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Glass sponges, as examples of natural biocomposites, inspire investigations aiming at both a better understanding of biomineralization mechanisms and novel developments in the synthesis of nanostructured biomimetic materials. Different representatives of marine glass sponges of the class Hexactinellida (Porifera) are remarkable because of their highly flexible basal anchoring spicules. Therefore, investigations of the biochemical compositions and the micro- and nanostructure of the spicules as examples of naturally structured biomaterials are of fundamental scientific relevance. Here we present a detailed study of the structural and biochemical properties of the basal spicules of the marine glass sponge Monorhaphis chuni. The results show unambiguously that in this glass sponge a fibrillar protein of collagenous nature is the template for the silica mineralization in all silica-containing structural layers of the spicule. The structural similarity and homology of collagens derived from M. chuni spicules to other sponge and vertebrate collagens have been confirmed by us using FTIR, amino acid analysis and mass spectrometric sequencing techniques. We suggest that nanomorphology of silica formed on proteinous structures could be determined as an example of biodirected epitaxial nanodistribution of amorphous silica phase on oriented fibrillar collagen templates. Finally, the present work includes a discussion relating to silica-collagen-based hybrid materials for practical applications as biomaterials.
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Hindawi Publishing Corporation
Journal of Nanomaterials
Volume 2008, Article ID 623838, 8pages
Research Article
Nanostructural Organization of Naturally Occurring
Composites—Part I: Silica-Collagen-Based Biocomposites
Hermann Ehrlich,1Sascha Heinemann,1Christiane Heinemann,1Paul Simon,2
Vasily V. Bazhenov,3Nikolay P. Shapkin,3Ren ´
e Born,1Konstantin R. Tabachnick,4
Thomas Hanke,1and Hartmut Worch1
1Max Bergmann Center of Biomaterials and Institute of Materials Science, Dresden University of Technology,
01069 Dresden, Germany
2Max Planck Institute of Chemical Physics of Solids, 01187 Dresden, Germany
3Institute of Chemistry and Applied Ecology, Far Eastern National University, 690650 Vladivostok, Russia
4P.P. Shirshov Institute of Oceanology, Russian Academy of Sciences, Nahimovsky pr. 36, 117997 Moscow, Russia
Correspondence should be addressed to Hermann Ehrlich,
Received 2 November 2007; Accepted 31 December 2007
Recommended by Donglu Shi
Glass sponges, as examples of natural biocomposites, inspire investigations aiming at both a better understanding of biomineral-
ization mechanisms and novel developments in the synthesis of nanostructured biomimetic materials. Dierent representatives of
marine glass sponges of the class Hexactinellida (Porifera) are remarkable because of their highly flexible basal anchoring spicules.
Therefore, investigations of the biochemical compositions and the micro- and nanostructure of the spicules as examples of nat-
urally structured biomaterials are of fundamental scientific relevance. Here we present a detailed study of the structural and bio-
chemical properties of the basal spicules of the marine glass sponge Monorhaphis chuni. The results show unambiguously that in
this glass sponge a fibrillar protein of collagenous nature is the template for the silica mineralization in all silica-containing struc-
tural layers of the spicule. The structural similarity and homology of collagens derived from M. chuni spicules to other sponge and
vertebrate collagens have been confirmed by us using FTIR, amino acid analysis and mass spectrometric sequencing techniques.
We suggest that nanomorphology of silica formed on proteinous structures could be determined as an example of biodirected
epitaxial nanodistribution of amorphous silica phase on oriented fibrillar collagen templates. Finally, the present work includes a
discussion relating to silica-collagen-based hybrid materials for practical applications as biomaterials.
Copyright © 2008 Hermann Ehrlich et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Glass sponges (Hexactinellida: Porifera) provide an abun-
dant source of unusual skeleton structures, which could be
defined as natural silica-based nanostructured composite
materials. They are intriguing research objects because of the
hierarchical organization of their spicules from the nanoscale
to the macroscale [13]. First observations reported by L´
et al. [4] on silica-based spicules of a Monorhaphis sponge
generated great interest because of their combination of
properties, namely, toughness combined with stiness, and
resilience. This sponge species synthesizes the largest biosil-
ica structures on earth [5]. Pencil-sized rod spicules, a me-
ter or more in length, could be bent into a circle without
breaking. When the load was released, the spicule recovered
its original shape. When the bending of the spicule rod was
compared with that of a synthetically derived pure silica rod,
the toughness of the spicule was found to be nearly an or-
der of magnitude higher [2]. Recently, the micromechani-
cal properties of biological silica in the giant anchor spicule
of Monorhaphis chuni were reported on [6]. Nanoidentation
showed a considerably reduced stiness of the spicule com-
pared to technical quartz glass with dierent degrees of hy-
dration. Moreover, stiness and hardness were shown to os-
cillate as a result of the laminate structure of the spicules.
Raman spectroscopic imaging showed that the organic lay-
ers are protein-rich and that there is an OH-enrichment in
silica near the central axial filament of the spicule. Small-
angle X-ray scattering revealed the presence of nanospheres
with a diameter of only 2.8 nm as the basic unit of silica.
2Journal of Nanomaterials
It was suggested that biogenic silica formed by glass sponges
possesses reduced stiness but substantially higher toughness
than technical glass due to its architecture, determined by
structure at the nanometer and the micrometer level [6]. Un-
fortunately, the nature and the origin of the protein matrix
were not investigated in this study.
There is no doubt that glass sponge anchoring spicules
are remarkable objects because of their size, durability, high
flexibility, and their exceptional fibre-optic properties, which
all together render them of interest as novel biomimetic ma-
terials [7]. Of course, the materials science aspects of glass
sponges can be studied by model systems, and utilized for
biomimetic engineering. However, we cannot mimic nature
with a view to designing novel biomaterials without knowl-
edge of the nature and origin of the organic nanostructured
matrices of corresponding natural biocomposites which are
present in these sponges. Therefore, the biggest shortcoming
common to all publications relating to mechanical [2], struc-
tural [3], and optical [8] properties of glassy sponge skeletal
formations is a lack of real information regarding the chem-
ical nature of corresponding organic matrices.
