Designing Fibrin Microthread Scaffolds for Skeletal Muscle Regeneration

描述(由申请人提供)：头部或颈部的骨骼肌损伤不仅会使人虚弱，还可能导致严重的心理损伤。虽然骨骼肌有一种由生长因子指导和卫星细胞介导的先天修复机制，但这种机制不能弥补因创伤相关事件(如车祸或癌症切除)而造成的大量肌肉体积损失。治疗这些缺陷的临床策略包括自体组织移植技术，需要大的供区和可能导致供区发病率和有限功能恢复的广泛的外科手术。因此，临床上需要一种现成的生物活性支架，引导患者的细胞在原位定位并分化为肌肉组织。我们实验室最近开发了纤维蛋白微线，这是一种类似于肌肉纤维的形态结构的组织结构，可以沿纤维长度纵向排列细胞。在伤口处，纤维蛋白在大量的酶如纤溶酶的存在下迅速降解。我们最近开发了一种交联策略来修饰纤维蛋白微线，以提高它们对酶降解的抵抗力。该项目的总体目标是利用这些微线设计一种新型的仿生支架，利用内源性骨骼肌再生途径来再生体积肌丢失。我们推测，纤维蛋白微线具有精确的降解特性，并与创伤愈合生长因子如肝细胞生长因子(HGF)和胰岛素样生长因子(IGF)-I结合，将通过内源性途径促进骨骼肌再生。为了系统地验证这一假设，我们将分析合成交联剂和天然交联剂对纤维蛋白微线结构和生化特性的影响。我们将分析卫星细胞对促进骨骼肌再生途径的纤维蛋白微线上的生长因子的剂量依赖性反应，并通过免疫组织化学分析确定这些因素如何影响细胞表型。我们将使用小鼠肌肉切除模型评估纤维蛋白微线对再生的贡献，以分析随着时间的推移伤口部位机械功能和胶原沉积的恢复情况。我们的最终目标是开发一种在骨骼肌再生高峰期(2-4周)持续存在的支架，具有临床相关的生长因子浓度，通过以下具体目标的研究指导体内机械功能组织的再生：(1)开发具有与天然骨骼肌相似的结构和生化特性的微线；(2)评估微线对体内骨骼肌再生的促进作用。通过结合微调的结构和机械性能，以及旨在促进和模拟骨骼肌再生的生长因子的结合，我们将创建一种仿生支架，在体内再生大型骨骼肌缺损。

DESCRIPTION (provided by applicant): Skeletal muscle trauma to the head or neck can not only be debilitating, but can result in severe psychological impairment. While skeletal muscle has an innate repair mechanism directed by growth factors and mediated by satellite cells, this mechanism cannot compensate for large, volumetric muscle loss as a result of trauma related events such as car accidents or cancer resection. Clinical strategies to treat these defects consist of autologous tissue transfer techniques, requiring large donor sites and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient's cell to align and differentiate into muscle tissue in situ. Our laboratory recently developed fibrin microthreads, a tissue construct that resembles the morphologic structure of a muscle fiber and can align cells longitudinally along the fiber length. In the wound site, fibrin degrades quickly i the presence of numerous proteinases such as plasmin. We recently developed a crosslinking strategy to modify fibrin microthreads to increase their resistance to enzymatic degradation. The overall goal of this project is to use these microthreads to design a novel biomimetic scaffold that will utilize the endogenous skeletal muscle regeneration pathway to regenerate volumetric muscle loss. We hypothesize that fibrin microthreads, with precisely tuned degradation properties and conjugated with wound healing growth factors such as hepatocyte growth factor (HGF) and insulin-like growth factor (IGF)-I, will enhance skeletal muscle regeneration of a large muscle defect via endogenous pathways. To systematically test this hypothesis, we will analyze the effects of synthetic and natural crosslinking agents on the structural and biochemical properties of fibrin microthreads. We will analyze the dose-dependent responses of satellite cells to growth factors on fibrin microthreads that contribute to the skeletal muscle regeneration pathway and determine how these factors affect cell phenotype through immunohistochemical assays. We will assess the contribution of fibrin microthreads to regeneration using a murine muscle excision model to analyze the recovery of mechanical function and collagen deposition within the wound site over time. Our ultimate goal is to develop a scaffold that will persist in siu throughout the peak time of skeletal muscle regeneration, 2-4 weeks, with clinically relevant growth factor concentrations to direct mechanically functional tissue regeneration in vivo through the study of the following Specific Aims: (1) To develop microthreads with structural and biochemical properties similar to native skeletal muscle, and (2) assess microthreads' enhancement of skeletal muscle regeneration in vivo. Through the combination of finely tuned structural and mechanical properties, and the conjugation of growth factors designed to promote and mimic skeletal muscle regeneration, we will create a biomimetic scaffold to regenerate large skeletal muscle defects in vivo.