Biomimetic Injectable Methacrylated Chondroitin Sulfate-Collagen Hydrogels Enhance Chondrocyte Function and Promote Osteochondral Regeneration

Abstract

The development of biomimetic scaffolds capable of promoting both cartilage and subchondral bone regeneration remains a major challenge in osteochondral tissue engineering. In this study, type I acidsoluble collagen (ASC) was successfully extracted from black flounder (Paralichthys olivaceus) skin and systematically characterized. The purified ASC retained its native triple-helical structure, as confirmed by SDS-PAGE, FTIR, CD, and XRD analyses, and exhibited favorable self-assembly behavior near physiological pH. Based on this natural matrix, photocrosslinkable methacrylated chondroitin sulfate (CSMA) was synthesized and combined with ASC or mineralized collagen (MC) to fabricate injectable composite hydrogels via UV-initiated polymerization. The resulting CSMA/COL and CSMA/MC hydrogels demonstrated tunable gelation times (90-120 s), high porosity, excellent swelling capacity, and superior mechanical strength (compressive modulus up to ~40 kPa). Rheological analysis revealed stable viscoelastic properties with G′ consistently exceeding G″. The composites also exhibited remarkable self-healing ability. In vitro, all hydrogel extracts displayed outstanding cytocompatibility, promoting primary chondrocyte adhesion, proliferation, and migration. Hydrogels containing collagen and MC (especially CS5M1) significantly enhanced alkaline phosphatase (ALP) activity and upregulated chondrogenic gene expression (COL II, Acan, and Sox9). In vivo implantation in a rat full-thickness cartilage defect model demonstrated that CSMA-based composite hydrogels facilitated seamless defect filling, enhanced proteoglycan and glycosaminoglycan deposition, and promoted subchondral bone remodeling. Among all formulations, CS5M1 achieved the most complete repair, regenerating hyalinelike cartilage integrated with surrounding tissue after 12 weeks. Collectively, these results demonstrate that the composite hydrogels provide a biomimetic, injectable, and photo-curable platform with excellent osteochondral regenerative potential.