# Nanostructural Organization of Naturally Occurring Composites—Part II: Silica-Chitin-Based Biocomposites

Authors: Hermann Ehrlich; Dorte Janussen; Paul Simon; Vasily V. Bazhenov; Nikolay P. Shapkin; Christiane Erler; Michael Mertig; René Born; Sascha Heinemann; Thomas Hanke; Hartmut Worch; John N. Vournakis
Journal: Journal of Nanomaterials
 (2008)

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## Abstract

Investigations of the micro- and nanostructures and chemical composition of the sponge skeletons as examples for natural structural biocomposites are of fundamental scientific relevance. Recently, we show that some demosponges (Verongula gigantea, Aplysina sp.) and glass sponges (Farrea occa, Euplectella aspergillum) possess chitin as a component of their skeletons. The main practical approach we used for chitin isolation was based on alkali treatment of corresponding external layers of spicules sponge material with the aim of obtaining alkali-resistant compounds for detailed analysis. Here, we present a detailed study of the structural and physicochemical properties of spicules of the glass sponge Rossella fibulata. The structural similarity of chitin derived from this sponge to invertebrate alpha chitin has been confirmed by us unambiguously using physicochemical and biochemical methods. This is the first report of a silica-chitin composite biomaterial found in Rossella species. Finally, the present work includes a discussion related to strategies for the practical application of silica-chitin-based composites as biomaterials.


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## Body

## 1. Introduction

This is the second paper on
naturally occurring silica-based biocomposites of sponges origin. The initial
paper [1] studied the presence of fibrillar collagen as a component of glassy
anchoring spicule of Monorphaphis sp.
glass sponge.Biocomposites of marine
origin including sponge skeletal formations are a constant source of inspiration
for finding solutions to a variety of technical challenges in bionics,
architecture, optics, engineering, as well as materials science and biomedicine
(reviewed in [2, 3]). The biomimetic potential of marine sponges seems to be a
goldmine to material scientists. Several main aspects relating to sponges as
biomaterials and biocomposites are recently described as follows [3]:(i)
hexactinellid
spicules as natural glass-based composites with specific mechanical properties [4, 5];(ii)
skeleton ofEuplectella sp. (Hexactinellida) as a
hierarchical natural structural material of remarkable design [6, 7];(iii)
basal spicules of
Hexactinellida as biological glass fibers with specific optical properties [8–10];(iv)
silicatein-based
biocatalitic formation of nanocomposite materials (reviewed in [11]);(v)
biomimetically
inspired hybrid materials based on silicified sponge collagen [12–14].In case of hexactinellid spicules, it is
reported that they are highly flexible and tough, possibly not only because of
their layered structure and the hydrated nature of the silica as suggested
earlier [15], but of the presence of collagen [16] or chitin [17]. According to
paleontological and molecular data, the sponge class Hexactinellida may be the
oldest metazoan taxon in earth’s history [18, 19]. Recently, we suggested that
silica-chitin scaffolds may be key templates for skeleton formation also in
ancestral unicellular organisms, rather than silica-protein composites [17].
From this point of view, we hypothesized that chitin molecules are probably
part of very old organic template system involved in a biosilicification
phenomenon, which was established a long time before the origin of glass
sponges and collagen as structural protein with respect to high templating activity
for biomineralization.The objective of the
current study was to test the hypothesis that chitin is an essential component
of the silica spicules of Antarctic glass spongeRossella fibulata (Figure
1(a)) as well, and if so, to unravel its involvement in the mechanical behavior
of these spicules. Nanomechanical properties, nanohardness and elastic modulus,
of a closely related sponge Rossella
racovitzea were determined previously by using a vertical indentation
system attached to an atomic force microscope [15] The Rossella spicules, known to have optical wave conduction
properties, are 10–20 cm long with a
circular cross-section of diameter 200–600μm. The
spicules are composed of 2–10μm-thick
layers of siliceous material that has no detectable crystallinity. Measurements
through the thickness of the spicules indicated uniform properties regardless
of layering. Both the elastic modulus and nanohardness values of the spicules
are about half of that of either fused silica or commercial glass optical fibers.
The fracture strength and fracture energy of the spicules, determined by
3-point bend tests, are several times those of silica rods of similar diameter.
The spicules offer bioinspired lessons for potential biomimetic design of
optical fibers with long-term durability that could potentially be fabricated
at room temperature in aqueous solutions [8]. Unfortunately, the nature and
origin of organic matrix were not investigated in these pioneering studies.Highly
flexible spicules of marine glass spongeRossella
fibulata (a). Pieces of clean spicules were placed in deionized water and
disrupted using magnet stirrer. Visual observations show that the water
solution became of a milky color typical for colloidal suspensions of silica (b).
Silica nanoparticles are associated around oriented organic matrix of
nanofibrillar nature (c). Transparent hard glassy spheres were obtained using
this fraction of colloidal suspension after dropping into 99% TMOS solution at
room temperature (d).
(a)(b)(c)(d)We decided also to
re-examine the results of some previously reported studies concerning the
presence of polysaccharides within silica-containing spicules of another
hexactinellid sponge. For example, Travis et al. [20] reported the presence of
parallel-oriented cellulose-like filaments with an average width of 1.9 nm
observed in organic matrix material after HF-based desilicification of the
spicules of hexactinellid Euplectella sp. These matrices also contain considerable amounts of hexosamine.In this study, we performed
structural, spectroscopic, and biochemical analysis of organic matrix isolated
from spicules ofR. fibulata. Finally,
the present work includes a discussion relating to strategies for the practical
application of silica-chitin- and silica- N-acethyl glucosamine (NAG)-based composites
as biomaterials.


