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

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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]. 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. Therefore, the biggest shortcoming common to all publications relating to mechanical [2], structural [3], and optical [8] properties of glassy sponge skeletal formations is a lack of real information regarding the chemical nature of corresponding organic matrices. The finding of collagen within basal spicules of the glass sponge Hyalonema sieboldi [9–11], as well as the occurrence of chitin within the framework skeleton of the glass sponges Farrea occa [12], and spicules of Euplectella aspergillum [7] as revealed by gentle desilicification in alkali, stimulated further attempts to search for materials of organic nature in skeletal structures of these unique deep-sea organisms. Consequently, the objective of the current study was to test our hypothesis that collagen is also an essential component of the giant anchoring silica spicules of Monorhaphis chuni,and if so, to unravel its involvement in the mechanical behavior of these formations, which was well investigated recently [6]. In the present work, we provide a detailed study confirming our hypothesis that the nanofibrillar organic matrix of collagenous nature within the giant spicules of M. chuni is responsible for their extraordinary mechanical properties. We performed structural, spectroscopic, and biochemical analyses of these glassy composites. Finally, this work includes a discussion relating to practical applications of silica-collagen composites artificially derived in vitro as biomaterials for use in biomedicine, engineering, and materials science.

2.1. Chemical etching of spicules and extraction of collagen
Monorhaphis chuni was collected by the R.V. “Vitiaz-2 (4),”voyage 17, St. 2601, 12◦ 31.5’–25.04’ S 48◦ 05.5’–08.0’ E, depth 700 m. Dried Monorhaphis basal spicules (length 120 cm, diameter 1.5–4.5 mm, Figure 1) were washed three times in distilled water, cut into 2–5 cm long pieces and placed in a solution containing purified Clostridium histolyticum collagenase (Sigma) to digest any possible collagen contamination of exogenous nature. After incubation for 24 hours at 15◦ C [13], the pieces of spicules were washed again three times in distilled water, dried and placed in 10 ml plastic vessels containing 5 ml of 2.5 M NaOH solution. The

Figure 1: (a) Marine glass sponge Monorhaphis chuni, a member of the hexactinellids, (b) the sponge consists of a giant basal spicule which anchors Monorhaphis to the sandy substratum.

vessel was covered, placed under thermostatic conditions at 37◦ C and shaken slowly for 14 days. The eﬀectiveness of the slow alkali etching was monitored using scanning electron microscopy (SEM) at diﬀerent locations along the spicules’ length and within the cross-sectional area.

2.2. Biochemical analysis of collagen
Alkali extracts of Monorhaphis spicules containing fibrillar protein were dialyzed against deionized water on Roth (Germany) membranes with a cut-oﬀ of 14 kDa. Dialysis was performed for 48 hours at 4◦ C. The dialyzed material was dried under vacuum conditions in a CHRIST lyophilizer (Germany). The approximate molecular weights of proteins in the lyophylizate were determined by gel electrophoresis in the presence of sodium dodecyl sulphate in 10% and 12% gel plates.The kit of molecular weight markers (Silver stain SDS molecular standard mixtures) from Sigma, USA, was used. Lyophylizates were dissolved in sample buﬀer (1 M Tris-HCl, pH 6.8, 2.5% SDS, 10% glycerine, 0.0125% bromphenol blue) incubated at 95◦ C for 5 minutes and then applied to 10% or 12% of SDS-polyacrylamide gels. After electrophoresis at 75 V for 1.5 hours, 10% gels were stained with GelCode SilverSNAP Stain Kit II (Pierce,USA), and 12% gels were stained with coomassie brilliant blue R250 to allow proteins to be visualized. To elucidate the nature of proteins isolated from glass sponge spicules, corresponding electrophoretic gels stained with Coomassie were used for the determination of the aminoacid sequence by the mass spectrometric sequencing technique (MALDI, Finnigan LTQ) as described earlier [14].

2.3. Structural analysis of spicule layers
Structural analysis of the glass sponge basal spicules and corresponding extracted proteinaceous components was performed using scanning electron microscopy (SEM)