by the infrared absorption, in which a typical peak at 890–892 cm−1 for α-chitin was observed [34, 35]. 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, highresolution electron micrographs were taken from particular sample regions (data not shown). The Fourier transform of the high-resolution micrograph revealed a spacing of 4.79 Å (a-axis), 10.2 Å (fiber axis), 3.73 Å, and 2.77 Å. 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. 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 ± 1.5 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. 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 glucosaminefragment of the chitin nanofibril and silica nanoparticles, which can be seen in Figure 5.
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
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.
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 (Figure 6) using TMOS and solgel 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
