新型可注射大孔支架携带hUCMSCs修复大面积骨骼肌缺损的实验研究

Macroporous scaffold is a disirable choice for tissue repair of large defects against cell death. Up to date, however, most methods only allow for fabrication of non-injectable macroporous scaffolds in case of in vivo usage. Therefore, their effects are not ideal because their application causes a significnat invasive trauma with insufficient plasticity. To slove the above problem, we recently developed a strategy of fabricating injectable macroporous hydrogel by the addition of fast degradable microbeads into fibrin gel. Both the viability and spreading of the encapsulated cells were greatly enhanced, suggesting that the novel scaffold is promising for promoting tissue engineering with high efficiency. Therefore, in the present project, we will further apply the new scaffold encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs) for volumetric skeletal muscle loss repair in nude mice. The strategies of releasing IGF-1/VEGF, and/or using PAX7 gene transfected hUCMSCs, will be adopted for the purpose of myogenic induction of hUCMSCs in vivo. By evaluation of skeletal muscle regeneration, cell apotosis and necrosis, vascularization and transplant safety, it aims to (1) confirm the advantages of the novel scaffold compared to the regular fibrin gels when used for skeletal muscle defect repair; (2) investigate a feasible strategy of inducing hUCMSCs towards skeletal muscle lineage in vivo; and (3) explore the mechanism of myogenic differentiation of hUCMSCs. The project will hopefully help to provide new ideas or methods for skeletal muscle tissue engineering in vivo.

大孔支架是修复大面积组织缺损避免再生细胞死亡的理想选择之一。但迄今大多数大孔材料只能以非注射方式用于体内，应用时创伤较大，可塑性有限，效果尚不理想。为解决上述问题，课题组前期在纤维蛋白凝胶内添加一种新研发的快速降解微球，成功制备出具备大孔结构、可注射的水凝胶，该新型支架大大增强了携带细胞的增殖活力和延展性，提示其在促进组织高效再生方面有一定应用前景。本项目拟进一步将该支架携带人脐带间充质干细胞（hUCMSCs）用于修复裸鼠大面积骨骼肌缺损，通过用支架缓释IGF-1/VEGF，和（或）将hUCMSCs转染PAX7基因，对骨骼肌再生、凋亡坏死发生、血管化以及移植安全性等进行检测比较。目的在于：①明确新型大孔支架与常规纤维蛋白支架相比用于骨骼肌缺损修复的优势；②寻求体内诱导骨骼肌定向分化的合适策略；③探讨hUCMSCs向骨骼肌转归的机制。结果可望为体内组织工程化骨骼肌的构建提供新的思路和方法。

大面积骨骼肌缺损的修复治疗是临床亟待解决的难点问题。肌肉组织工程的发展为这一难题的解决带来了希望，即通过选择理想的种子细胞和生物支架，借助一定的诱导分化策略进行肌肉再生，继而可望解决肌肉来源匮乏的难题。肌卫星细胞是肌肉组织中位于肌细胞膜和基膜之间的具有增殖分化潜力的前体细胞。其缺点是需要用手术创伤方式获取，在肌肉组织中分布少、来源有限，而且与其它种子细胞相比，其体外扩增潜力较低。肌卫星细胞体内移植修复骨骼肌实验表明其无需特殊干预即可向肌纤维分化。配对盒基因3（paired box gene 3, PAX3）是骨骼肌卫星细胞的特征性基因，对肌卫星细胞存活、增殖和自我更新等具有至关重要的作用；同时它也是强有力的成肌诱导因子，其调节作用位于 MyoD 上游。鉴于 PAX3发挥的重要作用，我们提出如下假设：由于肌卫星细胞数量稀少、来源困难，我们可将人脂肪干细胞（ADSCs）转染 PAX3 基因，使之具备肌卫星细胞的特性，继而利用体内微环境对其诱导，实现骨骼肌再生的目的。研究的目的是将ADSCs进行PAX3基因修饰，采用fibrin支架携带PAX3-ADSCs进行成肌分化研究。实验成功实现了ADSCs的PAX3基因转导。体外研究结果初步表明，PAX3过表达有效促进了ADSCs细胞的成肌分化。裸鼠肌肉缺损修复体内研究结果尚未完全得到。研究结果可望为利用自身干细胞构建组织工程化肌肉用于修复肌肉缺损提供新的方法。