Volumetric muscle loss (VML) overwhelms the native regenerative capabilities of skeletal muscle and has few effective treatments to regain lost muscle mass and function. Tissue engineered muscle constructs designed to promote neuromuscular regeneration are a promising therapeutic avenue. To date, there has been no engineered muscle construct for VML treatment that has incorporated a pharmacologic agent to promote neuromuscular regeneration. Here, we have modified electrospun fibrin microfiber bundles, which have demonstrated muscle regenerative potential, with the heparan sulfate proteoglycan, agrin, to stimulate innervation post-VML. Myoblasts cultured on microfiber bundles with either soluble or chemically tethered agrin demonstrated statistically significant increased clustering of acetylcholine receptors (AChRs) with soluble agrin displaying AChR clusters throughout the myofiber bundles, and tethered agrin displaying AChR clusters only at 10 μm from the substrate surface. Following implantation into murine VML defects for 4 weeks, constructs pre-treated with soluble or tethered agrin resulted in statistically significant increased neuromuscular junctions, regenerating myofibers, vascular infiltration, neural infiltration, and nuclear yes-associated protein (YAP) expression within the defect site compared to the control without agrin. The agrin-tethered microfiber bundles provided sustained agrin signaling within the regenerating site during the 4-week post-implantation periods and further augmented the density of regenerating myofibers in regenerated tissue with statistical significance compared to constructs with soluble agrin. These data demonstrate the neuromuscular regenerative potential of engineered muscle constructs pre-treated to induce AChR clustering with locally delivered agrin at the site of VML regeneration.

体积肌肉损失（VML）压倒了骨骼肌的天然再生能力，并且几乎没有有效的治疗方法来恢复损失的肌肉质量和功能。旨在促进神经肌肉再生的组织工程肌肉结构是一种有前途的治疗途径。迄今为止，还没有用于 VML 治疗的工程肌肉结构掺入了促进神经肌肉再生的药物。在这里，我们用硫酸乙酰肝素蛋白聚糖 agrin 修饰了静电纺丝纤维蛋白微纤维束，该纤维束已显示出肌肉再生潜力，可刺激 VML 后的神经支配。在具有可溶性或化学系留集聚蛋白的微纤维束上培养的成肌细胞显示，乙酰胆碱受体（AChR）的聚集在统计上显着增加，可溶性集聚蛋白在整个肌纤维束中显示AChR簇，而拴系集聚蛋白仅在距离基底表面10μm处显示AChR簇。植入小鼠 VML 缺陷 4 周后，用可溶性或系留的 agrin 预处理的构建体导致缺陷部位内神经肌肉接头、肌纤维再生、血管浸润、神经浸润和核 yes 相关蛋白 (YAP) 表达显着增加，具有统计学意义与没有集聚蛋白的对照相比。与具有可溶性集聚蛋白的构建体相比，集聚蛋白系留的微纤维束在植入后4周期间在再生位点内提供持续的集聚蛋白信号传导，并进一步增加了再生组织中再生肌纤维的密度，具有统计学显着性。 这些数据证明了经过预处理的工程肌肉结构的神经肌肉再生潜力，以在 VML 再生部位用局部递送的 agrin 诱导 AChR 聚集。