The finding of collagen within basal spicules of the glass
sponge Hyalonema sieboldi [911], as well as the occurrence
of chitin within the framework skeleton of the glass sponges
Farrea occa [12], and spicules of Euplectella aspergillum [7]as
revealed by gentle desilicification in alkali, stimulated further
attempts to search for materials of organic nature in skele-
tal structures of these unique deep-sea organisms. Conse-
quently, the objective of the current study was to test our hy-
pothesis that collagen is also an essential component of the
giant anchoring silica spicules of Monorhaphis chuni,and if
so, to unravel its involvement in the mechanical behavior of
these formations, which was well investigated recently [6].
In the present work, we provide a detailed study confirm-
ing our hypothesis that the nanofibrillar organic matrix of
collagenous nature within the giant spicules of M. chuni is re-
sponsible for their extraordinary mechanical properties. We
performed structural, spectroscopic, and biochemical analy-
ses of these glassy composites. Finally, this work includes a
discussion relating to practical applications of silica-collagen
composites artificially derived in vitro as biomaterials for use
in biomedicine, engineering, and materials science.
2.1. Chemical etching of spicules and extraction
of collagen
Monorhaphis chuni was collected by the R.V. “Vitiaz-2
(4),voyage 17, St. 2601, 1231.5’–25.04’ S 4805.5’–
08.0’ E, depth 700 m.DriedMonorhaphis basal spicules
(length 120 cm, diameter 1.5–4.5 mm, Figure 1) were washed
three times in distilled water, cut into 2–5 cm long pieces
and placed in a solution containing purified Clostridium his-
tolyticum collagenase (Sigma) to digest any possible colla-
gen contamination of exogenous nature. After incubation for
24 hours at 15C[13], the pieces of spicules were washed
again three times in distilled water, dried and placed in 10ml
plastic vessels containing 5 ml of 2.5 M NaOH solution. The
Figure 1: (a) Marine glass sponge Monorhaphis chuni, a member
of the hexactinellids, (b) the sponge consists of a giant basal spicule
which anchors Monorhaphis to the sandy substratum.
vessel was covered, placed under thermostatic conditions at
37C and shaken slowly for 14 days. The eectiveness of the
slow alkali etching was monitored using scanning electron
microscopy (SEM) at dierent locations along the spicules’
length and within the cross-sectional area.
2.2. Biochemical analysis of collagen
Alkali extracts of Monorhaphis spicules containing fibrillar
protein were dialyzed against deionized water on Roth (Ger-
many) membranes with a cut-oof 14 kDa. Dialysis was per-
formed for 48 hours at 4C. The dialyzed material was dried
under vacuum conditions in a CHRIST lyophilizer (Ger-
many). The approximate molecular weights of proteins in
the lyophylizate were determined by gel electrophoresis in
the presence of sodium dodecyl sulphate in 10% and 12% gel
plates.The kit of molecular weight markers (Silver stain SDS
molecular standard mixtures) from Sigma, USA, was used.
Lyophylizates were dissolved in sample buer (1 M Tris-HCl,
pH 6.8, 2.5% SDS, 10% glycerine, 0.0125% bromphenol
blue) incubated at 95C for 5 minutes and then applied to
10% or 12% of SDS-polyacrylamide gels. After electrophore-
sis at 75 V for 1.5hours, 10% gels were stained with GelCode
SilverSNAP Stain Kit II (Pierce,USA), and 12% gels were
stained with coomassie brilliant blue R250 to allow proteins
to be visualized. To elucidate the nature of proteins isolated
from glass sponge spicules, corresponding electrophoretic
gels stained with Coomassie were used for the determina-
tion of the aminoacid sequence by the mass spectrometric
sequencing technique (MALDI, Finnigan LTQ) as described
earlier [14].
2.3. Structural analysis of spicule layers
Structural analysis of the glass sponge basal spicules and
corresponding extracted proteinaceous components was
performed using scanning electron microscopy (SEM)
Hermann Ehrlich et al. 3
(ESEM XL 30, Philips) and transmission electron mi-
croscopy (TEM) (Zeiss EM 912). Additional transmission
electron microscopy experiments were carried out at the
Special Triebenberg Laboratory for electron holography
and high-resolution microscopy of the Technical University
Dresden.A field-emission microscope of the FEI company
(Endhoven, NL) CM200 FEG/ST-Lorentz was used equipped
with a 1 ×1 k CCD camera (multiscan, Gatan, USA). The
analysis of the TEM images was realized by means of the
Digital Micrograph software (Gatan, USA). Infrared spectra
were recorded with a Perkin Elmer FTIR Spectrometer
Spectrum 2000, equipped with an AutoImage Microscope
using the fourier transform infrared reflection absorption
spectroscopy (FT-IRRAS) technique. In the case of the
FTIR-analyses, calf skin collagen (Fluka) and Chondrosia
reniformis sponge collagen (Klinipharm GmbH, Germany)
were investigated as reference samples.
2.4. Silicification of collagen in vitro
Tetramethoxysilan (TMOS 98%, ABCR GmbH, Germany)
was chosen as a silica precursor and was hydrolysed for 24h
at 4C by adding water as well as HCl as a catalyst. This
procedure results in the soluble form of silica—orthosilicic
acid—whose further polycondensation reactions can be di-
vided into monomer polymerisation, nuclei growth, and ag-
gregation of particles. Hybridization—the combination of
silica and collagen—was performed by intensive mixing of
prehydrolysed TMOS and the homogeneous collagen sus-
pensions under ambient conditions as described in [15].
2.5. Biocompatibility of the silica-collagen
hybrid materials
was evaluated by cultivating human mesenchymal stem cells
on the material followed by induced dierentiation into
osteoblast-like cells [16].
