**5.** *Chondrosia* **Collagens**

Collagens from the demosponge *Chondrosia reniformis* (Nardo 1847) have received attention from researchers since 1970 [113,117–119] due to their diversity (type IV collagen, fibrillar and nonfibrillar collagens) [120] and interesting structural [121], physicochemical [122], and ecophysiological properties [123–127]. For example, slices of fibrillar collagen incubated with collagenase are not modified even after 48 h of incubation, and do not show any changes in the aspect, consistency, or fine structure of the fibrils. No kind of enzymatic damage was observed by electron microscope on the isolated collagen fibrils after collagenase treatment.

The mechanical properties of this collagen have been partially described by Garrone et al. [117]. The cortex of *Chondrosia* sponges is less resistant than calf skin, but has mechanical properties of the same order as those of bovine nasal cartilage (Young's modulus 150–250 kg/cm2 and 100–250 kg/cm2 respectively). Probably due to special mechanical features, the body of *Chondrosia* can slowly become flat and slide to avoid compression or stretch itself into a slender thread under continuous stress. Such *creeping* behavior of a fibrous and living material provides a remarkable example for the study of mechanical stresses as morphogenetic factors [117]. Although the nanomorphology of *C. reniformis* collagen fibrils has now been well investigated (Figure 7) [121], there is still a lack of knowledge about the relationship between the ultrastructural features of this collagen and its mechanical and physicochemical properties.

**Figure 7.** Schematic diagram of *C. reniformis* collagen fiber with numerous nanofibrils with characteristic nanotopography. Along the fibril, one characteristically thick segment (protrusion) about 28 nm in diameter is followed by two equal thinner and closer conjoined segments (interband) about 20 nm in diameter. The average distance between the protrusions is about 67–69 nm. The distance between two consecutive peaks of the interband regions or between a protrusion and an adjacent interband region is about 21–23 nm. The average step height between the protrusions and the interband regions was calculated to be about 4 nm (see for review [121]).

The well-known biocompatibility of *C. reniformis* fibrillar collagen has stimulated many studies on its possible applications in cosmetics and pharmacology (see for review [31]), including in transdermal drug delivery [128].

The production and selection of a triple transformed *Pichia pastoris* yeast strain expressing a stable P4H tetramer derived from *C. reniformis* sponge and a hydroxylated nonfibrillar procollagen polypeptide from the same organism have recently been reported by Giovine et al. [118]. The obtained recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets. It is suggested that the *Pichia* system could be used for the large-scale production of hydroxylated sponge- or marine-derived collagen polypeptides, which have high pharmacological potential [118].

The possibility of the application of *Chondrosia* fibrillar collagen as an organic template for in vitro silicification has been confirmed in several studies [121,129,130]. There are no doubts that the mechanical properties of biomimetically-inspired hybrid composites can be significantly improved with the presence of this special collagen.

#### **6. Glass Sponge Collagen**

Collagen is known as a universal template in biomineralization, including both calcification and silicification. It is proposed that this biopolymer functions as a fundamental template in biomineralization, inasmuch as it is very ancient from an evolutionary point of view and is common to many species and biological systems with a global distribution [131]. The identification of diverse collagens in demosponges as described above suggests that they may also be found within skeletal structures in the sister group, the glass sponges. Hexactinellida Schmidt (Porifera), with more than 700 species, consists exclusively of marine glass sponges. These are psychrophilic organisms which can produce huge biosilica-based skeletons and anchoring spicules at temperatures between −2 ◦C and 4 ◦C [132].

The challenging task of isolating and identifying collagen in the skeletal structures of diverse glass sponges was completed successfully only in 2010 [3], following numerous attempts at gentle demineralization [133,134]. Studies in this area have been motivated by the great flexibility of the glassy spicules, which allows researchers to tie a spicule into a bundle (Figure 8). It has been suggested that this peculiar feature of spicules in the hexactinellids must be due to the presence of a structural carcass of organic nature both on the surface (Figure 9) and within the spicules [133].

**Figure 8.** Photograph demonstrating the unique flexibility of the *H. sieboldi* anchoring spicule, and schematic view of the role of special hydroxylated collagen in silica condensation in this natural basilica structure (for details see [3]).

The organic phase has been identified as a highly hydroxylated fibrillar collagen which contains an unusual [Gly–3Hyp–4Hyp] motif predisposed for silica precipitation, and provides a novel template for biosilicification in nature [3]. This collagen presents a layer of hydroxyl groups that can undergo condensation reactions with silicic acid molecules with consequent loss of water. As a result, the initial layer of condensed silicic acid will be held fixed to the collagenous template in a geometric arrangement that will favor further polymerization of silicic acid. It therefore appears that collagen was a novel template for biosilicification that emerged at an early stage during metazoan evolution, and that the occurrence of additional trans-3-Hyp plays a key role in stabilizing silicic acid molecules and initiating the precipitation of silica.

Collagen has also been reported as the main organic component of the spicules of the glass sponge *Monorhaphis* sp. [135] (Figure 10). Results of the amino acid analysis of protein extracts isolated from demineralized spicules of this sponge showed an amino acid content typical for collagens of the same origin. Comparison with the Microsatellite Database (MSDB) protein database led to the identification of alpha 1 collagen in two high-MW bands. In contrast to its analog in *H. sieboldi*, collagen isolated and identified from *Monorhaphis* sp. was matched only to the type I collagen pre-pro-alpha (I) chain (COL1A1) from dog (AAD34619) (MW 139,74) [135].

**Figure 9.** SEM image of the nanofibrillar collagenous layer on the surface of an *H. sieboldi* glass sponge anchoring spicule.

**Figure 10.** High-resolution transmission electron microscope image of a fragment of *M. chuni* collagen nanofibril isolated from the glassy spicule (for details see [135]). The nanomorphology of such fibrils is similar to that from *H. sieboldi* glass sponge collagen [3], but different from the striated collagen fibrils from the demosponge *C. reniformis* [121].

The existence of naturally occurring collagen–silica-based composites in the form of spicules of glass sponges stimulated material scientists to develop analogous hybrid materials. Due to the limited available amounts of glass sponge collagen for the development of silica-based composite materials, fibrillar collagen from the demosponge *C. reniformis* has been successfully used as an alternative by the Ehrlich research group [121,129]. More recently, a new concept in biosilica material synthesis which does not require phosphate supplements and is based on the fusion of stabilized polysilicic acid into a fluidic precursor phase upon infiltration into polyamine-enriched collagen has been proposed by the Tay research group [136–138]. It has recently been shown that silicified collagen scaffolds produced by infiltrating collagen matrices with intrafibrillar amorphous silica exhibit angiogenic and osteogenic potential and can be used in tissue engineering [139]. In work by Aime et al. [140], collagen triple helices have been confined on the surface of sulfonate-modified silica particles in a controlled manner. This gives rise to hybrid building blocks with well-defined surface potentials and dimensions. Additionally, oligomeric collagen-fibril matrices with tunable microstructural properties have been used to template and direct the formation of biocompatible mesoporous sol–gel silica to develop nanostructured hybrid organic–inorganic composites [141]. It was experimentally confirmed that silica mineralization kinetics are critical for the precision-tuning of properties of the hybrid materials, including porous microstructure, mechanical strength, depth of silica penetration, and mass transport properties. It has also been shown that microstructural properties of the collagen-fibril template are preserved in the silica surface of hybrid materials [142]. Such novel silica-collagen hybrid materials may be useful, for example, in the regeneration of bone tissue or in cellular microencapsulation [141].
