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）损伤是导致肌肉功能永久丧失的衰弱性创伤。 此外，VML损伤通常由包括结缔组织和神经组织在内的多个组织的损伤而加重。 组织.周围神经系统损伤可导致去神经支配，这限制了力的产生，而 大多数肌肉损伤发生在肌腱连接处（MTJ）， 消融肌肉产生的力向骨骼系统的转移。不幸的是，许多治疗 VML的方法仅关注骨骼肌，忽略了对VML至关重要的邻近组织。 功能尽管有这种明确的临床需求，但缺乏治疗VML/MTJ联合损伤的疗法。因此，我们认为， 该建议的中心目标是应用模拟MTJ结构的组织工程支架来促进 VML/MTJ损伤的神经支配功能再生。我们将采取创新的生物材料为基础的方法， 它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， 糖胺聚糖（CG）支架，其概括了各向异性细胞外基质（ECM）组织 和天然骨骼肌的电兴奋性。我们假设一种在空间上- 明确的微环境提示与生肌细胞和神经细胞的生物反应器预处理配对， 使临床相关的VML/MTJ损伤再生。我们将通过两个目标来检验这一假设：1） 确定3D支架排列和电导率驱动体外肌生成的组合能力 肌源性细胞（MDC）和神经干细胞（NSC）共培养物的能力，以及2）确定3D多细胞共培养物的能力。 本发明的方法包括使用具有共培养的MDC和NSC的隔室支架来引导MTJ VML损伤的修复。我们将首先建立 最近的工作表明，共同培养神经和肌肉祖细胞的效用，以改善体外 通过确定仿生支架线索，包括3D结构排列和电 导电性，可以进一步放大这个过程。我们将评估MDC和NSC的活力，增殖，细胞骨架， 组织，以及支架内的肌管和神经肌肉接头（NMJ）形成， 电刺激和/或机械刺激。具有空间限定的电导率的各向异性CG支架 和机械来概括MTJ界面的生物物理性质， 在无生物反应器预处理的MDCs和NSC的情况下，在大鼠胫骨前VML/MTJ缺陷中。修复指标将 包括免疫组织化学、力产生量化和24岁以上步态生物力学分析 周我们的建议直接解决了具有挑战性和临床相关的VML损伤的治疗， 回答以前难以解决的生物学问题，包括理解支架的结构和 电信号可以协同地促进肌生成。总的来说，我们的方法将建立一个创新的 用于再生多组织界面和神经支配电响应组织的范例。

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.