Construction of pre-vascularized bone-like tissue by incorporation of mesodermal progenitor cells through simulating endochondral ossification

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BACKGROUND: Pre-vascularized cartilage enhances bone regeneration (intrachondral osteogenesis) and accelerates bone defect healing. While current strategies focus on co-culturing chondrocytes with endothelial cells, mature endothelial cells inhibit the necessary osteogenic transformation. To overcome this, we implemented a co-differentiation strategy using human induced pluripotent stem cells (hiPSCs)-derived mesodermal progenitor cells (iMPCs) instead. We generated pre-vascularized cartilage aggregates by 3D co-culture of these iMPCs with hiPSC-derived chondrocytes (Chos) and evaluated their osteogenic potential. This approach offers new insights and potential strategies for repairing bone defects. METHODS: HiPSCs were differentiated into iMPCs (characterized by FLK1(VEGF-R) expression, CD31/vWF immunofluorescence, tube formation, flow cytometry) and Chos (confirmed by SOX9/COL2/ACAN qRT-PCR, Alcian blue staining, VEGF165 ELISA). Pre-vascularized aggregates were generated by 3D co-culture of iMPCs and Chos in ultra-low attachment plates (monocultures as controls). After 14 days, aggregates were assessed in vitro for vascularization (CD31 immunofluorescence), gene/protein expression (qRT-PCR/immunofluorescence for IHH, ALP, COL1A1), and mineralization (Alizarin Red). In vivo osteogenesis was evaluated by implanting aggregates into rat calvarial defects, analyzing healing at 4/8 weeks via Micro-CT, histology (H&E, Masson's trichrome), and neovascularization (CD31 immunohistochemistry). RESULTS: IMPCs exhibited high endothelial potential, with a 52.84% induction rate and the ability to form tube-like structures. Co-culture aggregates developed extensive CD31⁺ vascular networks in vitro. Pre-vascularization significantly promoted chondrocyte hypertrophy (increased IHH expression), early osteogenesis (elevated ALP activity and COL1A1 expression), and enhanced in vitro mineralization compared to controls (*p < 0.05). In vivo, the pre-vascularized group demonstrated improved bone defect repair, as shown by increased bone volume in micro-CT analysis, histological evaluation (H&E and Masson's trichrome), enhanced host-derived vascular integration (CD31⁺ staining), and advanced bone maturation relative to control groups (*p < 0.05). CONCLUSION: Our findings indicate that this vascularization approach utilizing mesodermal cells successfully achieves pre-vascularization of cartilage aggregates in vitro, while also facilitating cartilage hypertrophy and early osteogenic transformation. In vivo study revealed that pre-vascularized cartilage aggregates exhibited more nascent vasculature and enhanced bone formation compared to cartilage aggregates alone.

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