基于定向光控细胞片层技术的血管化组织工程肌肉的构建及机制研究

Skeletal muscle tissue engineering (SMTE) aims to repair or regenerate defective skeletal muscle tissue result from traumatic injury, tumor ablation or muscular disease. However, the engineering of large functional skeletal muscle in the laboratory still remains a great challenge, for lacking of ordered tissue structure and effective blood supply as in natural muscle. In this study, we introduced a novel light induced cell alignment and detachment technology to harvest anisotropic cell sheets. Through observation of filopodia and detection of fascin-1, cofilin-1 and related signal pathways in cell alignment, we want to illustrate the mechanism how cells react to external environment for orienting themselves, to lay a foundation for induced cell alignment technology. Then we developed a novel technology to construct functional tissue engineered muscle layer-by-layer assembly with three dimension reticular formation of vessels by modified light-induced cell sheet engineering, of which the MPCs and ECs were derived from human ESCs. Besides, we designed a new perfusion bioreactor system for culturing 3-D cell sheets on collagen-gel fabricated with imbedded micro-channels to mimic a subcutaneous vascular structure. The vascularized tissue surrogates would be evaluated both in vitro and in vivo. This project proposes several new design and creation concepts, promising significant advances to both preclinical and clinical applications of muscle tissue engineering.

骨骼肌组织工程旨在修复由于创伤、肿瘤和手术等造成的软组织缺损。然而目前体外构建大体积、功能化肌肉组织依然是组织工程研究的难点，主要原因在于无法有效模拟天然肌肉组织有序的结构和丰富的血液供应。本研究开创性地采用紫外光调控的细胞片层技术，使细胞定向生长和整层脱附，并通过对细胞定向排列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，为诱导细胞定向技术提供理论依据。在此基础上，联合人胚干细胞定向分化、细胞片层转移和层层组装技术、类基底循环灌注系统，以阶段性复合培养的方式构建大体积血管化具有功能的肌肉组织，通过体外长期培养评价其生物活性和功能指标，利用动物肌肉瘢痕模型检测其修复效果，为实现临床前及临床应用提供实验依据。本研究为组织工程技术提供了新的理念及方法，对促进肌肉组织工程发展具有重要意义。

骨骼肌组织工程旨在修复由于创伤、肿瘤和手术等造成的软组织缺损。然而目前体外构建大体积、功能化肌肉组织依然是组织工程研究的难点，主要原因在于无法有效模拟天然肌肉组织有序的结构和丰富的血液供应。本研究开创性地采用紫外光调控的细胞片层技术，使细胞定向生长和整层脱附，并通过对细胞定向排列过程中细胞伪足的观察和骨架系统以及相关通路的检测，探讨了细胞感受图案化信号调控细胞行为和命运可能是整合素-黏着班蛋白激酶（FAK）介导的MAPK通路的作用，丰富了我们对材料界面调控细胞行为的认识，有助于更好的设计基板材料用于组织工程修复。在此基础上，通过联合光敏凝胶细胞片辅助系统，细胞片预血管化技术及类基底循环灌注系统，课题组构建了血管化功能化的肌肉组织块，体内体外评价其功能和修复效果，为实现临床前及临床应用提供实验依据。本研究为组织工程技术提供了新的理念及方法，对促进肌肉组织工程发展具有重要意义。