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

摘要 容积性肌肉丧失(VML)损伤是一种导致肌肉功能永久性丧失的衰弱创伤。 此外，VML损伤常常因多种组织的损伤而加重，包括结缔组织和神经。 组织。周围神经系统损伤会导致失神经，从而限制力量的产生，而 肌肉损伤最多的部位--肌腱连接处(MTJ)的肌肉纤维断裂可能会进一步 切断肌肉产生的力量向骨骼系统的转移。不幸的是，许多治疗性的 VML的治疗方法只关注骨骼肌，而忽略了邻近组织对 功能。尽管有这种明确的临床需求，但缺乏治疗VML/MTJ联合损伤的治疗方法。因此， 这项提议的中心目标是应用一种模仿MTJ结构的组织工程支架来促进 神经支配的VML/MTJ损伤的功能再生。我们将采取以生物材料为基础的创新方法 这是建立在我们团队最近开发的3D排列和导电胶原蛋白的基础上的- 重组各向异性细胞外基质(ECM)组织的糖胺聚糖(CG)支架 以及天然骨骼肌的电兴奋性。我们假设一种具有空间结构的工程生物材料- 明确的微环境线索与肌源性细胞和神经细胞的生物反应器预适应将 使临床相关的VML/MTJ损伤能够再生。我们将通过两个目标来检验这一假设：1) 测定3D支架排列和电导率联合驱动体外成肌的能力 肌源性细胞(MDC)和神经干细胞(NSC)共培养的能力；2)决定3D多细胞培养的能力 与MDCs和NSCs共培养的隔室支架用于指导MTJ VML损伤的修复。我们将首先建立 关于证明共培养神经和肌肉前体细胞在体外改进的有效性的最新工作 通过确定仿生支架线索，包括3D结构对齐和电信号，实现肌肉发生 导电性，可以进一步放大这一过程。我们将评估MDC和NSC的活性、增殖、细胞骨架 支架内的组织、肌管和神经肌肉接头(NMJ)的形成 电和/或机械刺激。具有空间电导率的各向异性CG支架 然后，将植入用于概括MTJ界面的生物物理属性的机械装置，或 未经生物反应器预处理的骨髓间充质干细胞和神经干细胞移植于大鼠胫骨前肌VML/MTJ缺损区。维修指标将 包括免疫组织化学、力量生成量化以及24岁以上步态生物力学分析 几周。我们的建议直接针对具有挑战性和临床相关的VML损伤的治疗，而 回答以前棘手的生物学问题，包括了解支架结构和 电信号可以协同促进肌肉发生。总体而言，我们的方法将建立一个创新的 再生多组织界面和神经电响应组织的范例。

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.