## 2. Experimental

### 2.1. Chemical Etching of Glass Sponge Skeletons

The object of our study wasRossella fibulata Schulze &
Kirkpatrick, 1910 (Hexactinellida: Porifera), collected in 2005 in the Scotia Sea,
Antarctic, at a depth of 200 m.Spicules ofR. fibulata were treated according to
the following procedure. Sponge material of R.
fibulata was stored for several days in fresh sea-water. The sponge was
dried afterwards for 4 days at 45°C. Finally, the sponge skeleton was cleaned
in 10% H2O2 and dried again at 45°C. Tissue-free dried
sponge material was washed three times in distilled water, cut into 3 cm long
pieces and placed in a solution containing purified Clostridium histolyticum collagenase (Sigma Aldrich, Saint Louis, USA) to digest any possible collagen
contamination of exogenous nature. After incubation for 24 hours at 15°C, the
pieces of glass sponge skeleton were again washed three times in distilled
water, dried, and placed in a 15 mL vessel containing chitinase solution (as
described below) to digest any possible exogenous chitin contaminations. After
incubation for 48 hours at 25°C, fragments of skeleton were again washed,
dried, and placed in 10 mL plastic vessel containing 8 mL 2.5 M NaOH solution.
The vessel was covered and placed under thermostatic conditions at 37°C without
shaking.The effectiveness of the
alkali etching was also monitored using optical and scanning electron
microscopy (SEM) at different locations along the length of the spicular
material and within the cross-sectional area. The colourless alkali-insoluble
material obtained after alkali treatment of the glass sponge samples was washed
with distilled water five times and finally dialysed against deionized water on
Roth (Germany)
membranes with a MWCO of 14 kDa. Dialysis was performed for 5 days at 4°C. The
dialyzed material was dried at room temperature and used for structural and analytical
investigations.


### 2.2. Development of the Silica Nanoparticular Fraction Using Mechanical Disruption of CleanedR. fibulata Spicules

The
spicules ofR. fibulata were treated
using enzymes as described above, cleaned, dialysed, and cut in 3 cm long
pieces. These fragments of clean spicules were placed in deionized water and
disrupted mechanically using glass covered stirring bars (25×6 mm) and magnet
stirrer during 24 hours at 25°C. After centrifugation (1500× g) for 5 minutes
the debris of mechanically disrupted spicules was collected and milky colour
supernatant was carefully decanted. Obtained debris-free suspension (Figure 1(b)) was used for SEM and TEM
investigations.


### 2.3. FTIR Spectroscopy

IR spectra were recorded with a Perkin
Elmer FTIR Spectrometer Spectrum 2000, equipped with an AutoImage Microscope
using the FT-IRRAS technique (Fourier transform infrared reflection absorption spectroscopy)
as described previously [21].


