BIOENGINEERED SKELETAL MUSCLES

颅面部骨骼肌缺陷发生在先天性畸形中，如半面部巨大症和面部/腭裂；发生在某些肌肉疾病中，如肌营养不良；以及口腔癌或创伤的外科手术。最近在报告颅面畸形患者的软组织轮廓方面取得了进展，包括半面部巨大症和唇腭裂。肌肉移植和存活的困难限制了修复或替代肌肉缺损区的成功。一种可能的解决方案是开发组织工程方案，其中成肌细胞直接注射以增强现有的肌肉纤维，或者，替代地，骨骼肌可以从收获的卫星成肌细胞体外产生并植入缺损处。这样的方案需要分离肌肉卫星细胞，它们的增殖和向肌纤维的分化，以及重要的是肌腱连接(MTJ)的形成，MTJ将肌肉产生的力传递到结缔组织附着部位。另一个重要的应用是引入成肌细胞进行基于细胞的基因治疗。这项研究的长期目标是设计分离骨骼肌卫星细胞的方法，优化其培养和转化为肌纤维，并在体外表征MTJ的形成。α7beta1整合素的表达受发育调控，对骨骼肌及其前体卫星细胞具有组织特异性。α7beta1整合素的表达受发育调控，对骨骼肌及其前体卫星细胞具有组织特异性。我们已经证明，α7整合素不仅在卫星细胞上表达，而且在终末分化的肌管中也集中在MTJ处。我们建议研究α7受体在调节MTJ形成中的功能，并将该受体用作分离骨骼肌卫星细胞的组织特异性标记。我们的具体目标是：1)检测在MTJ形成过程中，α7整合素的功能是否受到β1D亚基伴侣的调节。2)设计从分离的卫星细胞衍生的组织工程化的三维骨骼肌肌纤维有机体。3)克隆人α7整合素基因，为分离人卫星细胞制备特异性抗体。这些研究将加深对MTJ组装的了解，并提出组织工程学的新方法，以促进疾病、创伤或外科手术引起的头面部骨骼肌缺损的重建/增强。

Craniofacial skeletal muscle defects occur in congenital deformities, such as hemifacial microsomia and facial/palatal clefts: in certain myopathies, such as muscular dystrophy; and as a result of surgical procedures for oral cancer or trauma. Recent advances have been made in reporting soft-tissue contour in patients with craniofacial anomalies, including hemifacial microsomia and clefts of lip and palate. Success in the repair or replacement muscle defects is limited by difficulty in muscle transplantation and survival. A potential solution would be to develop tissue engineering protocols in which myoblasts are directly injected to augment existing muscle fibers or, alternatively, in which skeletal muscle could be generated in vitro from harvested satellite myoblasts and implanted in the defective site. Such protocols would require isolation of muscle satellite cells, their proliferation and differentiation to myofibers, and, importantly, formation of the myotendinous junction (MTJ) that transduces force generated by muscle to its connective tissue attachment site. Another important application is the introduction of myoblasts for cell-based gene therapy. The long-term objective of the proposed studies is to devise methods for isolating of skeletal muscle satellite cells, to optimize their culture and conversion to myofibers, and to characterize the formation of the MTJ in vitro. Expression of the alpha7beta1 integrin is developmentally regulated and is tissue-specific for skeletal muscle and its precursor satellite cells. Expression of the alpha7beta1 integrin is developmentally regulated and is tissue-specific for skeletal muscle and its precursor satellite ells. We have shown that the alpha7 integrin is not only expressed on satellite cells but is also concentrated at the MTJ in terminally differentiated myotubes. We propose to examine the function of the alpha7 receptor in mediating MTJ formation and to use this receptor as a tissue-specific marker for the isolation of skeletal muscle satellite cells. Our specific aims are: 1) Test whether the function of alpha7 integrin is regulated by beta1D subunit partner during formation of the MTJ. 2) Design tissue-engineered 3-D skeletal muscle myofiber organoids derived from sorted satellite cells. 3) Clone the human alpha7 integrin cDNA and generate specific antibodies for the isolation for human satellite ells. These studies will enhance understanding of the assembly of the MTJ and suggest new approaches in tissue engineering to promote reconstruction/augmentation of craniofacial skeletal muscle defects caused by disease, trauma, or surgical procedures.