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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). Spicule layers are connected among each other by nanostructured protein fibres (arrows) (b), (d). Micrograph (e) shows a silica distribution on the surface of nanofibrils in the form of nanopearl necklets (arrows).

for further analysis using LTQ and MALDI peptide finger printings.A comparison to the MSDB protein database [27] led to the identification of collagen alpha 1 in two high MW bands.In contrast to H. sieboldi [9], collagen isolated and identified by the same way from Monorhaphis sp. was matched only to type I collagen pre-pro-alpha (I) chain (COL1A1) from dog (AAD34619) (MW 139,74). To our best knowledge, this work is the first study which confirms the presence of collagen within the spicules of Monorhaphis sponge and not only on their surface in the form of a collagen net which covers spicules as recently described by Müller et al. [5]. We also used highly sensitive FTIR methods for the identification of collagen isolated from spicules of M. chuni. Spectra obtained from this collagen, calf skin collagen type I and C. reniformis collagen standards were compared to each other in order to elucidate changes in protein secondary structure. The results obtained from the FTIR study (data not shown) show that collagen derived from this glass sponge exhibited spectra very similar to those from calf skin and C. reniformis collagens [28]. The presence of collagen fibrils in alkali solution is no surprise. Hattori et al. [29] investigated the resistance of collagen to alkali treatment at a concentration range of between 3 and 4% NaOH at 37◦ C in vitro. The results ob-

Figure 3: SEM and TEM nanoimagery of the fibrillar organic matrix within partially demineralized spicule. (a) Axial filament is an organization of microfibrils with a diameter of approximately 25– 30 nm covered with a silica-containing layer and distributed along the axis of spicule. (b) Nanolocalization of amorphous silica particles (arrows) on the surface of partially demineralized protein fibrils using HRTEM. (c), (d) Collagen fibrils’ orientation within spicule possesses a twisted plywood architecture (arrows).

tained indicated that the triple helical conformation and the helicity of the collagen molecule were maintained throughout the period of the alkaline treatment. 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. Spiral twisting of the collagen fibril orientation was found in several biological tissues and described for diﬀerent organisms including cuticular collagens of polychaete, vestimentifera, scale collagens of primitive and bony fishes, and finally collagen fibers inside bone (all reviewed in [21]). According to the model proposed by Giraud-Guille, adjacent lamellae have diﬀerent orientations; either longitudinal (with the collagen fibers along the long axis of the lamellar sheet) or transverse (with the collagen fibers perpendicular to the long axis). From a mechanical point of view, helicoidal structures have certain advantages in resisting mechanical loads compared to orthogonal plywood structures since the twisted orientation enables a higher extensibility in tension and compression [32]. The twisted plywood architecture of collagen fibrils within basal spicules of Monorhaphis visible after alkali treatment (Figure 3) is very