二甲双胍介导AMPK通路调控iPSCs促进组织工程骨血管化的研究

The sufficient and rapid vascularization of tissue-engineered bone grafts in the recipient is an important guarantee for the seed cells to obtain sufficient blood oxygen supply to achieve repair of maxillofacial bone defects. However, the current vascularization strategies have problems such as instability or degeneration of initial vascular structures, and it is difficult to form a functional neovascularization by matching with the blood circulation system of the recipient. To solve the above problems，in this project we choose metformin (MET) as a key factor to promote the maturation and stability of early microvessel-like structures, synthesis and optimization of calcium phosphate cement-microfiber (CPC-AMF) scaffold materials, induced differentiation of human induced pluripotent stem cells (hiPSCs) into vascular endothelial cells (hiPSCECs) and human iPSC-derived mesenchymal stem cells (hiPSCMSCs) in vitro，create the construction of CPC-AMF vascularized tissue engineering bone loaded with co-culture system, explore the role of MET in promoting tissue engineering bone vascularization, and to further investigate the role of MET-mediated activation of the AMPK pathway in hiPSCECs. The possible mechanism of angiogenesis and stabilization of early vasculature in vitro and the effect of MET on the replacement effect of vascularized tissue engineering bone in vivo were investigated. This study provides a new idea for further promoting bone vascularization in tissue engineering, and has important theoretical and practical significance for improving the therapeutic effect of bone tissue engineering.

组织工程骨移植物在受体内充分快速的血管化是其内种子细胞获得充足血氧供应进而实现颌面部骨缺损修复的重要保障。但目前的血管化策略存在初始脉管样结构不稳定甚至退化等问题，难以与受体血液循环系统吻合形成功能性新生血管。本项目针对上述难题，引入二甲双胍(MET)作为促进早期微脉管样结构成熟稳定的关键因子，合成并优化磷酸钙骨水泥-微纤维(CPC-AMF)支架材料，将人诱导性多能干细胞(hiPSCs)体外诱导分化为血管内皮细胞(hiPSCECs)与间充质干细胞(hiPSCMSCs)，构建负载双细胞共培养体系的CPC-AMF血管化组织工程骨，深入探讨MET介导AMPK通路对hiPSCECs促血管生成及稳定早期脉管结构可能的作用机制，并通过体内模型评价MET对血管化组织工程骨骨替代效果的影响。本研究有望为组织工程骨血管化策略提供新思路，为hiPSCs在骨组织工程精准个体化临床应用提供强有力的实验依据。

组织工程骨移植物在受体内充分快速的血管化是其内种子细胞获得充足血氧供应进而实现颌面部骨缺损修复的重要保障。但目前的血管化策略存在初始脉管样结构不稳定甚至退化等问题，难以与受体血液循环系统吻合形成功能性新生血管。本项目针对上述难题，引入二甲双胍(MET)作为促进早期微脉管样结构成熟稳定的关键因子，合成并优化磷酸钙骨水泥-微纤维(CPC-AMF)支架材料，首先将不同体积分数的AMF与CPC混合，对CPC-AMF支架材料进行进一步优化，按照AMF/(AMF+CPC)=30%，40%，50%，60%，70%，80%，90%为研究对象，研究表明AMF/(AMF+CPC)=60，70%组别有望在保证材料骨生物力学性能的基础上，最大程度有效携带活性种子细胞，进而提高材料的骨替代效果。随后，申请人利用hiPSCs作为干细胞唯一组织来源，合成并优化CPC-AMF支架材料，应用Live/Dead染色技术，对比观察注射前与注射后细胞的活性，实验结果表明，注射前与注射后的细胞活性无显著差异。接下来，申请人将人诱导性多能干细胞(hiPSCs)体外诱导分化为血管内皮细胞(hiPSCECs)与间充质干细胞(hiPSCMSCs)，构建负载双细胞共培养体系的CPC-AMF血管化组织工程骨，深入探讨MET介导AMPK通路对hiPSCECs促血管生成及稳定早期脉管结构可能的作用机制，并通过体内模型评价MET对血管化组织工程骨骨替代效果的影响。本研究有望为组织工程骨血管化策略提供新思路，为hiPSCs在骨组织工程精准个体化临床应用提供强有力的实验依据。