### 2.4. Transmission Electron Microscopy (TEM)

Conventional
transmission electron microscopy [1] was performed with a Philips CM200 FEG∖ST
Lorentz electron microscope at an acceleration voltage of 200 kV. For electron
microscopy, a drop of the water suspension containing the sample was placed on
the electron microscopy grid. After one minute, the excess was removed using
blotting paper and thereafter dried in air. The electron microscopy grids (Plano, Germany)
were covered with a holey carbon film.


### 2.5. Scanning Electron Microscopy (SEM) Analysis

The
samples were fixed in a sample holder and covered with carbon, or with a gold
layer for 1 minute using an Edwards S150B sputter coater. The samples were then placed in an
ESEM XL 30 Philips or LEO DSM 982 Gemini scanning electron microscope.


### 2.6. Chitinase Digestion and Test

Dried
20 mg samples of purified sponge spicules, previously pulverized to a fine
powder in an agat mortar, were suspended in 400μL of 0.2 M phosphate buffer at
pH 6.5. Positive control was prepared by solubilizing 0.3% colloidal chitin in
the same buffer. Equal amounts of 1 mg/mL of three chitinases (EC 3.2.1.14 and
EC 3.2.1.30): N-acetyl-β-glucosaminidase from Trichoderma viride (Sigma-Aldrich) number C-8241, and two poly (1,4-β-[2-acetamido-2-deoxy-D-glucoside]) glycanohydrolases
from Serratia marcescens (Sigma-Aldrich)
number. C-7809 and Streptomyces griseus (Sigma-Aldrich) number. C-6137, respectively, were suspended in 100 mM sodium
phosphate buffer at pH 6.0. Digestion was started by mixing 400μL of the
samples and 400μL of the chitinase-mix. Incubation was performed at 37°C and
stopped after 114 hours by adding 400μL of 1% NaOH, followed by boiling for 5
minutes. The effectiveness of the enzymatic degradation was monitored using
optical microscopy (Zeis, Axiovert). The Morgan-Elson assay was used to
quantify the N-acetylglucosamin released after chitinase treatment as described
previously [22]. The
sample which contains chitinase solution without substrate was used as a control.


### 2.7. Preparation ofα-Chitin

Alpha-chitin
was prepared from a commercially available crab shell chitin (Fluka). The
material was purified with aqueous 1 M HCl for 2 hours at 25°C and then
refluxed in 2 M NaOH for 48 hours at 25°C. The resulting α-chitin was washed in
deionized water by several centrifugations until neutrality was reached. The whole procedure was repeated
twice. α-chitin was also
used as a standard for FTIR and for Fourier transform (HRTEM) studies.


### 2.8. Preparation of Colloidal Chitin

Ten
grams ofα-chitin (Fluka) was mixed with 500 mL of 85% phosphoric acid and
stirred for 24 hours at 4°C. The suspension was poured into 5 L of distilled
water (DW) and centrifuged (15000× g for 15 minutes). The resulting
precipitate was washed with DW until the pH reached 5.0 and then neutralized by
addition of 6 N NaOH. The suspension was centrifuged (15000× g for 15 minutes)
and washed with 3 L of DW for desalting. The resulting precipitate was
suspended in DW and dialyzed. The chitin content in the suspension was determined by drying a sample.


### 2.9. Silicification of Colloidal Chitin and NAG

In the first step, 0.93 g of
colloidal chitin or NAG (Sigma-Aldrich, Miss,
USA) previously
suspended in 34.6 g of methanol was added to 51.8 mL of deionized water. The suspension
pH was then raised above 10 with the addition of 100μL of 1 N
NaOH solution. Finally, 169μL of tetramethylorthosilicate (TMOS,
99 wt%, ABCR GmbH, Germany)
were added and the solution was stirred at room temperature. After 1 hour the
suspension was filtered and the recovered precipitate rinsed with deionized water,
then with methanol, and finally air-dried.


