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Background <p>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.</p> Methods <p>HiPSCs were differentiated into iMPCs (characterized by FLK1(VEGF-R) expression, CD31/vWF immunofluorescence, tube formation, flow cytometry) and Chos (confirmed by <i>SOX9/COL2/ACAN</i> 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 <i>IHH</i>,<i> ALP</i>,<i> COL1A1</i>), 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&amp;E, Masson’s trichrome), and neovascularization (CD31 immunohistochemistry).</p> Results <p>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 <i>IHH</i> expression), early osteogenesis (elevated ALP activity and <i>COL1A1</i> expression), and enhanced in vitro mineralization compared to controls (*<i>p</i> &lt; 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&amp;E and Masson’s trichrome), enhanced host-derived vascular integration (CD31⁺ staining), and advanced bone maturation relative to control groups (*<i>p</i> &lt; 0.05).</p> Conclusion <p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月24日

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  • Yian Guan,
  • Zixin Wang,
  • Hui Wang,
  • Rong Huang,
  • Jing Lin,
  • Yuan Wang,
  • Dongyang Ma,
  • Ping Zhou,
  • Liling Ren

摘要

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.