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Journal of Nanomaterials
Volume 2008, Article ID 623838, 8pages
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, firstname.lastname@example.org
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
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´
et al.  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 . 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 . Recently, the micromechani-
cal properties of biological silica in the giant anchor spicule
of Monorhaphis chuni were reported on . 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 . 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 . 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 , struc-
tural , and optical  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 , and spicules of Euplectella aspergillum 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 .
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
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, 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
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
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 .
2.5. Biocompatibility of the silica-collagen
was evaluated by cultivating human mesenchymal stem cells
on the material followed by induced diﬀerentiation into
osteoblast-like cells .
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 , 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
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 , and
present in all representatives of lyssacine Hexactinellida .
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 . We suggest that the
nanomorphology of silica on proteinous structures described
here could be determined as an example of biodirected epi-
taxial nanodistribution  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-
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 . 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
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 
led to the identiﬁcation of collagen alpha 1 in two high
MW bands.In contrast to H. sieboldi , 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. .
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 . The presence of collagen ﬁbrils in alkali solu-
tion is no surprise. Hattori et al.  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
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  for bothin vivo and in vitro 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 ).
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 . 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
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 .
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 . 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
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 , are
responsible for the formation of silica-based structures in
all sponges, we suggested that silicateins are associated with
collagen . 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 , 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 . The disc-like sam-
ples showed convincing homogeneity and mechanical stabil-
ity, enabling cell culture experiments for the ﬁrst time on
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
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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