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虚弱，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因车祸或癌症切除等创伤相关事件导致的大体积肌肉损失。治疗这些缺陷的临床策略包括自体组织移植技术，需要大的供体部位和广泛的外科手术，这可能导致供体部位的发病率和有限的功能恢复。因此，临床需要一种现成的、具有生物活性的支架来指导患者的细胞原位排列并分化成肌肉组织。我们的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形态结构的组织结构，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，以增加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仿生支架，该支架将利用内源性骨骼肌再生途径再生体积肌肉损失。我们假设纤维蛋白微线具有精确调节的降解特性，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)- 1）结合，将通过内源性途径促进骨骼肌大肌肉缺损的再生。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性能的影响。我们将分析卫星细胞对骨骼肌再生途径中纤维蛋白微线上生长因子的剂量依赖性反应，并通过免疫组化分析确定这些因子如何影响细胞表型。我们将使用小鼠肌肉切除模型来评估纤维蛋白微线对再生的贡献，以分析机械功能的恢复和伤口部位胶原沉积随时间的变化。我们的最终目标是通过以下具体目标的研究，开发一种能够在骨骼肌再生的高峰时间（2 -4周）内持续存在的支架，具有临床相关的生长因子浓度，以指导体内机械功能组织再生：(1)开发具有与天然骨骼肌相似的结构和生化特性的微线；(2)评估微线对体内骨骼肌再生的增强作用。通过精细调整结构和机械性能的组合，以及旨在促进和模仿骨骼肌再生的生长因子的结合，我们将创建一个仿生支架来再生体内的大型骨骼肌缺陷。

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.