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创伤不仅会使人衰弱，而且会导致严重的心理障碍。虽然骨骼肌具有由生长因子指导并由卫星细胞介导的先天修复机制，但这种机制不能补偿由于创伤相关事件（如车祸或癌症切除术）导致的大体积肌肉损失。治疗这些缺损的临床策略包括自体组织移植技术，需要大的供体部位和广泛的外科手术，这可能导致供体部位发病和功能恢复有限。因此，临床上需要一种现成的生物活性支架，其引导患者的细胞原位排列和分化成肌肉组织。我们的实验室最近开发了纤维蛋白微丝，一种组织结构，类似于肌肉纤维的形态结构，可以沿着纤维长度纵向排列细胞。在伤口部位，纤维蛋白在许多蛋白酶如纤溶酶的存在下快速降解。我们最近开发了一种交联策略来修饰纤维蛋白微丝，以增加其对酶降解的抵抗力。该项目的总体目标是使用这些微线程来设计一种新型仿生支架，该支架将利用内源性骨骼肌再生途径来再生体积损失的肌肉。我们假设，纤维蛋白微丝，具有精确调谐的降解性能，并与伤口愈合生长因子，如肝细胞生长因子（HGF）和胰岛素样生长因子（IGF）-I共轭，将通过内源性途径增强骨骼肌再生的大肌肉缺损。为了系统地检验这一假设，我们将分析合成和天然交联剂对纤维蛋白微丝的结构和生化性质的影响。我们将分析卫星细胞对纤维蛋白微丝上生长因子的剂量依赖性反应，这些生长因子有助于骨骼肌再生途径，并通过免疫组织化学测定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丝对再生的贡献，以分析随着时间的推移伤口部位内的机械功能和胶原沉积的恢复。我们的最终目标是开发一种支架，该支架将在整个骨骼肌再生的高峰时间（2-4周）持续siu，具有临床相关的生长因子浓度，以通过以下特定目的的研究在体内指导机械功能性组织再生：（1）开发具有与天然骨骼肌相似的结构和生化特性的微线程，以及（2）评估微线程在体内促进骨骼肌再生的作用。通过微调结构和机械性能的组合，以及旨在促进和模拟骨骼肌再生的生长因子的缀合，我们将创造一种仿生支架，以在体内再生大的骨骼肌缺损。

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.