Hermann Ehrlich

“ 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. ”

“ To test our hypothesis that alkali-insoluble residues of R. 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 of R. 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 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] . Unfortunately, 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 materials [7] . ”

“ Correspondingly, this kind of collagen fibril orientation could explain why sponge spicules exhibit specific flexibility and can be bent even to a circle as reported 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. ”

“ 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
Figure 2: TEM images of silica-organic matrix-based suspension obtained after mechanical disruption of spicules in deionized water (a) . ”

“ 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. ”

“ 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. ”

“ 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 knowledge of the nature and origin of the organic nanostructured matrices of corresponding natural biocomposites which are present in these sponges. ”

“ 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. ”

“ The procedure of alkali slow etching opens the possibility 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’ orientation within M. chuni spicules possesses twisted plywood architecture (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. ”

“ Therefore, it is not unreasonable to hypothesize that silicateins are proteins responsible for the reconstruction of collagen to form templates necessary for the subsequent silica formation. According to a dynamic model proposed by Müller 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 paper, we propose a model for the structure of the spicules of Monorhaphis sponges, including micro- and nanoaspects, which can be seen in Figure 5. ”

“ Crystallites of organic nature are embedded in amorphous silica (b) .
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 of H. 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. ”

“ Highly flexible spicules of marine glass sponge Rossella 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) . ”

“ 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 biosilification in vivo. ”

“ Experiments on biocompatibility of silica-chitin and silicaNAG composites derived in vitro are currently in progress.
4. CONCLUSION4. 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. ”

“ 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 material in-gel with trypsin to obtain tryptic peptide mixtures
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 mineralization (b) – (e) . ”

“ We detected 19.2 ± 1.5 μg N-acetyl-glucosamine per mg of spicule of R. fibulata. The finding of silica-chitin natural composites as the component of the R. fibulata spicules is in good agreement with results of in vitro experiments on silicification of a βchitin-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. ”

“ Moreover, stiffness and hardness were shown to oscillate as a result of the laminate structure of the spicules. Raman spectroscopic imaging showed that the organic layers are protein-rich and that there is an OH-enrichment in silica near the central axial filament of the spicule. Smallangle X-ray scattering revealed the presence of nanospheres with a diameter of only 2.8 nm as the basic unit of silica. ”

“ 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] . ”

“ H. Ehrlich, S. Heinemann, C. Heinemann, et al., “Nanostructiral organization of naturally occuring composites. Part I. Silica-collagen-based biocomposites,” Journal of Nanomaterials. In press.
[2] P. Fratzl, “Biomimetic materials research: what can we really learn from nature’s structural materials?” ”

“ C-6137, respectively, were suspended in 100 mM sodium phosphate buffer at pH 6.0. Digestion was started by mixing 400 μL of the samples and 400 μL of the chitinase-mix. Incubation was performed at 37◦ C and stopped after 114 hours by adding 400 μL 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] . ”