EXPLORING THE POTENTIAL OF MARINE RESOURCES: ECHINODERMS AS VALID MODELS FOR REGENERATION STUDIES AND BIOTECHNOLOGICAL APPLICATIONS

The marine ecosystems have always been exploited by humans as source of food, inspiration, bioactive compounds and biomaterials. Marine invertebrates especially caught the interests of scientists for their potential in basic research and applied biotechnology studies displaying the most spectacular variety of morphological, physiological and behavioural adaptations to diverse environmental conditions. Among them, echinoderms are interesting models for three main reasons: 1) their crucial phylogenetic position, since they are the second largest group of deuterostomes after chordates, 2) their striking regenerative abilities and 3) their peculiar dynamic connective tissues (Mutable Collagenous Tissues or MCTs) capable of rapidly changing their mechanical properties. These last two features are strongly related: indeed, echinoderm connective tissues are considered one of the key characteristics that allows their effective regeneration phenomena. In particular, the extracellular matrix (ECM), with both its fibrous (mainly collagen) and cellular components, is primarily involved during regeneration and can be regarded as a promising source of biomaterial (collagen) for regenerative medicine applications. Therefore, the present work followed two different but overlapping research lines whose main aims were to: a) describe echinoderm arm regeneration after traumatic amputation with a special focus on connective tissue and immune system in order to gain a better comprehension of this fundamental biological process and to compare it with other animals, especially with those with limited regenerative abilities (e.g. mammals), and b) explore the biotechnological potential of echinoderm connective tissues as source of fibrillar collagen to produce valuable tools for human biotechnological applications , such as regenerative medicine. The starfish Echinaster sepositus, the brittle star Amphiura filiformis, the sea urchin Paracentrotus lividus and the sea cucumber Holothuria tubulosa were selected as experimental models. For both research lines a multi-disciplinary approach was employed mainly including microscopic anatomy, gene expression, immunohistochemistry, ultrastructural and biomechanical characterisation and in vitro tests. Focusing on the first research line, starfish and brittle star were traumatically amputated and the regenerates at different time points were analysed with a specific focus on the ECM and immune system roles during the regenerative process. Our results showed that echinoderm emergency reaction and wound healing after injury are faster that those described in mammals and ECM fibrillar organisation at the wound site is delayed in comparison to them. Absence of fibrosis (i.e. over-deposition of collagen) is shown as well. In general, all these evidences can be regarded as key features to ensure their subsequent effective regeneration. Gene expression analyses showed that the identified collagen-like and ECM-related molecule genes in brittle star are differentially expressed during regeneration, thus indicating that different tissues are involved in collagen and ECM production/remodelling. The expression pattern of the collagen biosynthesis enzyme gene here identified indicated that in both experimental models the regenerating epidermis is involved in collagen production. Preliminary analyses on immune system molecules showed that in brittle star TNF-α-like presence is comparable to that of mammals during wound healing. Overall, the regenerative process is faster in brittle star than in starfish, mainly due to smaller arm size, but in both models leads to the complete restoration and functionality of the lost structures following the distalisation-intercalation model and a proximal-distal gradient of differentiation. Focusing on the second research line, echinoderm connective tissues can be regarded as eco-friendly sources of marine collagen since the starting material is coming e.g. from food industry wastes. Thanks to optimised extraction protocols we obtained fibrillar collagen suspensions used to produce two-dimensional collagen membranes (EDCMs) whose ultrastructural, biomechanical and biocompatibility features were characterised and compared to mammal-derived collagen membranes currently available in the market (commercial membranes or CMs) for biotechnological applications, e.g. Guided Tissue Regeneration (GTR). We showed that EDCMs are thinner, less porous and more resistant than CMs, all great advantages for GTR applications. In vitro tests using human skinderived fibroblasts showed that cells seeded on EDCMs present an elongated shape and few and short filopodial processes at the sides of th e cells and sea urchin-derived collagen membranes are the most promising in terms of cell number. Hence, EDCMs can be regarded as valuable marine-derived biomaterials for human applications. Overall, the main outcome of this research was that echinoderms can be considered valid models to explore both basic biological processes (i.e. regeneration) and biotechnological potential of marine-derived tissues. Further analyses will be necessary to continue investigating both these intriguing aspects, including highthroughput molecular analyses (transcriptome) of regenerating tissues and in vivo tests for EDCMs.