Learning from nature : sea urchin tissues as a source of inspiration for biomaterial design : The MIMESIS project

The natural world has often provided ideas and solutions (resulting from billions of years of trials, successes and failures) for different human applied fields such as architecture, engineering and medicine. In particular, biological tissues have been an immense source of inspiration, having been mimicked (biomimetic approach) and used for novel material design and production [1]. Among the best-known examples are velcro, inspired by the the burrs (seeds) of burdock and racing swimwear that mimics rough shark denticles to reduce water drag. 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, a bioresorbable weakly immunogenic and inextensible protein, widely used in pharmaceutics, cosmetics and biomedicine [2]. Currently, industrially available collagen is mainly of bovine origin which may carry a risk of transmission of serious diseases (BSE and TSE). Therefore alternative and safer sources of collagen are needed. Aquatic organisms are potentially such a source that has already begun to be exploited ([3-7]). The biomimetic approach represents a new strategy being pursued also in the field of human regenerative medicine. Nevertheless, existing biomaterials lack the inherent adaptability of natural tissues; in fact they do not mimic a structurally dynamic environment. Regenerating tissues, instead, are necessarily continuously dynamic environments: throughout the regenerative processes they have to constantly change both their mechanical and 3D structure as well as their composition. The regenerating tissue has to develop from a preliminary cell organization into a functional and well-defined structure. Incorporating the novel concept of dynamic self-assembly into a naturally derived (collagenous) scaffold would constitute a radical step beyond the existing intelligent matrices for tissue engineering and would represent progress towards more physiologically-responsive biomaterials for regenerative medicine. The MIMESIS project: objectives and approach The MIMESIS (Marine Invertebrates Models & Engineered Substrates for Innovative bio-Scaffolds) project, started in 2010 and funded by the Cariplo Foundation, was developed within this scientific context. The main idea behind this project is to use as a source of inspiration very common marine animals: the well-known sea urchins. These animals (as well as their close relatives starfish and sea cucumbers, belonging to the zoological group called “echinoderms”) possess peculiar and unique connective tissues, called Mutable Collagenous Tissues (MCTs) [8]. Echinoderm collagen resembles type-I collagen of mammals, in terms of structure and amino acid composition. MCTs represent a potential alternative source of collagen, economically advantageous and ecologically “friendly” since it can be obtained from “waste material” discarded by the sea urchin food industry. More intriguing, MCTs undergo extremely rapid, drastic and reversible/irreversible changes (independent of any muscular contribution) in their passive mechanical properties such as stiffness, tensile strength and viscosity [8]. There is evidence that MCTs are one of the key elements of the striking regenerative capacities found in echinoderms, since they provide optimal growth-promoting environments and “dynamic” structures for tissue repair and regeneration [8]. The ultimate challenge of the project is to explore the possible development of a new class of biomimetic materials inspired by sea urchin MCTs, which can be used for cell culture scaffolds and/or biomedical applications. The research work is now under development according to the project’s specific objectives: 1. To acquire the appropriate information on the model we want to get inspiration from MCTs, i.e. to understand how natural MCTs actually work and define their basic biology, particularly the key-components, their fundamental interactions and their evolutionary pathways. This is being achieved through morphological, biochemical, biomolecular and biomechanical characterization. 2. To find a new alternative and safe collagen source: extracted collagen is being used for the development of a first-level biomaterial, a very simple collagen scaffold (biofilm) for cell culture. 3. To develop a new (second-level) biomaterial simulating natural MCTs. Since reproducing the complex natural structure of MCT is virtually impossible, we will start manipulating simpler components in order to produce a composite with adjustable mechanical properties. The proposed project has a wide potential impact and scientific/technical benefits in different scientific fields, including animal biology, engineering and biomedicine. This project is based on a multidisciplinary approach: experts in echinoderm functional biology (University of Milan and Glasgow Caledonian University) are joining experts in biomaterial design (INEB) to guarantee the final success of the project. Historically, when such different disciplines as biology and material engineering have met, the obtained results have been outstanding (shark-inspired swimwear, gecko-inspired medical adhesive). Overview of the first MIMESIS outcomes Most research carried out during the first year of the project was focused on the characterization of natural MCTs and mutability mechanisms (objective 1). The development of a collagen biofilm has started recently (objective 2). Ultrastructural analyses showed that collagen fibril orientation was different in the different mechanical states of MCT: it was parallel in stiffened samples but less regularly distributed in compliant ones. Interfibrillar distance was reduced in stiffened samples, which may facilitate the mechanism that introduces stabilizing interfibrillar bridges. Tissue hydration and water diffusion might also assist this mechanism. Proteoglycans (PGs) were found regularly associated with collagen fibrils. In some (but not all) MCT analysed the glycosaminoglycan (GAG) content was similar in different mechanical states, thus suggesting that qualitative rather than quantitative changes in GAGs could be involved in the mutability mechanism. Biochemical carachterization of GAGs is now under investigation. Different chemical treatments (guanidine chloride and NaOH) revealed that covalently bound PGs predominate in this tissue. Biomechanical analyses suggested that the MCT under investigation behaves like a viscoelastic material in the different mechanical states. Further biochemical/biomechanical analyses showed that the mutability mechanism may evolved from an ancestral MMP-TIMP system. Collagen extraction from MCT was successfully carried out. As most of the sea urchin collagen is insoluble, specific protocols were employed and optimized. The presence of intact collagen fibrils in extracts was confirmed by TEM. The development of collagen biofilms and their biocompatibility with both sea urchin and human cell cultures is now under investigation. [1] Bar-Cohen Y. (2006). Introduction to biomimetics: the wealth of inventions in Nature as an inspiration for human Innovation. In: Biomimetics: Biologically Inspired Technologies, Bar-Cohen Y. (ed),CRC Press, 572 [2] Lee C.H., Singla A.and Lee Y. (2001) Biomedical applications of collagen, Int J Pharm 221, pp. 1–22 [3] Giraud-Guille M.-M., Besseau L., Chopin C., Durand P. and Herbage D. (2000) Structural aspects of fish skin collagen which forms ordered arrays via liquid crystalline states, Biomaterials 21, pp. 899–906. [4] Nagai T., Yamashita E., Taniguchi K., Kanamori N. and Suzuki N. (2001) Isolation and characterization of collagen from the outer skin waste material of cuttlefish (Sepia lycidas), Food Chem 72, pp. 425–429 [5] Li H., Liu, B.L. Gao L.Z. and Chen H.L. (2004) Studies on bullfrog skin collagen, Food Chem 84, pp. 65–69 [6] Ogawa M., Portier R.J., Moody M.W., Bell J., Schexnayder M.A. and Losso J.N. (2004) Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonia cromis) and sheepshead seabream (Archosargus probatocephalus), Food Chem 88, pp. 495–501. [7] Song E., Yeon Kim S., Chun T., Byun H.J., Lee Y.M. (2006). Collagen scaffolds derived from a marine source and their biocompatibility. Biomaterials, 27 (15): 2951-2961. [8] Wilkie I.C. (2005). Mutable collagenous tissues: overview and biotecnological perspective”, Matranga V.(ed). Progress in Molecular and Subcellular Biology, Subseries Marine Molecular Biotechnology, Echinodermata, Springer-Verlag, Berlin Heidelberg, pp:167-199.