3D-printed recombinant keratin-based dual-function biphasic scaffold for the treatment of osteochondral defects

Osteochondral defects (OCDs) are challenging to repair because of the structural and regenerative differences between cartilage and subchondral bone. To achieve synchronous regeneration of cartilage and bone, we constructed an acellular osteochondral scaffold, RK-C@GelMA, based on recombinant human hair keratin combined with photocrosslinking and 3D printing. Through screening of 17 recombinant keratin (RK) variants, RK81 was identified as exhibiting the strongest osteogenic activity, and RK84 was identified as possessing the strongest chondrogenic activity. These variants were loaded into the bone phase and cartilage phase, respectively, to induce osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In vitro experiments demonstrated that RK81@GelMA significantly upregulated osteogenesis-related gene and protein expression by activating the IL-17/MAPK signaling pathway, while RK84@GelMA markedly increased chondrogenesis-related marker expression by activating the PI3K/Akt/mTOR pathway. In a rat OCD model, the RK-C@GelMA scaffold facilitated the coordinated regeneration of cartilage and subchondral bone, restored the microarchitectural integrity of the defect site, and improved gait function without detectable systemic toxicity. Unlike traditional growth factor-loaded scaffolds, RK-C@GelMA combines tissue-specific inductive capability, appropriate mechanical performance, and a strong biosafety profile, representing a promising strategy for osteochondral interface regeneration with translational potential for the clinical treatment of OCD.