Aligned and electrically conductive collagen scaffolds for guiding innervated muscle-tendon junction repair of volumetric muscle loss injuries

对齐且导电的胶原蛋白支架，用于引导神经支配的肌肉肌腱连接修复体积性肌肉损失损伤

• 批准号: 10183865
• 负责人: Steven Caliari
• 金额: $ 38.29万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10183865
• 关键词: 3-Dimensional; Address; Animals; Antibodies; Biocompatible Materials; Biological; Biomechanics; Biomimetics; Bioreactors; Cell Maturation; Cell Survival; Cells; Cholinergic Receptors; Cicatrix; Clinical; Coculture Techniques; Collagen; Complex; Confocal Microscopy; Connective Tissue; Cues; Cytoskeletal Modeling; Defect; Denervation; Development; Electric Conductivity; Electric Stimulation; Engineering; Extracellular Matrix; Fibrosis; Gait; Generations; Glycosaminoglycans; Histologic; Image; Immunohistochemistry; Implant; In Vitro; Injury; Locomotion; Measurement; Measures; Mechanical Stimulation; Mechanics; Movement; Muscle; Muscle Fibers; Muscle function; Muscle satellite cell; Muscular Atrophy; Myosin Heavy Chains; Natural regeneration; Nerve; Nerve Tissue; Nervous System Trauma; Neuromuscular Junction; Neuronal Differentiation; Neurons; Operative Surgical Procedures; Patients; Peripheral; Peripheral Nerves; Peripheral Nervous System; Process; Rattus; Recovery of Function; Repair Complex; Signal Transduction; Skeletal Muscle; Skeletal muscle injury; Skeletal system; Structure; Technology; Tendon structure; Testing; Therapeutic; Tissue Engineering; Tissues; Trauma; Vascularization; Work; base; beta Tubulin; biophysical properties; clinically relevant; conditioning; electrical potential; experience; gait examination; immunocytochemistry; improved; in vivo; innovation; military patient; myogenesis; neglect; nerve stem cell; nerve supply; osteochondral tissue; preconditioning; relating to nervous system; repair strategy; repaired; restoration; scaffold; skeletal; stem cell differentiation; success; three dimensional structure; tibialis anterior muscle; tissue regeneration; tissue repair; transmission process; volumetric muscle loss

ABSTRACT Volumetric muscle loss (VML) injuries are debilitating traumas that result in permanent loss of muscle function. Moreover, VML injuries are often compounded by damage to multiple tissues including connective and nervous tissue. Peripheral nervous system damage can result in denervation that limits force generation while the disruption of muscle fibers at the musculotendinous junction (MTJ), where most muscle injuries occur, can further ablate the transfer of muscle-generated force to the skeletal system. Unfortunately, many therapeutic approaches for VML solely focus on skeletal muscle, neglecting neighboring tissues that are essential for function. Despite this clear clinical need, therapies to treat combined VML/MTJ injuries are lacking. Therefore, the central objective of this proposal is to apply a tissue engineering scaffold mimicking MTJ structure to promote innervated functional regeneration of VML/MTJ injuries. We will take an innovative biomaterials-based approach that builds on our team’s recent development of a 3D aligned and electrically conductive collagen- glycosaminoglycan (CG) scaffold that recapitulates both the anisotropic extracellular matrix (ECM) organization and electrical excitability of native skeletal muscle. We hypothesize that an engineered biomaterial with spatially- defined microenvironmental cues paired with bioreactor preconditioning of myogenic and neuronal cells will enable regeneration of clinically relevant VML/MTJ injuries. We will test this hypothesis through two aims: 1) Determine the combined ability of 3D scaffold alignment and electrical conductivity to drive in vitro myogenesis of muscle-derived cell (MDC) and neural stem cell (NSC) co-cultures, and 2) Determine the ability of 3D multi- compartment scaffolds with co-cultured MDCs and NSCs to guide repair of MTJ VML injuries. We will first build on recent work demonstrating the utility of co-culturing neural and muscle progenitor cells to improve in vitro myogenesis by determining if biomimetic scaffold cues, including 3D structural alignment and electrical conductivity, can further amplify this process. We will evaluate MDC and NSC viability, proliferation, cytoskeletal organization, and myotube and neuromuscular junction (NMJ) formation within scaffolds both with and without electrical and/or mechanical stimulation. Anisotropic CG scaffolds with spatially-defined electrical conductivity and mechanics to recapitulate the biophysical properties of the MTJ interface will then be implanted, with or without bioreactor preconditioned MDCs and NSCs, in rat tibialis anterior VML/MTJ defects. Repair metrics will include immunohistochemistry, quantification of force generation, and analysis of gait biomechanics over 24 weeks. Our proposal directly addresses the treatment of challenging and clinically relevant VML injuries while answering previously intractable biological questions, including understanding of how scaffold structural and electrical signals can synergistically promote myogenesis. Overall, our approach will establish an innovative paradigm for regenerating multi-tissue interfaces and innervating electrically-responsive tissue.

抽象的 体积肌肉损失（VML）损伤是使肌肉功能永久丧失的衰弱创伤。 此外，VML受伤通常会因多个时间因素（包括结缔组织和紧张）的损害而加重 组织。周围神经系统损害可能导致神经支配，从而限制了强迫产生 大多数肌肉损伤发生的肌肉束（MTJ）的肌肉纤维破坏可以进一步 消灭肌肉生成的力向骨骼系统的转移。不幸的是，许多治疗性 VML的方法仅专注于骨骼肌，忽略了相邻的组织 功能。尽管有明显的临床需求，但仍缺乏治疗VML/MTJ损伤的疗法。所以， 该提案的核心目的是应用模仿MTJ结构的组织工程脚手架来促进 VML/MTJ损伤的神经功能再生。我们将采取一种创新的基于生物材料的方法 这是基于我们团队最近的3D结盟和导电胶原蛋白的发展的基础 糖胺聚糖（CG）支架，概括了各向异性细胞外基质（ECM）组织 和原生骨骼肌的电气刺激性。我们假设一种具有空间的工程生物材料 定义的微环境提示与肌原性和神经元细胞的生物反应器预处理配对 使临床相关的VML/MTJ损伤能够再生。我们将通过两个目的检验这一假设：1） 确定3D支架比对和电导率的组合能力驱动体外肌发生 肌肉衍生的细胞（MDC）和神经干细胞（NSC）共培养，2）确定3D多数的能力 带有共培养的MDC和NSC的隔室支架，用于指导MTJ VML损伤的修复。我们将首先建立 在最近的工作中，证明了共同培养的神经和肌肉祖细胞的实用性，以改善体外 通过确定仿生型支架线索（包括3D结构比对和电气）是否存在肌发生 电导率可以进一步扩大此过程。我们将评估MDC和NSC生存力，增殖，细胞骨架 组织，Myotube和神经肌肉结（NMJ）在脚手架内有和不带有和没有 电气和/或机械模拟。各向异性CG支架具有空间定义的电导率 然后，将使用或 在大鼠胫骨前VML/MTJ缺陷中，没有生物反应器预处理的MDC和NSC。维修指标将 包括免疫组织化学，量化力的量化以及24多个步态生物力学的分析 几周。我们的建议直接解决了挑战和临床相关的VML伤害的治疗 回答以前棘手的生物学问题，包括了解脚手架结构和 电信号可以协同促进肌发生。总体而言，我们的方法将建立创新的 用于再生多组织界面和支配电响应组织的范式。