Synchronised regeneration of osteochondral defects via spatially patterned implants of stable and hypertrophic cartilage organoids

Repairing deep osteochondral defects remains clinically challenging due to the intrinsic inability of articular cartilage to self-repair and the need for integrated yet distinct regeneration of articular cartilage and subchondral bone. Here, we present a scaffold-free, modular strategy that spatially bioassembles iPSC-derived chondrocytes (iChon) organoids with human periosteum-derived cell (hPDC) organoids to engineer zonated osteochondral assembloids. The resulting iChon+hPDC assembloids exhibit intrinsic spatial organization, forming chondral- and osteo-like zones with an intermediate interface without exogenous scaffolds. In vitro characterization confirmed layered glycosaminoglycan-rich cartilage and collagen I-rich bone-like domains, with an intermediate interface emerging through self-directed matrix organization. Upon implantation into full-thickness osteochondral defects, iChon+hPDC assembloids promoted robust hyaline cartilage restoration, subchondral bone reconstruction, and partial restoration of collagen fiber anisotropy. Transcriptomic profiling revealed complementary biological programs, with iChon exhibited extracellular matrix remodeling and early developmental plasticity and hPDC in endochondral ossification pathways. This work provides a robust, scaffold-free platform for osteochondral regeneration that can be adapted toward patient-specific repair through integration of iPSC-derived cell sources and precise spatial assembly of zonated organoids.