## 2.1. Chemical Etching of Glass Sponge Skeletons

The object of our study wasRossella fibulata Schulze &
Kirkpatrick, 1910 (Hexactinellida: Porifera), collected in 2005 in the Scotia Sea,
Antarctic, at a depth of 200 m.Spicules ofR. fibulata were treated according to
the following procedure. Sponge material of R.
fibulata was stored for several days in fresh sea-water. The sponge was
dried afterwards for 4 days at 45°C. Finally, the sponge skeleton was cleaned
in 10% H2O2 and dried again at 45°C. Tissue-free dried
sponge material was washed three times in distilled water, cut into 3 cm long
pieces and placed in a solution containing purified Clostridium histolyticum collagenase (Sigma Aldrich, Saint Louis, USA) to digest any possible collagen
contamination of exogenous nature. After incubation for 24 hours at 15°C, the
pieces of glass sponge skeleton were again washed three times in distilled
water, dried, and placed in a 15 mL vessel containing chitinase solution (as
described below) to digest any possible exogenous chitin contaminations. After
incubation for 48 hours at 25°C, fragments of skeleton were again washed,
dried, and placed in 10 mL plastic vessel containing 8 mL 2.5 M NaOH solution.
The vessel was covered and placed under thermostatic conditions at 37°C without
shaking.The effectiveness of the
alkali etching was also monitored using optical and scanning electron
microscopy (SEM) at different locations along the length of the spicular
material and within the cross-sectional area. The colourless alkali-insoluble
material obtained after alkali treatment of the glass sponge samples was washed
with distilled water five times and finally dialysed against deionized water on
Roth (Germany)
membranes with a MWCO of 14 kDa. Dialysis was performed for 5 days at 4°C. The
dialyzed material was dried at room temperature and used for structural and analytical
investigations.


## 2.2. Development of the Silica Nanoparticular Fraction Using Mechanical Disruption of CleanedR. fibulata Spicules

The
spicules ofR. fibulata were treated
using enzymes as described above, cleaned, dialysed, and cut in 3 cm long
pieces. These fragments of clean spicules were placed in deionized water and
disrupted mechanically using glass covered stirring bars (25×6 mm) and magnet
stirrer during 24 hours at 25°C. After centrifugation (1500× g) for 5 minutes
the debris of mechanically disrupted spicules was collected and milky colour
supernatant was carefully decanted. Obtained debris-free suspension (Figure 1(b)) was used for SEM and TEM
investigations.


## 2.3. FTIR Spectroscopy

IR spectra were recorded with a Perkin
Elmer FTIR Spectrometer Spectrum 2000, equipped with an AutoImage Microscope
using the FT-IRRAS technique (Fourier transform infrared reflection absorption spectroscopy)
as described previously [21].


## 2.4. Transmission Electron Microscopy (TEM)

Conventional
transmission electron microscopy [1] was performed with a Philips CM200 FEG∖ST
Lorentz electron microscope at an acceleration voltage of 200 kV. For electron
microscopy, a drop of the water suspension containing the sample was placed on
the electron microscopy grid. After one minute, the excess was removed using
blotting paper and thereafter dried in air. The electron microscopy grids (Plano, Germany)
were covered with a holey carbon film.


## 2.5. Scanning Electron Microscopy (SEM) Analysis

The
samples were fixed in a sample holder and covered with carbon, or with a gold
layer for 1 minute using an Edwards S150B sputter coater. The samples were then placed in an
ESEM XL 30 Philips or LEO DSM 982 Gemini scanning electron microscope.


## 2.6. Chitinase Digestion and Test

Dried
20 mg samples of purified sponge spicules, previously pulverized to a fine
powder in an agat mortar, were suspended in 400μL of 0.2 M phosphate buffer at
pH 6.5. Positive control was prepared by solubilizing 0.3% colloidal chitin in
the same buffer. Equal amounts of 1 mg/mL of three chitinases (EC 3.2.1.14 and
EC 3.2.1.30): N-acetyl-β-glucosaminidase from Trichoderma viride (Sigma-Aldrich) number C-8241, and two poly (1,4-β-[2-acetamido-2-deoxy-D-glucoside]) glycanohydrolases
from Serratia marcescens (Sigma-Aldrich)
number. C-7809 and Streptomyces griseus (Sigma-Aldrich) number. C-6137, respectively, were suspended in 100 mM sodium
phosphate buffer at pH 6.0. Digestion was started by mixing 400μL of the
samples and 400μL of the chitinase-mix. Incubation was performed at 37°C and
stopped after 114 hours by adding 400μL of 1% NaOH, followed by boiling for 5
minutes. The effectiveness of the enzymatic degradation was monitored using
optical microscopy (Zeis, Axiovert). The Morgan-Elson assay was used to
quantify the N-acetylglucosamin released after chitinase treatment as described
previously [22]. The
sample which contains chitinase solution without substrate was used as a control.


