Synchronizing the Osteochondral Regeneration Process through Spatial Patterning of Stable and Hypertrophic Cartilage...

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 induced pluripotent stem cell (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 osteo-associated domains, with interface continuity emerging through self-directed matrix organization. Upon implantation into full-thickness osteochondral defects, iChon+hPDC assembloids promoted robust hyaline-like cartilage repair, supported subchondral bone formation with ongoing repair/remodeling, and partially reestablished collagen fiber anisotropy. Protein-level mapping further supported a surface-associated cartilage phenotype and remodeling-associated signatures in the deep compartment. Transcriptomic profiling revealed complementary biological programs, with iChon showing features suggestive of stable cartilage regulation and extracellular-matrix remodeling competence, and hPDC showing a transient hypertrophic program associated with endochondral ossification. This work provides a scaffold-free design framework for engineering zonated osteochondral implants through spatial assembly of lineage-specific organoid modules, with translational potential for future osteochondral repair strategies.