Nanostructural Organization of Naturally Occurring Composites Part I: Silica-Collagen-Based Biocomposites

Abstract

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.

Full text

Hindawi Publishing Corporation
Journal of Nanomaterials
Volume 2008, Article ID 623838, 8pages
doi:10.1155/2008/623838
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, hermann.ehrlich@tu-dresden.de
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. 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 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
cited.
1. INTRODUCTION
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 [1–3]. First observations reported by L´
evi
et al. [4] on silica-based spicules of a Monorhaphis sponge
generated great interest because of their combination of
properties, namely, toughness combined with stiffness, 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 stiffness of the spicule com-
pared to technical quartz glass with different degrees of hy-
dration. Moreover, stiffness 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 stiffness 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 [9–11], 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. EXPERIMENTAL
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, 12◦31.5’–25.04’ S 48◦05.5’–
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 15◦C[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
(a)
5mm
(b)
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
37◦C and shaken slowly for 14 days. The effectiveness of the
slow alkali etching was monitored using scanning electron
microscopy (SEM) at different 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-offof 14 kDa. Dialysis was per-
formed for 48 hours at 4◦C. 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 buffer (1 M Tris-HCl,
pH 6.8, 2.5% SDS, 10% glycerine, 0.0125% bromphenol
blue) incubated at 95◦C 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 4◦C 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 differentiation into
osteoblast-like cells [16].
3. RESULTS AND DISCUSSION
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. [9–11] developed novel,
slow etching methods, which use solutions of 2.5 M NaOH
at 37◦C 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 [9–11,21] 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
2μm
(a)
(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
M¨
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 37◦C in vitro. The results ob-
400 nm700 nm
700 nm 5 nm
(a) (b)
(c) (d)
Fibrils
Silica
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 different 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 different 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
(a)
(b)
Amino
acid
Monorhaphis
chuni collagen
Hyalonema
collagen [21]
Chondrosia
collagen [21]
6.1
4.9
10
0.1
9.5
25.5
1.4
7.1
3.2
4
1.6
2.5
0.4
2
6.7
4.4
5.2
1.2
4.8
Ala
Arg
Asx
Cys
Glx
Gly
His
H-LPro
Ile
Leu
L-HLys
Lys
Met
Phe
Pro
Ser
Thr
Tyr
Val
6.2
4.8
10.7
0.2
9.3
24.5
1.6
6.9
3.8
4.3
1.5
2
0.4
2.2
6.5
4.7
5.6
1.2
4.1
6.2
4.6
10.4
0
9
30.6
0.4
9.8
2.5
3.6
1.6
0.7
0.2
1.8
6.3
4.5
4.3
0.6
2.9
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 different sources [21].
Silicified
collagen
fibrils
Collagen net
Axial
filament
Axial
canal
Spicule
Collagenous
fibrillar matrix
(“glue”)
(a)
(b)
(c)
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
5mm
(a)
500 nm
(b)
100 μm
(c)
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¨
uller
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 different
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 differentiation 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.
4. CONCLUSION
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
scaffold for the deposition of a reinforcing mineral phase in
the form of silica. These findings allow us to discard differ-
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
ACKNOWLEDGMENTS
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.
REFERENCES
[1] J. Aizenberg, J. C. Weaver, M. S. Thanawala, V. C. Sundar,
D. E. Morse, and P. Fratzl, “Skeleton of Euplectella sp.: struc-
tural hierarchy from the nanoscale to the macroscale,” Science,
vol. 309, no. 5732, pp. 275–278, 2005.
[2] G. Mayer, “Rigid biological systems as models for synthetic
composites,” Science, vol. 310, no. 5751,pp. 1144–1147, 2005.
[3] J. C. Weaver, J. Aizenberg, G. E. Fantner, et al., “Hierarchi-
cal assembly of the siliceous skeletal lattice of the hexactinellid
sponge Euplectella aspergillum ,” Journal of Structural Biology,
vol. 158, no. 1, pp. 93–106, 2007.
[4] C. L´
evi, J. L. Barton, C. Guillemet, E. Le Bras, and P. Lehuede,
“A remarkably strong natural glassy rod: the anchoring spicule
of the Monorhaphis sponge,” Journal of Materials Science Let-
ters, vol. 8, no. 3, pp. 337–339, 1989.
[5] W.E.G.M
¨
uller, C. Eckert, K. Kropf, et al., “Formation of gi-
ant spicules in the deep-sea hexactinellid Monorphaphis chuni
(Schulze 1904): electron-microscopic and biochemical stud-