## 2.7. Preparation ofα-Chitin

Alpha-chitin
was prepared from a commercially available crab shell chitin (Fluka). The
material was purified with aqueous 1 M HCl for 2 hours at 25°C and then
refluxed in 2 M NaOH for 48 hours at 25°C. The resulting α-chitin was washed in
deionized water by several centrifugations until neutrality was reached. The whole procedure was repeated
twice. α-chitin was also
used as a standard for FTIR and for Fourier transform (HRTEM) studies.


## 2.8. Preparation of Colloidal Chitin

Ten
grams ofα-chitin (Fluka) was mixed with 500 mL of 85% phosphoric acid and
stirred for 24 hours at 4°C. The suspension was poured into 5 L of distilled
water (DW) and centrifuged (15000× g for 15 minutes). The resulting
precipitate was washed with DW until the pH reached 5.0 and then neutralized by
addition of 6 N NaOH. The suspension was centrifuged (15000× g for 15 minutes)
and washed with 3 L of DW for desalting. The resulting precipitate was
suspended in DW and dialyzed. The chitin content in the suspension was determined by drying a sample.


## 2.9. Silicification of Colloidal Chitin and NAG

In the first step, 0.93 g of
colloidal chitin or NAG (Sigma-Aldrich, Miss,
USA) previously
suspended in 34.6 g of methanol was added to 51.8 mL of deionized water. The suspension
pH was then raised above 10 with the addition of 100μL of 1 N
NaOH solution. Finally, 169μL of tetramethylorthosilicate (TMOS,
99 wt%, ABCR GmbH, Germany)
were added and the solution was stirred at room temperature. After 1 hour the
suspension was filtered and the recovered precipitate rinsed with deionized water,
then with methanol, and finally air-dried.