It was generally accepted that the skeletons of Hexactinel-
lida are composed of amorphous hydrated silica deposited
around a proteinaceous axial filament [17,18]. The nanolo-
calization of the proteinaceous component of the glass
sponge spicules was not investigated in detail because of lack
of a demineralization method which preserved the organic
matrix during desilicification. Up to now, the common tech-
nique for the desilicification of sponge spicules was based on
hydrogen fluoride solutions [5], however this kind of dem-
ineralization is rather aggressive chemical procedure which
could drastically change the structure of proteins [19,20]. To
overcome this obstacle, Ehrlich et al. [911] developed novel,
slow etching methods, which use solutions of 2.5 M NaOH
at 37C and take 14 days. Using these methods, it was shown
for the first time that the same class of proteins—collagen—
involved in cartilage and bone formation also forms the ma-
trix and deposition site of amorphous silica in H. sieboldi
glass sponge spicules [9,21]. It was suggested that the H.
sieboldi basal spicule is an example of a biocomposite con-
taining a silificated collagen matrix and that the high colla-
gen content is the origin of the high mechanical flexibility of
the spicules.
SEM investigations of the alkali-etched Monorhaphis
chuni spicules (Figure 2(a)) confirmed the multilayered silica
structure, well-known since the first microscopically investi-
gation of hexactinellid sponges by Schultze in 1860 [22], and
present in all representatives of lyssacine Hexactinellida [18].
We focused on the investigation of fibrillar components ob-
servable at the sites of interstitial layer fractures within par-
tially desilicified spicules. SEM investigations parallel to the
slow etching procedures reveal that a fibrillar organic matrix
is the template for silica mineralization. Typical fibrillar for-
mations were observed within the tubular silica structures
in all layers starting from the inner axial channel contain-
ing axial filament (Figure 3(a)) up to the outermost surface
layer of the spicules as shown in Figures 2(b) and 2(c). The
fibrils in each cylinder form individual concentric 2D net-
works with the curvature of the corresponding silicate lay-
ers. These layers of about 1 μm in thickness are connected
amongeachotherbyproteinfibres(Figure 2(a)), which pos-
sess a characteristic nanofibrillar organization (Figures 2(b)
and 2(d)). Partially desilicificated nanofibrillar organic ma-
trix observed on the surface of silica-based inner layers of
the demineralized spicule provides strong evidence that sil-
ica nanoparticles of diameter about 35 nm are localized on
the surface of corresponding nanofibrils (Figures 2(c), 2(e),
and Figure 3(b)). This kind of silica nanodistribution is very
similar to the silica distribution on the surface of collagen
fibrils in the form of nanopearl necklets, firstly observed by
us in the glass sponge H. sieboldi [21]. We suggest that the
nanomorphology of silica on proteinous structures described
here could be determined as an example of biodirected epi-
taxial nanodistribution [23] of the amorphous silica phase
on oriented organic fibrillar templates.
The nonsilicificated microfibrils of the M. chuni axial
filament with a diameter of approximately 20–30nm are
organized in bundles with a thickness of 1-2 μmoriented
along the axis of the spicule. They can be easily identified
by SEM (Figure 3(a)). The morphology of these microfib-
rils observed by TEM (Figures 4(a) and 4(b)) is very simi-
lar to nonstriated collagen fibrils isolated previously from H.
sieboldi [911,21] and examined using electron microscopy.
Except for collagen, there are some other possible candi-
dates (e.g., silicateins of axial filaments such as in Demospon-
giae [24,25]orasrecentlyreportedbyM
uller et al. [5,26]
in M. chuni) which would explain the nature and origin of
these fibrillar formations. Therefore, a thorough biochemi-
cal analysis of isolated fibrils was performed.
The results of the aminoacid analysis of protein extracts
isolated from demineralized spicules showed an aminoacid
content typical for collagens isolated from several sources
listed in Figure 4 and also reported previously [21]. The
same extracts were investigated using PAG-electrophoresis.
Corresponding electrophoretic gels stained with Coomassie
were used for the determination of the aminoacid sequence
by a mass spectrometric sequencing technique as described
above.We excised two main bands and digested protein ma-
terial in-gel with trypsin to obtain tryptic peptide mixtures
4Journal of Nanomaterials
400 nm700 nm
700 nm 400 nm
(b) (c)
(d) (e)
Figure 2: SEM images of multilayer constructed M. chuni spicule
(a) treated with alkali solution which provides strong evidence that
the multifibrillar organic matrix is the template for silica miner-
alization (b)–(e). Spicule layers are connected among each other
by nanostructured protein fibres (arrows) (b), (d). Micrograph (e)
shows a silica distribution on the surface of nanofibrils in the form
of nanopearl necklets (arrows).
for further analysis using LTQ and MALDI peptide finger
printings.A comparison to the MSDB protein database [27]
led to the identification of collagen alpha 1 in two high
MW bands.In contrast to H. sieboldi [9], collagen isolated
and identified by the same way from Monorhaphis sp. was
matched only to type I collagen pre-pro-alpha (I) chain
(COL1A1) from dog (AAD34619) (MW 139,74). To our
best knowledge, this work is the first study which confirms
the presence of collagen within the spicules of Monorhaphis
sponge and not only on their surface in the form of a col-
lagen net which covers spicules as recently described by
uller et al. [5].
We also used highly sensitive FTIR methods for the iden-
tification of collagen isolated from spicules of M. chuni.Spec-
tra obtained from this collagen, calf skin collagen type I and
C. reniformis collagen standards were compared to each other
in order to elucidate changes in protein secondary structure.
The results obtained from the FTIR study (data not shown)
show that collagen derived from this glass sponge exhibited
spectra very similar to those from calf skin and C. reniformis
collagens [28]. The presence of collagen fibrils in alkali solu-
tion is no surprise. Hattori et al. [29] investigated the resis-
tance of collagen to alkali treatment at a concentration range
of between 3 and 4% NaOH at 37C in vitro. The results ob-
400 nm700 nm
700 nm 5 nm
(a) (b)
(c) (d)
Figure 3: SEM and TEM nanoimagery of the fibrillar organic ma-
trix within partially demineralized spicule. (a) Axial filament is an
organization of microfibrils with a diameter of approximately 25–
30 nm covered with a silica-containing layer and distributed along
the axis of spicule. (b) Nanolocalization of amorphous silica parti-
cles (arrows) on the surface of partially demineralized protein fibrils
using HRTEM. (c), (d) Collagen fibrils’ orientation within spicule
possesses a twisted plywood architecture (arrows).
tained indicated that the triple helical conformation and the
helicity of the collagen molecule were maintained through-
out the period of the alkaline treatment.
