COLLAGEN AND ANTIOXIDANTS FROM SEA URCHINS: WASTE VALORIZATION CHALLENGES TOWARDS REGENERATIVE MEDICINE

Sea urchins (mainly Paracentrotus lividus) are well known worldwide as a delicacy that is processed in the food industry or consumed directly in restaurants. The gonads are the only edible part of these marine animals, while the rest (more than 90-95% of their total mass) is discarded as waste. From the point of view of the circular economy, there is now a great interest in food waste, pointing to the valorization of biomasses to obtain products with added value. In fact, it's already been shown that sea urchin waste contains highly valuable collagen derived from by-product tissues such as the peristomal membrane, which can be used as an innovative biomaterial for tissue engineering. The remaining part of the waste (test and spines) can be an additional source of biologically interesting compounds. Indeed, it contains potent natural antioxidants named polyhydroxynaphthoquinones (PHNQs). The research aims to address the dual challenge of waste reduction and valorization through the development of innovative bioactive biomaterials ranging from basic formulations to advanced technologies, with potential applications in tissue regeneration and wound healing. Given the wide biodiversity of edible sea urchins that are marketed annually, the first aim was to investigate the potential of different species that make up the global waste stream as sources of antioxidants and collagen, in order to increase the potential of the “sea urchin circular supply chain” by exploiting waste other than P. lividus. Therefore, a comparative study on the extraction of PHNQs and collagen was carried out between P.lividus and another Mediterranean species: Sphaerechinus granularis. PHNQs extraction yields were compared, pigments were quantified and identified, and antioxidant activities were assessed and correlated to specific PHNQ presence and abundances. Similarly, collagen extraction yields were evaluated, and the resulting collagen-based biomaterials were compared in terms of their ultrastructure, degradation kinetics, and resistance to compression. Results showed a partially similar PHNQs profile in both species, with significantly higher yield in P. lividus, while S. granularis exhibited better antioxidant activity. P. lividus samples showed higher collagen extraction yield, but S. granularis scaffolds showed higher stability. Subsequently, the study was focused on the development and characterization of collagen-based scaffolds added with PHNQs (Coll-PHNQ), successfully incorporated into biomaterials, enhancing scaffold stability and integrity and providing additional antioxidant functionalities. Water uptake, mechanical properties and degradation kinetics of the composite scaffolds were evaluated and compared with standard scaffolds (Coll). Results indicated that Coll-PHNQ scaffolds exhibited superior chemical stability and slower degradation rates, attributed to strong interactions between collagen and PHNQs, also verified through in silico investigations. Furthermore, it was confirmed that the antioxidant activity of PHNQs was retained in the composite scaffolds, providing additional therapeutic benefits. As a successive step, aiming at developing structurally stable hydrogels and improving the structural properties of the produced scaffolds, methacrylation of collagen was carried out. Methacrylated collagen hydrogels (CollMA- UV) demonstrated improved enzymatic resistance, thickness retention and lower swelling compared to the standard scaffolds (Coll), enhancing biomaterial structural performance. In addition, CollMA-UV exhibited a fibril network with interconnected pores and the characteristic collagen banding pattern, comparable to standard Coll. Further technologic advancements in this work involved the development of inks for 3D printing, combining sea urchins collagen with polysaccharides (such as alginate or ulvan) to achieve biomaterials printability. The printed hydrogels (Coll-P) allowed the production of shape- customizable scaffolds with interconnected pores. Coll-BioP hydrogels were characterized for their ultrastructure, degradation kinetics and structural stability and compared to the following sea urchins collagen-based scaffolds: standard scaffolds (Coll), stabilized scaffolds (Coll-UV), composite scaffolds (Coll-PHNQ), methacrylated scaffolds (CollMA-UV) and commercial bovine membrane (Integra®). The Coll-P hydrogel’s ultrastructure highlights a unique alignment of collagen fibrils, interconnected porosity and demonstrated area and thickness expansion when hydrated. Coll-P’s hydration properties closely resemble those of CollMA-UV, suggesting that it achieves stability without chemical modification. Finally, under enzymatic conditions, Coll-P hydrogel exhibited a degradation rate that balances structural integrity, comparable to established commercial materials like Integra®. In conclusion, by leveraging a circular economy approach, this study successfully demonstrated the feasibility of valorizing sea urchin waste from different species through PHNQs and collagen extraction, offering diverse applications in the biomedical field, according to specific technical requirements. In addition, the work introduced sustainable and innovative strategies for developing biomaterials suitable for tissue engineering and regenerative medicine. Future research should focus on extending the antioxidant activity to CollMA-UV and Coll-P, which has been demonstrated by adding PHNQs for Coll- PHNQ development, and in vivo evaluations to validate the efficacy and scalability of these biomaterials, ensuring their translation into real-world biomedical applications.