Engineering Vascularized Skeletal Muscle Tissues with Adipose-derived Stem Cells

项目摘要/摘要：该项目的目标是创建一种组织工程化的血管化骨骼 肌肉构造。容积性肌肉丢失(VML)是一种肌肉缺陷，影响超过20% 个体肌肉的体积，压倒自然修复机制，导致慢性功能 受影响肌肉的缺陷。目前VML的治疗选择受到供区发病率的限制，缺乏 捐献组织，以及对高技能外科团队的需求。血管化组织工程化材料的研制 骨骼肌结构将减轻这些并发症，并提供临床相关的治疗 VML。脂肪干细胞是骨骼肌组织工程的理想细胞来源，具有很高的 增殖率，可直接分化为成肌细胞，并逃避宿主免疫系统。ASCs将成为 生长在新的电纺纤维纤维上，通过提供肌样来模拟自然肌肉的环境 僵硬和地形对齐线索，以鼓励ASC肌肉发生在3D。这些纤维提供了许多 优点：它们模仿天然肌肉的结构和力学，由一种生物材料组成 但可以重复制造，并且它们与宿主组织结合良好，导致最小程度的纤维化。 拟议研究的目标是有效克服与以下方面相关的两个主要限制 ASC介导的组织工程化骨骼肌，使VML缺损处获得功能性再生。第一, ASC向骨骼肌成肌细胞分化的效率将提高。ASC是一种高度异质的 拟肌3D中的群体和将ASCs缩小为潜在的亲肌源性CD34+CD31-亚群 微环境可能导致成肌细胞分化水平显著提高，这将是必要的 做一次收缩肌肉移植。第二，一种使组织工程结构快速血管化的策略 最大限度地提高移植后细胞的存活率和工程移植物与宿主组织的整合 发展起来的。天然骨骼肌大量血管化，肌肉缺损处血管增多 已被证明可以促进肌肉再生。带血管的肌肉结构将由 将ASC来源的骨骼肌移植物与含有工程血管网络的纤维捆绑在一起 ASCs和内皮祖细胞(EPC)。血管化肌肉结构的发展可能会 进一步增强ASCs体内再生潜能及其临床可译性。在特定目标中1 CD34+CD31-ASC亚群与未分选ASCs在电纺纤维蛋白上的成肌能力比较 将对纤维进行比较。在特定的目标2中，将在体外开发一种3D血管化的纤维蛋白结构，并将其 进入宿主血管系统的研究将在体内进行。在特定的目标3中，有血管的肌肉 构建将在体外开发，其在VML缺损处促进体内肌肉再生的能力将 被评估。这项工作将使血管化骨骼肌结构的开发变得容易 可获得的细胞类型和现成的生物材料，将为VML提供临床相关的治疗。

Project Summary/ Abstract: The goal of this project is to create a tissue engineered vascularized skeletal muscle construct. Volumetric muscle loss (VML) is a muscle defect that impacts greater than 20% of the volume of an individual muscle, overwhelming the natural repair mechanisms and leading to chronic functional deficits in the affected muscle. Current treatment options for VML are limited by donor site morbidity, lack of donor tissue, and the need for a highly skilled surgical team. Developing a tissue engineered vascularized skeletal muscle construct would mitigate these complications and provide a clinically-relevant treatment for VML. An ideal cell source for skeletal muscle tissue engineering, adipose-derived stem cells (ASCs) have high proliferation rates, can directly differentiate into myoblasts, and evade the host immune system. ASCs will be grown on novel electrospun fibrin fibers that mimic the environment of native muscle by providing a muscle-like stiffness and topographic alignment cues to encourage ASC myogenesis in 3D. The fibers provide a number of advantages: they mimic the structure and mechanics of native muscle, are composed of a biological material yet can be manufactured reproducibly, and they integrate well with host tissue causing minimal fibrosis. The objective of the proposed study is to effectively overcome two major limitations associated with ASC-mediated tissue engineered skeletal muscle and enable functional regeneration of a VML defect. First, the efficiency of ASC differentiation into skeletal myoblasts will be increased. ASCs are a highly heterogeneous population and narrowing ASCs to a potentially pro-myogenic CD34+CD31- subpopulation in a myomimetic 3D microenvironment may result in significantly higher levels of myoblast differentiation, which will be necessary to obtain a contractile muscle graft. Second, a strategy to rapidly vascularize the tissue engineered constructs to maximize post-transplantation cell survival and integration of the engineered graft with the host tissues will be developed. Native skeletal muscle is heavily vascularized and increasing vasculature within muscle defects has been shown to improve muscle regeneration. Vascularized muscle constructs will be developed by bundling ASC-derived skeletal muscle grafts with fibers containing engineered vascular networks comprised of ASCs and endothelial progenitor cells (EPCs). The development of a vascularized muscle construct is likely to further enhance the in vivo regenerative potential of ASCs and their clinical translatability. In Specific Aim 1 the myogenic capabilities of CD34+CD31- ASC subpopulation versus unsorted ASCs on electrospun fibrin fibers will be compared. In Specific Aim 2 a 3D vascularized fibrin construct will be developed in vitro and its incorporation into host vasculature will be investigated in vivo. In Specific Aim 3 a vascularized muscle construct will be developed in vitro and its ability to enhance in vivo muscle regeneration in a VML defect will be assessed. This work will enable the development of a vascularized skeletal muscle construct with easily procurable cell types and off-the-shelf biomaterials that would provide a clinically-relevant treatment for VML.