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

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.