Three-dimensionally-printed biphasic PCL/β-TCP scaffold with spatially confined GelMA/CS hydrogel for coordinated osteochondral regeneration.

Osteochondral defects remain difficult to repair because articular cartilage and subchondral bone differ in structure and regenerative capacity, and stable interface integration is challenging. Here, we developed a structurally continuous yet functionally stratified biphasic scaffold via dual-temperature 3D printing, consisting of a poly(ε-caprolac tone)/β-tricalcium phosphate (PCL/β-TCP) bone-mimetic phase with larger macropores and a pure PCL cartilage-guiding phase with smaller pores. A GelMA/chondroitin sulfate (GelMA/CS) hydrogel was selectively infiltrated into the upper region by immersion and photocrosslinking, while a ∼0.5 mm hydrogel-free transitional zone was preserved as a structural transition region between the chondral hydrogel compartment and the subchondral scaffold. In vitro, GelMA/CS functionalization enhanced chondrocyte adhesion, promoted sGAG secretion and upregulated chondrogenic genes. Under osteochondral induction, the scaffolds supported osteo- and chondrogenic marker expression in human umbilical cord-derived mesenchymal stem cells. Transcriptomic profiling indicated enrichment of ECM- and mechanotransduction-related pathways, consistent with the involvement of TRPV4-associated mechanosensing and PI3K/AKT-related signaling. In a rabbit femoral osteochondral defect model, the GelMA/CS-functionalized scaffold enhanced early subchondral bone regeneration by micro-CT and promoted cartilage-like matrix deposition and chondrogenic marker staining compared with the unmodified scaffold and untreated control at 4 and 8 weeks. This hydrogel-integrated biphasic design offers a scalable strategy for coordinated osteochondral regeneration.