ies,” Cell and Tissue Research, vol. 329, no. 2, pp. 363–378,
2007.
[6] A. Woesz, J. C. Weaver, M. Kazanci, et al., “Microme-
chanical properties of biological silica in skeletons of deep-
sea sponges,” Journal of Materials Research, vol. 21, no. 8,
pp. 2068–2078, 2006.
[7] H. Ehrlich and H. Worch, “Sponges as natural composites:
from biomimetic potential to development of new biomate-
rials,” in Porifera Research: Biodiversity, Innovation & Sustain-
ability, M. R. Custodio, G. Lobo-Hajdu, E. Hajdu, and G.
Muricy, Eds., Museu Nacional, Rio de Janeiro, Brasil, 2007.
[8] J. Aizenberg, V. C. Sundar, A. D. Yablon, J. C. Weaver, and G.
Chen, “Biological glass fibers: correlation between optical and
structural properties,” Proceedings of the National Academy
of Sciences of the United States of America, vol. 101, no. 10,
pp. 3358–3363, 2004.
[9]H.Ehrlich,T.Hanke,P.Simon,etal.,“Demineralizationof
natural silica based biomaterials: new strategy for the isola-
tion of organic frameworks,” BIOmaterialien, vol. 6, no. 4,
pp. 297–302, 2005.
[10] H. Ehrlich, T. Hanke, H. Meissner, et al., “Nanoimagery and
the biomimetic potential of marine glass sponge Hyalonema
sieboldi (Porifera),” VDI Ber ichte, vol. 1920, pp. 163–166, 2005.
[11] H. Ehrlich, A. V. Ereskovskii, A. L. Drozdov, et al., “A mod-
ern approach to demineralization of spicules in glass sponges
(Porifera: Hexactinellida) for the purpose of extraction and
examination of the protein matrix,” Russian Journal of Marine
Biology, vol. 32, no. 3, pp. 186–193, 2006.
[12] H. Ehrlich, M. Krautter, T. Hanke, et al., “First evidence of
the presence of chitin in skeletons of marine sponges. Part
II. Glass sponges (Hexactinellida: Porifera),” JournalofExper-
imental Zoology Part B, vol. 308, no. 4, pp. 473–483, 2007.
[13] S. Kimura and M. L. Tanzer, “Nereis cuticle collagen. Isolation
and properties of a large fragment resistant to proteolysis by
bacterial collagenase,” Journal of Biological Chemistry, vol. 252,
no. 22, pp. 8018–8022, 1977.
[14] A. Shevchenko, M. Wilm, O. Vorm, and M. Mann, “Mass spec-
trometric sequencing of proteins from silver-stained polyacry-
lamide gels,” Analytical Chemistry, vol. 68, no. 5, pp. 850–858,
1996.
[15] S. Heinemann, H. Ehrlich, C. Knieb, and T. Hanke,
“Biomimetically inspired hybrid materials based on silicified
collagen,” International Journal of Materials Research, vol. 98,
no. 7, mbox pp. 603–608, 2007.
[16] S. Heinemann, C. Knieb, H. Ehrlich, et al., “A novel
biomimetic hybrid material made of silicified collagen: per-
spectives for bone replacement,” Advanced Engineering Mate-
rials, vol. 9, no. 12, pp. 1061–1068, 2007.
[17] J. C. Weaver and D. E. Morse, “Molecular biology of demo-
sponge axial filaments and their roles in biosilicification,” Mi-
croscopy Research and Technique, vol. 62, no. 4, pp. 356–367,
2003.
[18] M.-J. Uriz, X. Turon, M. A. Becerro, and G. Agell, “Siliceous
spicules and skeleton frameworks in sponges: origin, diversity,
ultrastructural patterns, and biological functions,” Microscopy
Research and Technique, vol. 62, no. 4, pp. 279–299, 2003.
[19] O. B¨
utschli, “Einige beobachtungen ¨
uber die kiesel- und kalk-
nadeln von spongien,” Zeitschrift f¨
ur Wissenschaftliche Zoolo-
gie, vol. 59, no. 2, mbox pp. 235–286, 1901.
[20] G. Croce, A. Frache, M. Milanesio, et al., “Fiber diffraction
study of spicules from marine sponges,” Microscopy Research
and Technique, vol. 62, no. 4, pp. 378–381, 2003.
[21] H. Ehrlich and H. Worch, “Collagen, a huge matrix in
glass-sponge flexible spicules of the meter-long Hyalonema
sieboldi,” in Handbook of Biomineralization. Vol.1. The Biol-
ogy of Biominerals Structure Formation,E.B
¨
auerlein, Ed., Wi-
ley VCH, Weinheim, Germany, 2007.
[22] M. Schultze, Die Hyalonemen. Ein Beitrag zur Naturgeschichte
der Spongien, Adolph Marcus, Bonn, Germany, 1860.
[23] T. Kondo, M. Nojiri, Y. Hishikawa, E. Togawa, D. Romanovicz,
and R. M. Brown Jr., “Biodirected epitaxial nanodeposition of
polymers on oriented macromolecular templates,” Proceedings
of the National Academy of Sciences of the United States of Amer-
ica, vol. 99, no. 22, pp. 14008–14013, 2002.
[24] J. N. Cha, K. Shimizu, Y. Zhou, et al., “Silicatein filaments
and subunits from a marine sponge direct the polymeriza-
tion of silica and silicones in vitro,” Proceedings of the National
Academy of Sciences of the United States of America, vol. 96,
no. 2, pp. 361–365, 1999.
[25] M. M. Murr and D. E. Morse, “Fractal intermediates in the
self-assembly of silicatein filaments,” Proceedings of the Na-
tional Academy of Sciences of the United States of America,
vol. 102, no. 33, pp. 11657–11662, 2005.
[26] X.-H. Wang, J.-H. Li, L. Qiao, et al., “Structure and character-
istics of giant spicules of the deep sea hexactinellid sponges
of the genus Monorhaphis (Hexactinellida: Amphidiscosida:
Monorhaphididae),” Acta Zoologica Sinica,vol.53,no.3,
pp. 557–569, 2007.
[27] “Sequence Database Setup: MSDB,” Imperial College London,
http://www.matrixscience.com.

