euhadra peliomphala. The shell shows crossed-lamellar structure (a

double staining, b no staining, bars: a = 5μm, b = 2μm)

Fig. 43.24 SEM and TEM of otoliths of mouse. (a) SEM of both ends shows triangular in shape,

and side view looks like cylindrical shape. (b) TEM of thin section without staining demonstrates

that ne road-like crystals are arranged in a radial pattern (bars: a = 1μm, b = 540nm)

43 Selected SEM andTEM Images by Late Dr. Hiroshi Nakahara

412

Fig. 43.25 TEM of apatite crystals of the rat tooth enamel. Central dark lines (arrows) do not cre-

ate two lattice lines after electron beam exposure, showing a different physical property of octacal-

cium phosphate (a before beam damage, b after beam damage, bar = 10nm)

Fig. 43.26 TEM

observations of shark

enameloid (a) and rat tooth

enamel (b) crystals. Cross

sections show two different

crystal characters,

indicating two different

mechanisms of crystal

formation. Arrow: central

dark line (CDL) (bar =

10nm)

M. Kakei

413

Fig. 43.27 TEM of bone

resorption by osteoclast in

rat. Arrows show that bone

crystals were resorbed by

endocytosis (bar = 2μm)

Fig. 43.28 TEM of

Malpighian tubule of

Drosophila melanogaster.

Minerals of calcospherite

are formed (bar = 1μm)

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as long as you give appropriate

credit to the original author(s) and the source, provide a link to the Creative Commons license and

indicate if changes were made.

The images or other third party material in this chapter are included in the chapter’s Creative

Commons license, unless indicated otherwise in a credit line to the material. If material is not

included in the chapter’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from

the copyright holder.

43 Selected SEM andTEM Images by Late Dr. Hiroshi Nakahara

Correction to: TEM Study oftheRadular

Teeth oftheChiton Acanthopleura japonica

MitsuoKakei, MasayoshiYoshikawa, andHiroyukiMishima

Correction to:

Chapter 2 in: K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_2

In the original version of chapter 2, references 5 and 12 were incorrect. In this ver-

sion, references 5 and 12 are corrected. The corrected references have now been

added in the Chapter which reads as follows:

Reference 5

Weaver JC, Wang Q, Miserez A, Tantuccio A, Stromberg R, Bozhilov KN, Maxwell

P, Nay R, Heier ST, DiMasi E, Kisailus D (2010) Analysis of an ultra hard magnetic

biomineral in chiton radular teeth. Materialstoday 13 (1–2):42–52

Reference 12

Nemoto M, Wang Q, Li D, Pan S, Matsunaga T, Kisailus D (2012) Proteomic analy-

sis from the mineralized radular teeth of the giant Pacic chiton, Cryptochiton stel-

leri (Mollusca). Proteomics 12:2890–2894

The updated online version of this chapter can be found at

https://doi.org/10.1007/978-981-13-1002-7_2

K. Endo et al. (eds.), Biomineralization,

https://doi.org/10.1007/978-981-13-1002-7_44

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J BIOMED MATER RES A

Electrospinning, a technique used to fabricate fibrous scaffolds, has gained popularity in recent years as a method to produce tissue engineered grafts with architectural similarities to the extracellular matrix. Beyond its versatility in material selection, electrospinning also provides many tools to tune the fiber morphology and scaffold geometry. Recent efforts have focused on extending the capabilities of electrospinning to produce scaffolds that better recapitulate tissue properties and enhance regeneration. This review highlights these advancements by providing an overview of the processing variables and setups used to modulate scaffold architecture, discussing strategies to improve cellular infiltration and guide cell behavior, and providing a summary of electrospinning applications in tissue engineering. This article is protected by copyright. All rights reserved.

Calcium Biomineralizations Associated with Bioclastic Deposits in Coastal Pedostratigraphic Sequences of the Southeastern Pampean Plain, Argentina

Chapter

May 2017

The biomineralization process is genetically controlled, and it is the result of the metabolic activity of different organisms . Microorganisms , plants and animals produce calcium biomineralizations , calcium oxalates and carbonates being the most representative. The Quaternary pedosedimentary sequences of the southeastern coast of Buenos Aires province evolved from bioclastic and loess sediments, reworked by water and/or wind action . Calcium biomineralizations play an important role in the development of soils and contribute to differentiate sedimentary levels affected by pedogenesis . This work aims to characterize calcium biomineralizations in bioclastic , loessic and fluvio-eolian pedosedimentary sequences , typical of coastal environments of the southeastern Buenos Aires province . Modal soil profiles were defined in pedosedimentary sequences of the fluvio-eolian and coastal plains , in which disturbed and undisturbed samples were analyzed. Samples were analyzed at different scales of resolution: mesoscopic , microscopic and submicroscopic , using optical microscopy and scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDX) . Organic matter content , pH , particle size distribution , mineralogy and calcium content were also quantified. Three geopedological units were differentiated in a sequence developed on loess mantle deposits: dunes and interdunes , coastal lagoon/paleo-coastal lagoon and shell ridges . In soils with incipient development, calcium biomineralizations are scarce, mainly associated with isolated bioclasts . In soils with more pedological development , bioclasts are affected by bioerosion through microorganism action (fungi and algae ), and subsequently calcium re-precipitated as secondary oxalates and carbonates (biomineralizations ). These biomineralizations also weakly add or bind skeletal components , incorporating themselves into the matrix of soils and sediments . The type and diversity of calcium biomineralizations increase directly in relation with time and pedogenetic evolution ; so, these biomineralizations have been determinant in the origin, evolution and resistance to natural and anthropic degradation of the late Quaternary pedosedimentary sequences of southeastern Buenos Aires province , Argentina .

Biomineralization From Molecular and Nano-structural Analyses to Environmental Science: From Molecular and Nano-structural Analyses to Environmental Science

Abstract

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