## 3. Results and Discussion

Most of the glass sponges inhabit soft
muddy substrates. One of the strategies of survival under such conditions is the
formation of root structures that prevent the body of the animal from sinking
into the ground [23]. Due to their preferred deep-sea habitat, the Hexactinellida
have been poorly investigated with respect to their general biology [24, 25] and
the nature of organic components which build their skeletal structures. It was
generally accepted that their skeletons are composed of concentric layers of amorphous
hydrated silica, containing varying amounts of organic material [26, 27]
deposited around a proteinaceous axial filament [28, 29].The finding of collagen
within basal spicules ofH. sieboldi [12, 16] and chitin in skeletons
of F. occa [17] and in spicules of E. aspergillum [3], stimulated our
attempts to find materials of organic nature in other species of glass sponges.
In the case of R. fibulata, we have
not observed any visible signs of demineralization of these materials using optical
microscopy and SEM after 14 days and at the similar experimental conditions as
in the study on H. sieboldi and Monorhaphis sp. On the contrary, spicules
of R. fibulata show high resistance
to alkali treatment even after 3 months of demineralization. This was similar
the resistance observed for E.
aspergillum [3]. This phenomenon led us to the assumption that siliceous skeletons
of investigated sponges possess a material which protects amorphous silica from
dissolution in alkali, and is highly resistant to alkali digestion. It is well
known that chitin in alkali is stable with respect to degradation [30].
Correspondingly, in our experiments, chitin was the first candidate for a
biomaterial with this property.Initially, we performed experiments
on mechanical disruption of cleanedR.
fibulata spicules (Figure 1(a)) in deionized water as described above. This
method of desintegration of spicules was very effective. Visual observations
show that water solution became of a milky color typical for silica colloidal
suspensions (Figure 1(b)) even after 6 hours. Debris-free suspensions obtained
in this way were stable during 3-4 days. SEM of the suspensions confirmed their
nanoparticular structure (Figure 1(c)). Silica nanoparticles of diameter
between 20 and 35 nm are associated around oriented organic matrix of
nanofibrillar nature as shown in Figure 1(c). To verify whether this kind of
silica-organic matrix obtained from the colloidal suspension mimic the
biosilicification, we carried out in
vitro experiments in which we
exposed it to silicic acid solution derived from TMOS. We developed hard and
transparent glassy spheres (Figure 1(d)) which were stable in water and in air
during several months.TEM investigation of these colloidal
suspensions (Figure2(a)) used for the development of spherical glassy
materials clearly revealed that organic crystallites of approximately 3 nm in diameter
are embedded in amorphous silica matrix. Observed HRTEM image (Figure 2(b)) is
highly similar to previously reported HRTEM images of chitin nanocrystallites
of the same diameter [31]. Therefore, in following experiments it was decided
to isolate organic matrix from silica-containing spicules of R. fibulata using a desilicification
procedure based on alkali treatment [16, 17].TEM images
of silica-organic matrix-based suspension obtained after mechanical disruption
of spicules in deionized water (a). Crystallites of organic nature are embedded
in amorphous silica (b).
(a)(b)To test our hypothesis
that alkali-insoluble residues ofR.
fibulata spicules are of chitinous nature, we carried out different highly
sensitive structural and biochemical analysis as described below.FTIR observation of purified,
dialysed, and dried samples of the alkali-insoluble organic matrix isolated
after demineralization ofR. fibulata spicules (Figure 3) confirmed occurrence of pyranose rings and β-1, 4 linkages,
with peaks at 1156 cm−1 and at 894 cm−1 very similar to
those of α-chitin. It was reported previously that the spectral feature between
1153 and 1157 cm−1 is mainly associated with an ether bond in a
pyranose ring [32, 33]. The β-linkage was also indicated by the infrared
absorption, in which a typical peak at 890–892 cm−1 for α-chitin was observed [34, 35].Figure 3
FTIR
spectra of organic matrix isolated after desilicification ofR. fibulata spicules show strong
evidence for β-1, 4-glycosodic linkage at 890–896 cm−1 and for ether bond in pyranose ring at 1153–1157 cm−1 (arrows).There is no evidence for the presence of Si–O–Si bonds.In recent years, high-resolution
electron microscopy has proved to be an important tool for analysis of the structure
of fibrous crystalline polysaccharides, such as cellulose and chitin [36–39]. Therefore, the samples of organic
matrix used for FTIR were subsequently submitted to HR-TEM analyses in order to
examine the crystalline nature of this material and the plausible additional
occurrence of chitin. HRTEM and AFM
studies (Figures 4(a) and 4(b), resp.) of the organic matrix residue obtained after demineralization of R. fibulata spicules revealed the presence of
nanocrystallites having a diameter of 2 nm. These structures were extremely
similar to those previously reported by TEM observations of chitinous skeletal
formations in insects, crustaceans, and arachnid species [40–42]. For further
examination, high-resolution electron micrographs were taken from particular
sample regions (data not shown).The Fourier transform of the high-resolution
micrograph revealed a spacing of 4.79Å (a-axis), 10.2Å (fiber axis), 3.73Å,
and 2.77Å. Such distances, corresponded to [(100) (040)], (001), [(130), (050)
], and [(103), (043) (113)] reflections, proving the orthorhombic structure
typical for α-chitin, as described in detail by Carlström [34] and Minke and Blackwell [42]. These measurements confirm our earlier observations [17, 21]
that chitin in marine sponges appear to be consistently in the alpha
modification.High-resolution transmission electron microscopy image of the fragment of isolated
chitin nanofibril (a); the arrows indicate the presence of crystallite-like
structures with a diameter which corresponds to that of chitin crystallites (2 nm). AFM micrograph of chitin nanofibrillar matrix (b).
