基于光控MSCs细胞片的血管化组织工程骨的构建及其促血管机制研究

The pre-vascularization of biological bone tissue is significant for repairing and rebuilding of bone tissue. In previous studies, we have clarified the mechanism of the cell sheet technology and developed a novel technology for tissue engineering to construct tissue engineered bone layer-by-layer assembly by using light-responsived cell sheet engineering, we also discussed the mechanism of the inner cells metabolism and vascularization in tissue engineered bone. Based on this, we designed to construct tissue engineered bone with three dimension reticular formation of vessels by using hMSCs cell sheets composited with ECs and SMCs derived from human ESCs. Then, we designed a new perfusion bioreactor system for culturing 3-D cell sheets on gel fabricated with imbedded microchannels, which enabled production of thick tissue dense constructs with pre-integrated and vessel-populated architectures. Besides, we would focus on studying the mechanism of the inner cells metabolism and vascularization regulated through specific integrin-dependent signal transduction pathways by the presence of a collagen-rich or fibrinectin-rich ECM from the MSCs sheets, which would help us further explain the molecular mechanisms of vascularization. This strategy is presented to contribute several new design and creation concepts, promising significant advances to both preclinical and clinical applications of tissue engineering.

生物移植骨组织的早期血管化对于骨缺损的修复及重建具有重要的意义，已成为研究的难点与热点。前期研究中，项目组阐明了紫外光致脱离技术的机理，通过光控细胞薄层培养及层层组装技术构建了三维仿生组织工程骨，并对其内层细胞片代谢形式及血管化形成机制进行了初探。在此基础上，本课题拟采用hESCs来源的MSCs细胞片复合ECs及SMCs制备具有血管网状结构的叠层化物，并于其基底构建微流控仿生灌注系统，探讨体外培养规模更大的血管化组织工程骨的可能性；同时，重点研究叠层化物内部细胞代谢方式，并就细胞片外ECM中Ⅰ型胶原或纤连蛋白所介导的整合素相关信号传导通路促进成血管作用的分子机制进行深入研究，进一步阐明MSCs细胞片诱导ECs血管化的分子机制，并为组织再生技术的发展提供新的理念及方向，最终为实现其临床前及临床应用提供实验依据。

生物移植骨组织的早期血管化对于骨缺损的修复及重建具有重要的意义，然而具备良好血管化的体外组织工程骨的构建及后者具体的促血管机制仍是现阶段研究的难点。本研究进一步阐明了紫外光致脱离技术的机理，基于光控细胞薄层培养及层层组装技术，采用hESCs来源的MSCs细胞片复合ECs及SMCs制备具有血管网状结构的叠层化物，并于其基底构建微流控仿生灌注系统，体外构建了血管化组织工程骨；重点研究叠层化物内部细胞代谢方式，就细胞片外ECM中Ⅰ型胶原或纤连蛋白所介导的细胞骨架相关信号传导通路促进成血管作用的分子机制进行深入研究，并通过对细胞运动过程中细胞伪足的观察和fascin-1、cofilin-1以及相关通路的检测，探讨了细胞感受外界环境实现方向性排列的机制；同时，发现间充质干细胞可能通过分泌促血管生成因子如ANG1等，并经PDGF-BB/PDGFR-β通路分化为周细胞进而实现双重促血管作用，而良好的血管化可进一步上调VEGF以促进骨再生作用。本研究为组织再生的发展提供新的技术手段及方向，最终为实现其临床前及临床应用提供实验依据。