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

Abstract

ABSTRACT 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.