Nanopatterned 3D Vascularized Functional Muscle Patch

纳米图案 3D 血管化功能性肌肉贴片

• 批准号: 8636918
• 负责人: Deok-Ho Kim
• 金额: $ 19.14万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8636918
• 关键词: Adult; Affect; Architecture; Biochemical; Biocompatible Materials; Blood Vessels; Bungarotoxins; Cell Culture Techniques; Cell Survival; Cell Therapy; Cell fusion; Cells; Cessation of life; Chemistry; Complex; Confocal Microscopy; Development; Disease; Duchenne muscular dystrophy; Dystrophin; Endothelial Cells; Engineering; Engraftment; Extracellular Matrix; Fiber; Fibrin; Fibrin Tissue Adhesive; Geometry; Goals; Immunocompromised Host; Immunohistochemistry; Implant; Intramuscular; Isolectin; Length; Limb structure; Longevity; Measures; Modality; Molecular; Mus; Muscle; Muscle Cells; Muscle Contraction; Muscle Fibers; Muscle Tension; Muscle function; Muscular Dystrophies; Myography; Myopathy; Nanotopography; Natural regeneration; Neuromuscular Junction; Patients; Pattern; Play; Polymers; Process; Property; Role; Skeletal Muscle; Staining method; Stains; Stem cells; Structure; Supporting Cell; System; Techniques; Testing; Tissue Engineering; Tissues; Transgenic Mice; Transplantation; United States; Vascularization; Weight-Bearing state; Western Blotting; Workload; angiogenesis; base; cell motility; controlled release; cytochemistry; disability; functional restoration; implantation; inorganic phosphate; intravital imaging; male; mdx mouse; mouse model; muscle form; muscle regeneration; muscular dystrophy mouse model; muscular structure; myogenesis; nanoengineering; nanometer; nanopattern; nanostructured; neurofilament; public health relevance; reconstruction; release of sequestered calcium ion into cytoplasm; repaired; satellite cell; scaffold; sphingosine 1-phosphate; stem cell therapy; success; therapy development

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.

描述(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名男性中就有一人受到影响，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、保存率低、植入度不佳，肌肉内移植的成功非常有限。骨骼肌由高度各向异性排列的高度横纹细胞组成，附着在由许多纳米级特征尺寸的纤维组成的细胞外基质(ECM)上，高保真地延伸到不同长度的尺度上。我们之前的研究表明，为细胞提供基于ECM的支持，模仿天然的肌肉ECM纳米拓扑学，可以为干细胞提供一个生理上相关的微环境，使其排列并融合在一起，形成成熟的肌肉。我们还发现并证明了1-磷酸马钱子苷(S1P)作为一种有效的血管生成和肌肉生成因子的作用。这项提议的总体目标是开发纳米级的、各向异性的3D肌肉组织贴片，接近模拟天然骨骼肌结构，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。我们将结合基于纳米拓扑学的组织工程方法、细胞板操作技术和S1P疗法来生成血管肌肉组织的无支架3D贴片。表达Pax7Cre/Flox GCaMP3的原代肌肉内皮细胞和卫星细胞将被培养在纳米地形定义的、具有可调几何结构的温度响应性底物上，以促进肌肉发生并产生各向异性细胞片。这些片材将被堆叠起来，形成3D肌肉结构，在移植之前将进行分子、结构和功能分析。我们将测试我们的3D组织构建物与营养不良肌肉整合的能力，提供干细胞库，恢复肌营养不良症小鼠的肌营养不良蛋白表达，并增强四肢肌肉。具体目标包括：(1)利用纳米细胞片操作技术开发血管化的3D肌肉贴片；以及(2)植入并评估带有1-磷酸鞘氨醇结合纤维蛋白的3D肌肉贴片，以恢复Duchenne肌营养不良小鼠模型的肌肉功能。