Development and Characterization of a Sardine Scale‐Chitosan Biomaterial: In Vivo Evaluation for Bone Regeneration in a Rat Osteoporosis Model

The development of functional materials for osteoporosis is essential for effective bone remodeling. In this context, the extraction of biocompatible implantable biomaterials from bio-waste emerges as a valuable strategy, addressing both environmental challenges and promoting human health. The objective of this work was to evaluate the physicochemical properties of the added-value by-product biomaterial (SS-90), extracted from sardine scales (Sardina Pilchardus) and combined with chitosan (SS-90-CH). Besides, the efficacy of both biomaterials for bone regeneration was evaluated through in vitro and in vivo tests. The physicochemical characteristics of the biomaterials were demonstrated by ICP-OES, TGA, XRD, FTIR, and SEM-EDS analyses. Their characteristic features were compared with pure commercial hydroxyapatite (HA_syn) and associated with chitosan (HA_syn-CH). ICP-OES analysis evidenced the presence of Ca, P, Mg, Na, Sr, and Zn in SS-90 with a molar ratio (Ca/P) of 1.84 near to that of synthetic hydroxyapatite (1.67). The FTIR spectrum confirmed the presence of carbonate and phosphate functional groups in SS-90, which is similar to healthy rat bone (HRB). In vitro, SS-90 and SS-90-CH biomaterials demonstrated no cytotoxicity, maintaining cell viability between 80% and 100% for SaOS-2, L929, and LIG cells after 72 h of incubation. Furthermore, these biomaterials were implanted into bone defects in femoral condyles of osteoporotic rats to evaluate their effectiveness in bone fracture repair under osteoporotic conditions. Physicochemical, biochemical, and histological studies conducted at different time intervals after implantation indicated that the biomaterials could effectively promote bone regeneration. In conclusion, the present study highlights that SS-90 and SS-90-CH biomaterials are promising solutions for repairing bone defects or fractures under osteoporotic conditions, combining the valorization of marine bio-waste with biomedical applications.