The procedure of alkali slow etching opens the possibil-
ity to observe the forms of collagen fibrils located within
silica layers of spicules and their distribution. The results
obtained by SEM observations of the desilicified spicular
layers provide strong evidence that collagen fibrils’ orienta-
tion within M. chuni spicules possesses twisted plywood ar-
chitecture (Figures 3(c) and 3(d)). The twisted plywood or
helicoidal structure of collagen fibrils is well-described by
Giraud-Guille [30] for bothin vivo and in vitro [31]systems.
Spiral twisting of the collagen fibril orientation was found in
several biological tissues and described for dierent organ-
isms including cuticular collagens of polychaete, vestimen-
tifera, scale collagens of primitive and bony fishes, and finally
collagen fibers inside bone (all reviewed in [21]).
According to the model proposed by Giraud-Guille, ad-
jacent lamellae have dierent orientations; either longitu-
dinal (with the collagen fibers along the long axis of the
lamellar sheet) or transverse (with the collagen fibers per-
pendicular to the long axis). From a mechanical point of
view, helicoidal structures have certain advantages in re-
sisting mechanical loads compared to orthogonal plywood
structures since the twisted orientation enables a higher ex-
tensibility in tension and compression [32]. The twisted ply-
wood architecture of collagen fibrils within basal spicules of
Monorhaphis visible after alkali treatment (Figure 3)isvery
Hermann Ehrlich et al. 5
20 nm
100 nm
chuni collagen
collagen [21]
collagen [21]
Figure 4: (a) High-resolution transmission electron microscopy image of the fragment of M. chuni collagen microfibril; (b) the arrows
indicate the presence of nanofibrillar structures with a diameter which corresponds to that of collagen triple helices (1.5nm). The results of
aminoacid analysis (right) of these microfibrills showed an aminoacid content typical for collagens isolated from dierent sources [21].
Collagen net
fibrillar matrix
Figure 5: proposed model of micro- and nanostructural organisation of the basal spicule of M. chuni with respect to the organic matrix. (a)
Collagen nets, surrounding the spicules, showed a tight mat of nanofibrils. Schematic view (b) shows a collagenous fibrillar matrix which
could function as a glue between concentric layers. Image (c) represents the region of the axial canal and axial filament. The axial canal of M.
chuni possesses a characteristic quadratic opening (c) and contains oriented bundles of unsilicified collagenous nanofibrils. The base material
of the walls of the axial canal and concentric layers distributed above it consists of silicified collagen fibrils with a twisted plywood orientation.
This kind of fibrillar architecture could be responsible for the remarkable micromechanical properties of the spicule as a biocomposite.
similar to that reported for lamellar bone and thus could
also confirm the Girauld-Guille model in the case of biosili-
fication in vivo. Correspondingly, this kind of collagen fib-
ril orientation could explain why sponge spicules exhibit
specific flexibility and can be bent even to a circle as re-
ported previously [2,4,21]. From this point of view, basal
spicules of Monorhaphis sponges could be also defined as
natural plywood-like silica-ceramics organized similarly to
the crossed-lamellar layers of seashells [33]. Thus, we suggest
that the matrix of the M. chuni anchoring spicule is silificated
fibrillar collagen rather than collagen-containing silica which
is the reason for their remarkable mechanical flexibility.
6Journal of Nanomaterials
500 nm
100 μm
Figure 6: Rod-like collagen-silica-based biomaterial derived in vitro (a) shows morphological similarity to M. chuni basal spicule (a, left).
SEM image (b): nanoparticles of amorphous silica deposited in vitro from silicic acid solution on sponge collagen fibrils replicate the nanos-
tructure of glass sponge spicules (Figure 2(e)). SEM micrograph (c) of the surface of silica-collagen hybrid material after 14 days of cultivation
of human mesenchymal stem cells, which shows high biocompatibility on this substrate.
Contrary to the postulate that silicateins, as the major
biosilica-forming enzymes present in demosponges [34], are
responsible for the formation of silica-based structures in
all sponges, we suggested that silicateins are associated with
collagen [21]. From our point of view, silicateins resemble
cathepsins, which are known to be collagenolytic and capa-
ble of attacking the triple helix of fibrillar collagens. There-
fore, it is not unreasonable to hypothesize that silicateins
are proteins responsible for the reconstruction of collagen
to form templates necessary for the subsequent silica for-
mation. According to a dynamic model proposed by M¨
and his team [5], collagen guides the silicatein(-related)
protein/lectin associates concentrically along the spicules of
M. chuni. On the basis of the results presented in this pa-
per, we propose a model for the structure of the spicules
of Monorhaphis sponges, including micro- and nanoaspects,
which can be seen in Figure 5.
Recently, we confirmed that silicification of sponge colla-
gen in vitro occurs via selfassembling, nonenzymatic mech-
anisms [15,21]. To verify whether the collagenous matrix
shapes the morphology of the spicules, we carried out in-
vitro experiments in which we exposed collagen to silicic acid
solution (Si(OH)4). We obtained rod-like structures of sev-
eral mm in diameter and demonstrated their similarity to the
sponge spicules (Figure 6(a)). The ultrastructural analysis of
these selfassembled, collagen-silica composites demonstrates
that amorphous silica is deposited on the surface of collagen
fibrils in the form of nanopearl necklets (Figure 6(b)), closely
resembling the nanoparticulate structure of natural M. chuni
spicules (Figures 2(e) and 3(a)).
Bridging the nano- and microlevel, we used dierent
techniques to create a wide spectrum of macroscopic silica-
collagen-based hybrid materials. These are highly biocom-
patible, as demonstrated by the successful cultivation and os-
teogenic dierentiation of human mesenchymal stem cells
on our materials (Figure 6(c)), and potentially useful for
technical and biomedical applications. On the basis of the
results reported above, we also developed an advanced pro-
cedure for the biomimetically inspired production of mono-
lithic silica-collagen hybrid xerogels [16]. The disc-like sam-
ples showed convincing homogeneity and mechanical stabil-
ity, enabling cell culture experiments for the first time on
such materials.
