Nanopatterned 3D Vascularized Functional Muscle Patch

描述（由申请人提供）：杜氏肌营养不良症（DMD）是一种致命的疾病，在美国每3500名男性中就有一人患病，导致严重残疾和死亡。尽管卫星细胞具有内源性再生骨骼肌的能力，但由于细胞存活率低、保留率低和次优植入，肌内移植的成功率非常有限。骨骼肌由以高度各向异性的方式排列的高度横纹细胞组成，附着于由许多特征尺寸在纳米范围内的纤维组成的细胞外基质（ECM），以高保真度延伸各种长度尺度。我们以前的研究表明，为细胞提供基于ECM的支持，模仿天然肌肉ECM纳米形貌，可以为干细胞提供生理相关的微环境，以对齐和融合在一起形成成熟的肌肉。我们还确定并证明了鞘氨醇1-磷酸（S1 P）作为一种有效的血管生成和肌生成因子的作用。该提案的总体目标是开发纳米图案化的各向异性3D肌肉组织贴片，这些贴片紧密模仿天然骨骼肌结构，然后在DMD小鼠模型中测试它们整合和恢复肌肉功能的能力。我们将结合联合收割机的纳米地形学为基础的组织工程方法与细胞片操作技术和S1 P治疗，以产生无支架的血管化肌肉组织的3D补丁。表达Pax 7 Cre/flox GCaMP 3的原代肌肉内皮细胞和卫星细胞将在具有可调几何形状的纳米地形学定义的温敏基底上培养，以促进肌生成并产生各向异性细胞片。这些薄片将被堆叠以形成3D肌肉结构，这些肌肉结构将在移植前进行分子、结构和功能分析。我们将测试我们的3D组织构建体与营养不良肌肉整合的能力，提供干细胞库，恢复肌营养不良蛋白表达并加强肌肉萎缩症小鼠的肢体肌肉。具体目标包括：（1）使用纳米图案化的细胞片操作技术开发血管化的3D肌肉贴片;和（2）植入和评估具有鞘氨醇1-磷酸缀合的纤维蛋白的3D肌肉贴片，以恢复杜氏肌营养不良症小鼠模型中的肌肉功能。

DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD) is a lethal disease that affects one in every 3500 males in the United States, causing severe disabilities and death. Despite the capacity of satellite cells to endogenously regenerate skeletal muscle, intramuscular transplantation efforts have shown very limited success due to poor cell survival, low retention, and suboptimal engraftment. Skeletal muscle consists of highly striated cells arranged in a highly anisotropic manner, attached to an extracellular matrix (ECM) composed of many fibers of feature sizes in the nanometer range, extending for various length scales with high fidelity. Our previous studies suggest that providing an ECM-based support for cells, mimicking the native muscle ECM nanotopography, can provide a physiologically relevant microenvironment for the stem cells to align and fuse together to form mature muscle. We also identified and demonstrated the role of sphigosine 1-phosphate (S1P) as a potent angiogenic and myogenic factor. The overall goals of this proposal are to develop nanopatterned, anisotropic 3D muscle tissue patches that closely mimic native skeletal muscle structure and then to test their ability to integrate and restore muscle function in a mouse model of DMD. We will combine a nanotopography-based tissue engineering approach with cell sheet manipulation techniques and S1P therapy to generate scaffold free 3D patches of vascularized muscle tissues. Primary muscle endothelial and satellite cells expressing Pax7Cre/flox GCaMP3 will be cultured on nanotopographically-defined, thermoresponsive substrates with tunable geometries to promote myogenesis and create anisotropic cell sheets. These sheets will be stacked to form 3D muscle constructs that will undergo molecular, structural, and functional analyses prior to transplantation. We will test our 3D tissue construct's ability to integrate with dystrophic muscle provide a stem cell reservoir, restore dystrophin expression and strengthen limb muscles in muscular dystrophy mice. Specific aims include: (1) to develop vascularized 3D muscle patches using nanopatterned cell sheet manipulation techniques; and (2) to implant and evaluate 3D muscle patches with sphingosine 1-phosphate conjugated fibrin to restore muscle function in a mouse model of Duchenne muscular dystrophy.