Fish Collagen‐Based Bilayer Composite Scaffold Functionalized With Fibrin/Hydroxyapatite/Sodium Citrate for Osteochondral Tissue Engineering—In Vitro and In Vivo Studies

ABSTRACT Osteochondral defects (OCDs) present significant clinical challenges, necessitating scaffolds that effectively regenerate both cartilage and subchondral bone. We developed a bilayer scaffold using fish collagen extracted from Catla catla skin to overcome the limitations of conventional biomaterials, such as mammalian collagen and synthetic polymers, which often suffer from immunogenic risks, poor bioactivity, or inadequate structural integration. The scaffold is comprised of collagen/fibrin (CC/FIB) for the articular cartilage layer and collagen/sodium citrate/hydroxyapatite (CC/NAC/HAP) for the subchondral bone layer, which is cross‐linked with citric acid. Physicochemical characterization confirmed scaffold integration, enhanced thermal stability, and a porous architecture. The scaffold demonstrated optimal porosity (63.12%), degradation (62.08% over 28 days), superior swelling potential, and enhanced bio‐mineralization in simulated body fluid. In vitro studies using MG‐63 osteoblast‐like cells and MC3T3‐E1 cells showed high biocompatibility, increased alkaline phosphatase activity, and enhanced calcium deposition (33.73 ± 0.53 μg/mg of protein at 21 days). Gene expression analysis revealed upregulation of osteogenic (COL I ~23‐fold, RUNX‐2 ~15‐fold, OCN ~8‐fold) and chondrogenic (COL II ~12‐fold, SOX‐9 ~10‐fold, ACAN ~6‐fold) markers, confirming osteochondral regeneration potential. In vivo studies involving the implantation of 3 mm femoral trochlear OCDs in albino Wistar rats (n = 3 per group) resulted in substantial bone and cartilage regeneration, with complete defect closure by 12 weeks. Radiographic and histological assessments at 4, 8, and 12 weeks confirmed well‐organized osteochondral repair, demonstrating superior regenerative capability compared to control groups. This study establishes the novelty of the fish collagen‐based bilayer scaffold as a promising candidate for osteochondral tissue engineering, supporting effective cartilage and subchondral bone regeneration in OCD treatment.