BIOENGINEERED SKELETAL MUSCLES

颅面骨骼肌缺陷发生在先天性畸形中，如半面短小和面/腭裂;发生在某些肌病中，如肌营养不良;以及由于口腔癌或创伤的外科手术。近年来，颅颌面畸形患者软组织轮廓的研究取得了一些进展，包括半侧颜面矮小和唇腭裂。修复或替代肌肉缺损的成功受到肌肉移植和存活困难的限制。一个潜在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加现有的肌纤维，或者，骨骼肌可以在体外从收获的卫星成肌细胞中产生并植入缺陷部位。这样的方案将需要分离肌肉卫星细胞，其增殖和分化为肌纤维，并且重要的是，形成将肌肉产生的力传递到其结缔组织附着位点的肌腱连接（MTJ）。另一个重要的应用是引入成肌细胞用于基于细胞的基因治疗。提出的研究的长期目标是设计用于分离骨骼肌卫星细胞的方法，以优化其培养和向肌纤维的转化，并表征MTJ在体外的形成。α 7 β 1整联蛋白的表达受发育调节，并且对骨骼肌及其前体卫星细胞具有组织特异性。α 7 β 1整合素的表达是发育调节的，并且对于骨骼肌及其前体卫星细胞是组织特异性的。我们已经表明，α 7整联蛋白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上。我们建议检查α 7受体介导的MTJ形成的功能，并使用这种受体作为骨骼肌卫星细胞分离的组织特异性标记。我们的具体目标是：1）测试在MTJ形成过程中，α 7整合素的功能是否受到β 1 D亚基伴侣的调节。2)设计源自分选卫星细胞的组织工程化3D骨骼肌肌纤维类器官。3)克隆人α 7整合素cDNA，制备特异性抗体，用于分离人卫星细胞。这些研究将增强对MTJ组装的理解，并提出组织工程的新方法，以促进疾病、创伤或手术引起的颅面骨骼肌缺陷的重建/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.