Single BMSC-derived cartilage organoids for gradient heterogeneous osteochondral regeneration by leveraging native vascular microenvironment

Heterogeneous osteochondral regeneration remains a significant challenge due to the distinct microenvironments across the cartilage, calcified cartilage, and subchondral bone layers. The natural gradient of vascularization from the superficial to deep layers of osteochondral tissue plays a critical role in guiding the differentiation of bone marrow stem cells (BMSCs) into chondrocytes and osteoblasts. In this study, we propose a strategy for gradient heterogeneous osteochondral regeneration using cartilage organoids derived from single BMSCs, leveraging the natural vascularization gradient within osteochondral tissue. We successfully isolated BMSCs from rabbits and generated cartilage organoids via in vitro three-dimensional chondrogenic culture. To mimic the pro-vascular microenvironment, we introduced vascular endothelial growth factor, which promoted the hypertrophic differentiation of the cartilage organoids. We then prepared cartilage organoid/GelMA complexes, with or without the anti-vascular drug Axitinib, and implanted them subcutaneously in nude mice. The vascularized subcutaneous microenvironment induced osteogenic differentiation, while Axitinib treatment created an anti-vascular microenvironment, inhibiting osteogenesis and preserving chondrogenesis within the complexes. Both in vitro and in vivo data demonstrated the crucial role of the vascular microenvironment in regulating osteogenic differentiation of cartilage organoids. Finally, organoid/GelMA cylinders were implanted into a rabbit osteochondral defect, where the gradient vascularization at the defect site guided the organoids to differentiate into both cartilage and bone. This single BMSC-derived cartilage organoid approach enables precise gradient heterogeneous osteochondral regeneration, guided by the natural microenvironment within the osteochondral defect site, representing a significant advancement for clinical applications.