Recently, interest in biomaterial properties of silica-
containing structures made by living sponges has grown.
In order to exploit the mechanisms for the synthesis of
advanced materials and devices, an investigation of the
nanoscopic structure of the three-dimensional networks of
these remarkable biomaterials needs to be performed [35
38]. Understanding the composition, hierarchical structure,
and resulting properties of glass sponge spicules gives im-
petus for the development of equivalents designed in vitro.
We showed for the first time that the silica skeletons of
hexactinellids represent examples of biological materials in
which a collagenous or chitinous organic matrix serves as a
scaold for the deposition of a reinforcing mineral phase in
the form of silica. These findings allow us to discard dier-
ent speculations about materials, which have previously been
defined as organic structures (layers, filaments, surfaces) of
unknown nature, and open the way for detailed studies on
sponge skeletons and spicules as collagen- and/or chitin-
based nanostructured biocomposites with high potential for
practical applications.
Hermann Ehrlich et al. 7
This work was partially supported by a joint Russian-
German program “DAAD–Mikhail Lomonosov.” We thank
Professor H. Lichte for the possibility to use the facilities
at the Special Electron Microscopy Laboratory for high-
resolution and holography at Triebenberg, TU Dresden, Ger-
many. The authors are deeply grateful to Patrice Waridel and
Andrei Shevchenko (Max Planck Institute of Molecular Cell
Biology and Genetics, Dresden) for the identification of col-
lagen in the composition of spicules, and also to Timothy
Douglas, Heike Meissner, Gert Richter, Axel Mensch, and Or-
trud Trommer for helpful technical assistance.
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... 8]. The collagen nature of this net was recognized by Ehrlich et al. [63,64] who also demonstrated that it is hydroxylated fibrillary collagen [65]. The fact that it can be easily detached from the basal spicule surface, that small body spicules occur between (Figs. 11b, 12b, 13a), and that there is no direct relation between the meshes of the net and the tubercles and ridges on the spicule surface, suggest that the net is not directly participating in the process of basal spicule biomineralization, which must instead be controlled by the sclerosyncytium (multinucleate scleroblast masses) [42]. ...
... In our opinion, the net is better considered as a structural element of the sponge body keeping it connected with the basal spicule. Apart from the exterior collagen net that covers the spicule surface, organic substances permeate the spicule silica (Fig. 14b) and our analysis indicates that these are proteins which were already reported-silicatein and/or galectin [2,28], or collagen [3,64,65]. The ability to precipitate silica by much more simple synthetic minicollagens has been demonstrated [66]. ...
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Background A basal spicule of the hexactinellid sponge Monorhaphis chuni may reach up to 3 m in length and 10 mm in diameter, an extreme case of large spicule size. Generally, sponge spicules are of scales from micrometers to centimeters. Due to its large size many researchers have described its structure and properties and have proposed it as a model of hexactinellid spicule development. Thorough examination of new material of this basal spicule has revealed numerous inconsistencies between our observations and earlier descriptions. In this work, we present the results of detailed examinations with transmitted light and epifluorescence microscopy, SEM, solid state NMR analysis, FTIR and X-ray analysis and staining of Monorhaphis chuni basal spicules of different sizes, collected from a number of deep sea locations, to better understand its structure and function. Results Three morphologically/structurally different silica layers i.e. plain glassy layer (PG), tuberculate layer (TL) and annular layer (AL), and an axial cylinder (AC) characterize adult spicules. Young, immature spicules display only plain glassy silica layers which dominate the spicule volume. All three layers i.e. PG, TL and AL can substitute for each other along the surface of the spicule, but equally they are superimposed in older parts of the spicules, with AL being the most external and occurring only in the lower part of the spicules and TL being intermediate between AL and PG. The TL, which is composed of several thinner layers, is formed by a progressive folding of its surface but its microstructure is the same as in the PG layer (glassy silica). The AL differs significantly from the PG and TL in being granular and porous in structure. The TL was found to display positive structures (tubercles), not depressions, as earlier suggested. The apparent perforated and non-perforated bands of the AL are an optical artefact. The new layer type that we called the Ripple Mark Layer (RML) was noted, as well as narrow spikes on the AL ridges, both structures not reported earlier. The interface of the TL and AL, where tubercles fit into depressions of the lower surface of the AL, represent tenon and mortise or dovetail joints, making the spicules more stiff/strong and thus less prone to breaking in the lower part. Early stages of the spicule growth are bidirectional, later growth is unidirectional toward the spicule apex. Growth in thickness proceeds by adding new layers. The spicules are composed of well condensed silica, but the outermost AL is characterized by slightly more condensed silica with less water than the rest. Organics permeating the silica are homogeneous and proteinaceous. The external organic net (most probably collagen) enveloping the basal spicule is a structural element that bounds the sponge body together with the spicule, rather than controlling tubercle formation. Growth of various layers may proceed simultaneously in different locations along the spicule and it is sclerosyncytium that controls formation of silica layers. The growth in spicule length is controlled by extension of the top of the axial filament that is not enclosed by silica and is not involved in further silica deposition. No structures that can be related to sclerocytes (as known in Demospongiae) in Monorhaphis were discovered during this study. Conclusions Our studies resulted in a new insight into the structure and growth of the basal Monorhaphis spicules that contradicts earlier results, and permitted us to propose a new model of this spicule’s formation. Due to its unique structure, associated with its function, the basal spicule of Monorhaphis chuni cannot serve as a general model of growth for all hexactinellid spicules.
... Similar cylindrical layered architectures have also been found in spicules from a number of related sponge species [17][18][19][20] . Images of spicules from other Hexactinellid species that are partially dissolved in alkali solution reveal that the silica layers also contain a fibrillar organic matrix similar to the interlayers [21][22][23] . Thus, this organic matrix serves both as a scaffold within the layers and a glue between them 24 . ...