(a)(b)To quantify chitin in our samples,
we measured the amount of N-acetyl glucosamine released by chitinases using a
Morgan-Elson colorimetric assay [22], which is the most reliable method for the
identification of alkali-insoluble chitin because of its specificity [43]. We
detected 19.2±1.5μg N-acetyl-glucosamine per mg of spicule of R. fibulata.The finding of silica-chitin natural
composites as the component of theR. fibulata spicules is in good
agreement with results of in vitro
experiments on silicification of a β-chitin-containing cuttlebone-derived
organic matrix as reported by Ogasawara et al. [44]. These authors suggest that
silicate ions and silica oligomers preferentially interact with glycopyranose
rings exposed at the β-chitin surface, presumably by polar and H-bonding
interactions. We believe that chitin is acting as an organic template for
silica mineralization in Rossella species in a very similar fashion as in F.
occa [17] and E. aspergillum [3].
On the basis of the results presented in this work, we propose a model for the
nanostructure of the naturally occurring silica-chitin composite unit,
including interaction between poly-N-acetyl glucosamine-fragment of the chitin
nanofibril and silica nanoparticles, which can be seen in Figure 5.Figure 5
Proposed
model of nanostructural organization of the naturally occurring silica-chitin
composite unit (a) isolated from spicules ofR. fibulata. Silica nanoparticles tightly surround chitinous
nanofibrils (b). Schematic view (c) shows a possible nanodistribution of silica
on the surface of chitinous nanofibril. Image (d) represents the hypothetic
scheme of interaction between silica and poly-N-acetyl glucosamine-fragment of
the chitin nanofibril and formation of the corresponding hydrogen bonds.Because sponges are
often regarded as the most ancient metazoans (630 to 542 My) [45, 46], the finding of chitin within skeletal
formations of these organisms is of major scientific significance, since it
gives important indications to the basic pattern of the Metazoa. As chitin also
serves as a template for calcium carbonate deposition in sponges [21], this
suggests that the evolution of mineralized skeletons in early metazoans share a
common origin with respect to chitin as a unified template for
biomineralization, similar to collagen as common structural protein in nature
[12]. This feature may be considered a basic metazoan character and thus also
has implications for the question of establishing the monophyletic status of
the taxon Metazoa.A comprehensive
understanding of silica-chitin-based sponge skeletons with respect to chemical
composition and structure may prove to be a novel model for the biomimetic
synthesis also of N-acetyl glucosamine (NAG) and poly-NAG-based composites
analogous to well established chitosan-silica hybrid materials [47, 48] with
very attractive bioactive properties for applications in biomedicine. It was
reported [49] that silicon was found to be a constituent of certain
glycosaminoglycans. It was concluded that Si is present as silanolate, that is,
an ether (or ester-like) derivative of orthosilicic acid, and that R1–O–Si–O–R2 bridges play a role in the structural organisation of glycosaminoglycans. Thus
Si may function as a biological cross-linking agent and contribute to
architecture and resilience of connective tissue [49].To test our hypothesis
that also NAG as monomer unit of poly-NAG and
chitin could be used as substrate for silicification, we obtained silica-NAG-based
materials in the form of rods or spheres (Figure6) using TMOS and sol-gel
techniquesin vitroas described
in Section 2. The diameter of these spheres
could be varied between 2 and 10 mm. SEM investigations on micro- and
nanostructural organization of silica-NAG composites revealed strong evidence
that oriented nanocrytals of NAG (Figure 6(a)) could be also observed in form
of nanocrystals compactly embedded within amorphous silica matrix (Figures 6(b)
and 6(d)). Probably this kind of NAG nanodistribution is responsible for
observed high mechanical stability and resistance of these composite materials
to swelling and following dissolution in water containing solutions (Figure 6(c)).
These properties could be probably of interest for technical purposes similar to
intercalated chitosan/layered silicate nanocomposites prepared to develop
robust and stable sensors useful for anionic detection in aqueous media as
reported on [50, 51].Figure 6
Crystals of N-acetyl
glucosamine obtained from solution (a) could be also visualized using SEM even
if being included into amorphous silica matrix (b). SEM image (d) revealed
strong evidence that oriented crystals of NAG are observed in form of
nanocrystals compactly embedded within this matrix. Light micrograph (c) of the
silica-NAG spherical composites, which are highly stable in water-containing
solutions.We suggest that silica-chitin
and silica-NAG (-poly NAG) composites could be highly optimized biocompatible structures
that would support and organize functional tissues if applied in tissue
engineering of bone and cartilage replacements similar to silica-chitosan-based
biomaterials [52]. Experiments on biocompatibility of silica-chitin and
silica-NAG composites derived in vitro
are currently in progress.


## 4. Conclusion

Chitin and poly-N-acetyl glucosamine
are well investigated materials of biological origin with wide fields of
application in biomedicine because of their unique multifunctional engineering
mechanical properties and biocompatibility [53–56]. With respect
to polysaccharides, including sponge chitin, it is theoretically possible that
inorganic Si binds after the macromolecular structure has been formed. An
alternative, more plausible from stereochemical considerations [49], would
consist in the incorporation of preformed mono-or disaccharide Si derivatives
during the synthesis of the polysaccharide chain. The finding of nanostructured
silica-chitin biocomposites as structural scaffolds of glass sponge skeletons
introduces a new aspect into the discussion surrounding the chemistry,
diversity, and nanolocalization of these materials. Chitin as a template for
biomineralization probably belongs to the basic pattern of the Metazoa.



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*Source: 670235-2008-03-30.xml*