Endothelialized callus organoids drive rapid regeneration of critical-size segmental long bone defects.

Large bone defects remain a major clinical challenge, as current treatments cannot effectively regenerate them and fail to restore functionality. To recapitulate the cellular complexity of the early fracture callus, we engineered human endothelialized callus organoids (hECOs) by co-culturing periosteum-derived skeletal progenitors with endothelial cells. This co-culture resulted in cellular self-assembly, leading to spatially organized, callus-like structures. Endothelial cells promoted skeletal progenitor cell expansion, extracellular matrix maturation, and progression toward hypertrophic cartilage, hallmarks of endochondral ossification. Multi-omics analyses identified endothelial cell-mediated activation of regenerative programs associated with skeletal maturation, matrix remodeling, and angiogenesis. Following brief in vitro differentiation, macroscale aggregates of hECOs supported rapid host vascularization and regeneration of critical-size tibial defects in immunocompromised mice. Donor-derived cells actively contributed to early regeneration but were progressively replaced during remodeling, consistent with hECOs functioning as transient biological templates that guide host-mediated bone regeneration.