... It is believed that this organic matrix acts as a template for cellassisted silica mineralization during the spicule's growth process [21][22][23]25 . However, little is known about the growth process of Ea. spicules 25 . ...
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The layered architecture of stiff biological materials often endows them with surprisingly high fracture toughness in spite of their brittle ceramic constituents. Understanding the link between organic–inorganic layered architectures and toughness could help to identify new ways to improve the toughness of biomimetic engineering composites. We study the cylindrically layered architecture found in the spicules of the marine sponge Euplectella aspergillum. We cut micrometer-size notches in the spicules and measure their initiation toughness and average crack growth resistance using flexural tests. We find that while the spicule’s architecture provides toughness enhancements, these enhancements are relatively small compared to prototypically tough biological materials, like nacre. We investigate these modest toughness enhancements using computational fracture mechanics simulations. Nacre is a biological composite whose architecture greatly enhances its toughness. Here, the authors report on the toughness enhancement in the spicules of a marine sponge. The spicules display similar architecture to nacre; however, their architecture does not lead to similar toughness enhancement.
... The latest approach is connected with findings of organic matrix inside sponge silica spicules (different from the axial filament which organic nature has long been known) after solution and dissociation (usually long-term) using NaOH. The organic remnants of the spicule after dissociation of the amorphic silica were found to be various: collagen is found in basal spicules of Hyalonema sieboldi (Ehrlich et al. 2006), Monorhaphis chuni (Ehrlich et al. 2008a, and α-chitin was documented in Farrea occa , Euplectella aspergillum , Rossella fibulata (Ehrlich et al. 2008b). The both types of organic matrix are considered to be templates for amorphous silica biomineralization, and chitin also serves as a template for multiphase biomineralization of both silica and crystalline aragonite . ...
... Both types of mineralization are known from Verongida sponges, but that from Caulophacus (Hexactinellida) is found to include minute calcitic reinforcements of the silica joints (Ehrlich et al. 2011). Experimental silicification of colloidal chitin (Ehrich and Worch 2007) and Rossella fibulata (Ehrlich et al. 2008a) was performed at room temperature, the collagen (from Chondrosia reniformis Demospongiae origin) silica mineralization was created at 20°C (Heinemann et al. 2007). ...
Chitin is recognized as an evolutionarily ancient and fundamental skeletal construct, commonly found in diverse uni‐ and multicellular (mostly invertebrate) organisms across the globe. This chapter discusses the occurrence and structural peculiarities of chitin from sponges (Porifera), as well as methods for isolating this structural aminopolysaccharide from these organisms. It describes trends for the application of this unique, naturally pre‐fabricated, three‐dimensional (3D) chitin towards biomedical (tissue engineering) and technological (biomaterials‐inspired science) goals. Sponges of the class Hexactinellida, with their biosilica‐based skeletal structures, may be the oldest metazoan taxon in Earth's history. The chitin isolated from demosponges has a 3D, tubular and fibrous morphology, with numerous channels and chambers having the ability to swell. Demineralisation can be achieved using various acids, most commonly diluted hydrochloric acid, acetic acid or sulphuric acid. Microwaves are electromagnetic waves and are a type of non‐ionising radiation.
... Acidic solutions are subsequently used for dissolving the demineralized collagen to complete the extraction process [104]. Collagen extracted from marine invertebrate, especially those extracted from glass sponges, are excellent organic templates for in vitro silicification [105]. Marine glass sponge-derived collagen accounts for the most abundant naturally-silicified biomaterials on earth. ...
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Marine resources have tremendous potential for developing high-value biomaterials. The last decade has seen an increasing number of biomaterials that originate from marine organisms. This field is rapidly evolving. Marine biomaterials experience several periods of discovery and development ranging from coralline bone graft to polysaccharide-based biomaterials. The latter are represented by chitin and chitosan, marine-derived collagen, and composites of different organisms of marine origin. The diversity of marine natural products, their properties and applications are discussed thoroughly in the present review. These materials are easily available and possess excellent biocompatibility, biodegradability and potent bioactive characteristics. Important applications of marine biomaterials include medical applications, antimicrobial agents, drug delivery agents, anticoagulants, rehabilitation of diseases such as cardiovascular diseases, bone diseases and diabetes, as well as comestible, cosmetic and industrial applications.
... One approach is the use of biomimetic methods, for example to use naturally occurring biocomposites. For instance, instead of spin-coating chitin on a silicon surface [23], silica-chitin based biocomposites, or other biocomposites, could be used and it is available from various sources and can even be obtained invitro [24][25][26][27][28]. A newly emerging area is the use of natural sources from environments where life is found under extreme heat or pressure, also referred to as extreme biometrics [5]. ...
Chitin, abundant in nature, is a renewable resource with many possible applications in bioengineering. Biosensors, capable of label-free and in-line evaluation, play an important role in the investigation of chitin synthesis, degradation and interaction with other materials. This work presents a comparative study of the usefulness of a chitin surface preparation, either on gold (Au) or on polystyrene (PS). In both cases the most common method to dissolve chitin was used, followed by a simple spin-coating procedure. Multi-parametric surface plasmon resonance (MP-SPR), modeling of the optical properties of the chitin layers, scanning electron microscopy, and contact angle goniometry were used to confirm: the thickness of the layers in air and buffer, the refractive indices of the chitin layers in air and buffer, the hydrophobicity, the binding properties of the chitin binding domain (CBD) of Bacillus circulans , and the split-intein capture process. Binding of the CBD differed between chitin on Au versus chitin on PS in terms of binding strength and binding specificity due to a less homogenous structured chitin-surface on Au in comparison to chitin on PS, despite a similar thickness of both chitin layers in air and after running buffer over the surfaces. The use of the simple method to reproduce chitin films on a thin polystyrene layer to study chitin as a biosensor and for chitin binding studies was obvious from the SPR studies and the binding studies of CBD as moiety of chitinases or as protein fusion partner. In conclusion, stable chitin layers for SPR studies can be made from chitin in a solution of dimethylacetamide (DMA) and lithium chloride (LiCl) followed by spin-coating if the gold surface is protected with PS.
