BIOLOGY OF THE DYNAMIC CONNECTIVE TISSUE (MCTS) IN INVERTEBRATE MARINE MODELS:AN INTEGRATED APPROACH

Animal tissues are an immense source of inspiration for humans which actually mimic (biomimetic approach) and use them for novel material design and production. Connective tissue is the most important animal structural material and it (or its components) is often used as source of inspiration/model for different applications. Its main extracellular matrix (ECM) component is collagen. Currently, industrially available collagen is mainly of bovine origin that, however, carries a risk of transmission of serious diseases (bovine spongiform encephalopathy, BSE, and transmissible spongiform encephalopathy, TSE). Therefore, alternative and safer sources of collagen are required for regenerative medicine and one of the safer and recently exploited source are aquatic organisms. The marine invertebrates that I used in this project (echinoderms, in particular sea urchins) possess peculiar and unique connective tissues, called Mutable Collagenous Tissues (MCTs), which could actually represent an alternative source of collagen. Moreover, MCTs undergo extremely rapid, drastic and reversible changes (completely independent from any muscular contribution) in their passive mechanical properties such as stiffness, tensile strength and viscosity under nervous control. Several evidences suggest that MCTs are probably one of the key elements of the striking regenerative capacities found in echinoderms, since they directly help the regenerative process, exante creating the conditions and ex-post providing optimal growth-promoting environment and “dynamic” structures for tissue healing and regeneration. MCTs could therefore represent a valuable source of inspiration for biomaterial design addressed to biomedical applications. The main general aim of this work was to acquire the appropriate knowledge of the model we want to get inspiration from (MCTs) and to understand how natural MCTs actually work. In particular, the specific objective was to define the basic biology of natural MCTs, particularly the key-components and their fundamental interactions; this will be achieved through morphological, biochemical, biomolecular and biomechanical characterizations. This thesis is part of the MIMESIS Project financed by CARIPLO Foundation (2009). The very ultimate challenge of the project is to explore the possible development of a new class of biomimetic materials inspired to echinoderm MCT to be used for scaffolds for tissue regeneration and cell colture studies. The first approach consisted in the investigation of the MCT structural key-components, including fibrillar proteins, proteoglycans (PGs) and glycosaminoglycans (GAGs) in order to deeply investigate how the natural tissue works. The transmission electron microscopy technique was used to obtain micro-scale view to understand the micro-organization of the ECM components. With this detailed investigation the current knowledge of the structural organization of MCT ECM was expanded. The biochemical analysis with SDS-PAGE and Western blot analysis on collagen and PGs/GAGs showed how complex MCTs are. We found that the fibrillar collagen has strong similarities with collagen type I and that all the PGs/GAGs families are represented in MCTs but with differences in quality and quantity according to the tissues analysed or to the related mechanical state. Another major focus of this work was the biomolecular approach related to a presumptive key effector protein, tensilin. This factor, previously found and characterised in other echinoderms (holothurians), is considered as responsible for mutability phenomena. In our study, attention was addressed to “tensilin” possible presence and function in two common sea urchin species, Strongylocentrotus purpuratus and Paracentrotus lividus. We have successfully found and produced tensilin in S. purpuratus and performed fibril aggregation assays. On the basis of our results we can conclude that the S. purpuratus tensilin does not react with collagen like that isolated from holothurians and that the specific MCT environment is fundamental for its activity. The last approach was addressed to extract effector molecules directly from fresh MCT tissues with chromatography and to characterize them with rheology tests. Very important data were collected: 1) the rheological characterization of insoluble and soluble echinoderm collagens in comparison with the ultrapure bovine sample showed that echinoderm collagen possesses peculiar mechanical properties that must be taken in account in view to build and produce an MCT inspired biomaterial; 2) moreover, some chromatographic fractions showed the capability to modify the standard collagen properties.