Gradient intrafibrillar mineralized collagen scaffold promotes osteochondral regeneration by enhancing differentiation of SCRG1+ progenitor cells.

Osteochondral defects arising from trauma, joint diseases, or aging remain a major challenge in orthopedics. Native osteochondral tissue possesses a highly ordered biphasic gradient architecture; however, current biomimetic scaffolds often fail to simultaneously recapitulate its macro- and microstructural features. Inspired by the mineralized transition zone at the natural osteochondral interface, we developed an integrated biomimetic mineralized hydrogel scaffold with gradient mineral distribution and intrafibrillar mineralization via a bottom-up strategy combining ultrasmall calcium phosphate polymer-induced liquid precursor (CaP-PILP) and collagen molecular self-assembly. Transmission electron microscopy and energy-dispersive X-ray spectroscopy revealed a gradient mineralization profile of collagen fibers within an approximately 20 μm interface, with calcium content gradually increasing from 5.13% to 24.05%. The scaffold supported the viability of human bone marrow mesenchymal stromal cells (hBMSCs) and effectively promoted their osteochondral lineage differentiation in vitro. RNA sequencing identified the calcium-mediated ERK signaling pathway as a key regulator of this process. In a knee osteochondral defect model of adult New Zealand White rabbits, micro-CT and histological analyses demonstrated that the IMC group significantly enhanced subchondral bone formation (BV/TV: 23.40 ± 5.63% and Tb.Sp: 0.112 ± 0.056 mm at 12 weeks) and cartilage regeneration (with markedly increased expression of COLLAGEN II and AGGRECAN) compared with the pure collagen and extrafibrillar mineralization control. Mechanistically, the scaffold activated the calcium-mediated ERK/TGF-β signaling pathway in vivo, directing SCRG1+ progenitor cells toward osteochondral differentiation and thereby facilitating tissue repair. By integrating physical support with biological induction, this gradient mineralized scaffold offers a promising strategy for osteochondral defect regeneration.