Page:Nanostructural Organization of Naturally Occurring Composites Part I.pdf/3

 Hermann Ehrlich et al.

(ESEM XL 30, Philips) and transmission electron microscopy (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 24 h 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 divided into monomer polymerisation, nuclei growth, and aggregation of particles. Hybridization—the combination of silica and collagen—was performed by intensive mixing of prehydrolysed TMOS and the homogeneous collagen suspensions 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 Hexactinellida are composed of amorphous hydrated silica deposited around a proteinaceous axial filament [17, 18]. The nanolocalization 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 technique for the desilicification of sponge spicules was based on hydrogen fluoride solutions [5], however this kind of demineralization 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 matrix 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 collagen 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 investigation 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 observable at the sites of interstitial layer fractures within partially desilicified spicules. SEM investigations parallel to the slow etching procedures reveal that a fibrillar organic matrix is the template for silica mineralization. Typical fibrillar formations were observed within the tubular silica structures in all layers starting from the inner axial channel containing 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 networks with the curvature of the corresponding silicate layers. These layers of about 1 μm in thickness are connected among each other by protein fibres (Figure 2(a)), which possess a characteristic nanofibrillar organization (Figures 2(b) and 2(d)). Partially desilicificated nanofibrillar organic matrix observed on the surface of silica-based inner layers of the demineralized spicule provides strong evidence that silica 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 epitaxial 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 20–30 nm are organized in bundles with a thickness of 1-2 μm oriented along the axis of the spicule. They can be easily identified by SEM (Figure 3(a)). The morphology of these microfibrils observed by TEM (Figures 4(a) and 4(b)) is very similar 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 candidates (e.g., silicateins of axial filaments such as in Demospongiae [24, 25] or as recently reported by Müller et al. [5, 26] in M. chuni) which would explain the nature and origin of these fibrillar formations. Therefore, a thorough biochemical 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 material in-gel with trypsin to obtain tryptic peptide mixtures