... The use of natural materials as scaffolds is beneficial, and recently it has been shown that certain marine organisms are a promising bio-source to obtain collagen for scaffold formation in a variety of biomedical applications. The great interest in the field is highlighted by a series of studies for biomaterial isolated from different marine species allow assorted applications as biomedical devices [2][3][4][5][6][7][8][9][10][11]. ...
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Scaffold material is essential in providing mechanical support to tissue, allowing stem cells to improve their function in the healing and repair of trauma sites and tissue regeneration. The scaffold aids cell organization in the damaged tissue. It serves and allows bio mimicking the mechanical and biological properties of the target tissue and facilitates cell proliferation and differentiation at the regeneration site. In this study, the developed and assayed bio-composite made of unique collagen fibers and alginate hydrogel supports the function of cells around the implanted material. We used an in vivo rat model to study the scaffold effects when transplanted subcutaneously and as an augment for tendon repair. Animals’ well-being was measured by their weight and daily activity post scaffold transplantation during their recovery. At the end of the experiment, the bio-composite was histologically examined, and the surrounding tissues around the implant were evaluated for inflammation reaction and scarring tissue. In the histology, the formation of granulation tissue and fibroblasts that were part of the inclusion process of the implanted material were noted. At the transplanted sites, inflammatory cells, such as plasma cells, macrophages, and giant cells, were also observed as expected at this time point post transplantation. This study demonstrated not only the collagen-alginate device biocompatibility, with no cytotoxic effects on the analyzed rats, but also that the 3D structure enables cell migration and new blood vessel formation needed for tissue repair. Overall, the results of the current study proved for the first time that the implantable scaffold for long-term confirms the well-being of these rats and is correspondence to biocompatibility ISO standards and can be further developed for medical devices application.
... The present study is focused on bio-inspired composites based on silica and collagen as biphasic materials as well as triphasic composites based on silica, collagen and a further mineral phase. These composite materials for bone substitution are inspired by marine glass sponge spicules due to their excellent mechanical properties 7 . To produce multiphasic silica/collagen xerogels, fibrillar bovine collagen was used as template for silica mineralization. ...
Full-text available
Multiphasic silica/collagen xerogels are biomaterials designed for bone regeneration. Biphasic silica/collagen xerogels (B30) and triphasic xerogels (B30H20 or B30CK20) additionally containing hydroxyapatite or calcite were demonstrated to exhibit several structural levels. On the first level, low fibrillar collagen serves as template for silica nanoparticle agglomerates. On second level, this silica-enriched matrix phase is fiber-reinforced by collagen fibrils. In case of hydroxyapatite incorporation in B30H20, resulting xerogels exhibit a hydroxyapatite-enriched phase consisting of hydroxyapatite particle agglomerates next to silica and low fibrillar collagen. Calcite in B30CK20 is incorporated as single non-agglomerated crystal into the silica/collagen matrix phase with embedded collagen fibrils. Both the structure of multiphasic xerogels and the manner of hydroxyapatite or calcite incorporation have an influence on the release of calcium from the xerogels. B30CK20 released a significantly higher amount of calcium into a calcium-free solution over a three-week period than B30H20. In calcium containing incubation media, all xerogels caused a decrease in calcium concentration as a result of their bioactivity, which was superimposed by the calcium release for B30CK20 and B30H20. Proliferation of human bone marrow stromal cells in direct contact to the materials was enhanced on B30CK20 compared to cells on both plain B30 and B30H20.
This book covers recent trends in all aspects of basic and applied scientific research on marine skeletal proteins and biopolymers (e.g., chitin, collagen), and their derivatives. Some recent innovations of marine proteins have been incorporated in this book that could be potentially applied in scientific and industrial research. Due to their broad array of biological functions in biopolymer- and protein-based drugs, such as anticancer, antimicrobial, bone tissue regeneration, antioxidant, and anti-aging functions, bioactive skeletal proteins and biopolymers have recently attracted a great amount of interest in the pharmaceutical, nutraceutical, and cosmeceutical industries (including anti-aging drugs).
Collagens represent the suprafamily of more than 28 main types which have been isolated and described in diverse marine organisms including invertebrates. Especially collagens from glass sponges (Hexactinellida) and horny sponges (Demospongiae) attract attention because of their very ancient origin and unique structural features. Domains typical for collagen have been detected as main structural segments in other structural marine proteins including spongin, gorgonin, byssus, conchiolin. Collagen is to be found as crucial player in diverse mineral-based biocomposites as well as within non-mineralized, cartilage-like tissues of invertebrates. Due to their biocompatibility, marine collagens remain to be biological materials in trend for applications in biomedicine, regenerative medicine, wound healing, cartilage and hard tissue engineering. Soft corals collagens have been recently recognized as a new potential group pf marine collagens for biomedicine.
Skeletons of only marine hexactinellids (glass sponges) display an amazing amount of sizes, complexity and diversity due to their ability to produce silica-based spicules of triaxonic (cubic) and mostly hexactinic symmetry. These structural repetitive motifs are to be found in up to 2 m large and highly hierarchical structured skeletons of selected hexactinellid species. Recent data confirmed, however, the presence of crystalline phases of calcium carbonates origin within glassy spicules of some hexactinellids. Hexactinnelds are still in trend as objects of investigations which are carried out by experts in materials science, architecture, photonics and biomimetics oriented scientific directions.
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Es wurden Basal-Spiculen des Glasschwammes Hyalonema sieboldi untersucht. Sie sind bemerkenswert hinsichtlich ihrer Größe, hohen Flexibilität und faseroptischen Eigenschaften, was sie zu einem interessanten neuartigen Material für die Biomimetik macht. Diese Spiculen sind bekannt für ihre hierarchische Strukturierung. Die bioorganische Grundsubstanz, an die sich die Silikate beim Aufbau der Strukturen anlagern, konnte jedoch bisher nicht geklärt werden, da die übliche Desilikatisierung mittels Fluorwasserstoff zu einer drastischen Strukturänderung der beteiligten Proteine führt. Um dieses Hindernis zu umgehen, entwickelten wir eine neue, langsame Ätzmethode, die mit wässriger 2,5 M NaOH-Lösung bei 37 °C über 14 Tage vorgenommen wird. Wir zeigen zum ersten Mal, dass dieselbe Klasse von Proteinen (Kollagene), die bei der Knorpel- und Knochenbildung eine Rolle spielt, auch die Matrix für die Abscheidung amorphen Silikates in Schwamm-Spiculen bildet. Unsere Entdeckung eröffnet neue Möglichkeiten für die Entwicklung neuartiger, biomimetisch hergestellter, auf Kollagen basierender Silikat-Komposite mit Anwendungen in der Materialwissenschaft, der Biomedizin und weiteren modernen Technologien.