8Journal of Nanomaterials
[28] S. Heinemann, H. Ehrlich, T. Douglas, et al., “Ultrastruc-
tural studies on the collagen of the marine sponge Chon-
drosia reniformis nardo,” Biomacromolecules, vol. 8, no. 11,
pp. 3452–3457, 2007.
[29] S. Hattori, E. Adachi, T. Ebihara, T. Shirai, I. Someki, and S.
Irie, “Alkali-treated collagen retained the triple helical confor-
mation and the ligand activity for the cell adhesion via α2β1
integrin,” Journal of Biochemistry, vol. 125, no. 4, pp. 676–684,
1999.
[30] M. M. Giraud-Guille, “Twisted plywood architecture of colla-
gen fibrils in human compact bone osteons,” Calcified Tissue
International, vol. 42, no. 3, pp. 167–180, 1988.
[31] M. M. Giraud-Guille, G. Mosser, C. Helary, and D. Eglin,
“Bone matrix like assemblies of collagen: from liquid crystals
to gels and biomimetic materials,” Micron, vol. 36, no. 7-8,
pp. 602–608, 2005.
[32] W. Wagermaier, H. S. Gupta, A. Gourrier, et al., “Spiral twist-
ing of fiber orientation inside bone lamellae,” Biointerphases,
vol. 1, no. 1, pp. 1–5, 2006.
[33] B. Pokroy and E. Zolotoyabko, “Microstructure of natural
plywood-like ceramics: a study by high-resolution electron
microscopy and energy-variable X-ray diffraction,” Journal of
Materials Chemistry, vol. 13, no. 4, pp. 682–688, 2003.
[34] H. C. Schr ¨
oder, D. Brandt, U. Schloßmacher, et al., “Enzymatic
production of biosilica glass using enzymes from sponges: ba-
sic aspects and application in nanobiotechnology (material
sciences and medicine),” Naturwissenschaften, vol. 94, no. 5,
pp. 339–359, 2007.
[35] C. W. P. Foo, J. Huang, and D. L. Kaplan, “Lessons from
seashells: silica mineralization via protein templating,” Tr ends
in Biotechnology, vol. 22, no. 11, pp. 577–585, 2004.
[36] C. Sanchez, H. Arribart, and M. M. Giraud-Guille,
“Biomimetism and bioinspiration as tools for the design
of innovative materials and systems,” Nature Materials, vol. 4,
no. 4, pp. 277–288, 2005.
[37] E. Pouget, E. Dujardin, A. Cavalier, et al., “Hierarchical
architectures by synergy between dynamical template self-
assembly and biomineralization,” Nature Materials, vol. 6,
no. 6, pp. 434–439, 2007.
[38] P. Fratzl, “Biomimetic materials research: what can we really
learn from nature’s structural materials?” Journal of the Royal
Society Interface, vol. 4, no. 15, pp. 637–642, 2007.