Page:Nanostructural Organization of Naturally Occurring Composites Part II.pdf/2

 2 Figure 1: 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). Transparent hard glassy spheres were obtained using this fraction of colloidal suspension after dropping into 99% TMOS solution at room temperature (d).

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 sponge Rossella 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 200–600 μm. The spicules are composed of 2–10 μm-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. 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 hexactinel-

lid 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 of R. fibulata. Finally, the present work includes a discussion relating to strategies for the practical application of silicachitin- and silica- N-acethyl glucosamine (NAG)-based composites as biomaterials.

2.1. Chemical etching of glass sponge skeletons
The object of our study was Rossella fibulata Schulze & Kirkpatrick, 1910 (Hexactinellida: Porifera), collected in 2005 in the Scotia Sea, Antarctic, at a depth of 200 m. Spicules of R. 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% H2 O2 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