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Glass sponges of the class Hexactinellida are a group of the most ancient multicellular animals, whose fossil remnants from the early Proterozoic have been registered. In order to demineralize the skeletal structures of the glass sponge Hyalonema sieboldi, we have used for the first time a strategy of slow leaching of the silicon-bearing component, based on the usage of alkaline solutions of sodium hydroxide, sodium dodecyl sulfate, and an anionic biosurfactant of a rhamnolipid nature. The obtained data unequivocally corroborate the presence of a fibrillar protein matrix functioning as a basis for silicon biomineralization in the basal spicules of H. sieboldi. Also, it has been found for the first time that the protein matrix is constructed of a collagenous protein. The technical approach proposed here might appear important for the study of the structural organization of skeletons in other silicon-bearing animals and, in an applied aspect, to work out new biomaterials for implantology and biocomposites, in order to use the latter as bioactive additives.
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Nanoscale control of the polymerization of silicon and oxygen determines the structures and properties of a wide range of siloxane-based materials, including glasses, ceramics, mesoporous molecular sieves and catalysts, elastomers, resins, insulators, optical coatings, and photoluminescent polymers. In contrast to anthropogenic and geological syntheses of these materials that require extremes of temperature, pressure, or pH, living systems produce a remarkable diversity of nanostructured silicates at ambient temperatures and pressures and at near-neutral pH. We show here that the protein filaments and their constituent subunits comprising the axial cores of silica spicules in a marine sponge chemically and spatially direct the polymerization of silica and silicone polymer networks from the corresponding alkoxide substrates in vitro, under conditions in which such syntheses otherwise require either an acid or base catalyst. Homology of the principal protein to the well known enzyme cathepsin L points to a possible reaction mechanism that is supported by recent site-directed mutagenesis experiments. The catalytic activity of the “silicatein” (silica protein) molecule suggests new routes to the synthesis of silicon-based materials.
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The silica skeleton of the deep-sea sponge Euplectella aspergillum was recently shown to be structured over at least six levels of hierarchy with a clear mechanical functionality. In particular, the skeleton is built of laminated spicules that consist of alternating layers of silica and organic material. In the present work, we investigated the micromechanical properties of the composite material in spicules of Euplectella aspergillum and the giant anchor spicule of Monorhaphis chuni. Organic layers were visualized by backscattered electron imaging in the environmental scanning electron microscope. Raman spectroscopic imaging showed that the organic layers are protein-rich and that there is an OH-enrichment in silica near the central organic filament of the spicule. Small-angle x-ray scattering revealed the presence of nanospheres with a diameter of only 2.8 nm as the basic units of silica. Nanoindentation showed a considerably reduced stiffness of the spicule silica compared to technical quartz glass with different degrees of hydration. Moreover, stiffness and hardness were shown to oscillate as a result of the laminate structure of the spicules. In summary, biogenic silica from deep-sea sponges has reduced stiffness but an architecture providing substantial toughening over that of technical glass, both by structuring at the nanometer and at the micrometer level.
The three-dimensional microstructure of a Strombus decorus persicus seashell was studied by means of high-resolution electron microscopy and energy-variable X-ray diffraction on synchrotron beam lines. Energy variation in small steps allows the X-ray penetration depth to be changed precisely and, on this basis, for a non-destructive microstructural analysis with depth resolution to be developed. This technique enabled determination of depth-resolved microstructural parameters, such as the degree of the preferred orientation, the lamella size, and average microstrain fluctuations in both the prismatic and the crossed-lamellar layers of these seashells. The X-ray results were in good agreement with direct observations made by electron microscopy. A detailed study of the shell microstructure shed additional light on the relationship between the structural characteristics and superior mechanical properties of seashells.
The internal skeletons of sponges of the class Hexactinellida comprise a meshwork of six-rayed spicules of ca. 1 mm diameter made from solid silica (SiO2-nH2O). Their hierarchical construction from the nanometer to the centimeter scale has been elucidated, but as yet the nature of the organic template on which silica is deposited from dissolved silicic acid Si(OH)4 has eluded identification. In order to investigate the structure of the organic matrix associated with silica, we studied spicules from the stalk of the glass rope sponge (Hyalonema sieboldi). These anchoring spicules are remarkable for their size, durability, high flexibility and their exceptional fiber-optic properties which together render them of interest as a novel natural material. Among these investigations, we present a study confirming our hypothesis that an organic matrix of collagenous nature within the H. sieboldi spicules is responsible for their extraordinary mechanical properties. Hexactinellida sponges are organisms that date back to the Cambrium period (600 million years ago); consequently, it can be assumed that the evolutionary history of collagen is at least equally long. Furthermore, collagen also serves as a template for calcium phosphate and carbonate deposition in bone, suggesting that the evolution of silica and bone skeletons share a common origin with respect to collagen as a unified template for biomineralization.
A novel biomimetic hybrid material made of silicified collagen was analyze for bone replacement. The evolution of silica and bone skeletons shared a common origin with respect to collagen as a unified template for biomineralization. The sol-gel route was used to mimic biological processing as it works under ambient conditions and allows the formation of a mineral phase for solution and the integration of a second component. A paste of fibrillar bovine collagen was used as an organic template for silicification in vitro. The reinforcement of the hybrid xerogels due to synergetic effects between the silica and the collagen network enabled their direct usage as substrates for cell culture experiments. Results show that the silica-collagen hybrid materials exhibit proper biocompatibility by supporting the adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells.