Engineering Vascularized Skeletal Muscle for Treatment of Volumetric Muscle Loss

容积性肌肉损失(VML)的特征是相当大一部分的肌肉损失 骨骼肌损伤，导致肌肉结构和功能的永久性损伤。 VML是由严重的创伤性损伤引起的，而且越来越频繁 因路边爆炸、枪伤和汽车造成的退伍军人 车辆相撞。VML有助于长期残疾和4000亿美元的经济 美国每年的负担。导致VML的创伤性损伤与 内源性肌肉再生和血运重建能力受损。当前 外科手术如肌瓣移植或疤痕组织清创术 与严重的供体部位发病率和功能缺陷有关。实验 使用脱细胞细胞外基质支架的方法在 肌肉恢复。因此，一种能够恢复正常的组织工程系统 对于VML的治疗，骨骼肌的结构和功能仍然缺乏。自.以来 骨骼肌一般由一束平行排列的肌纤维组成。 穿插着为肌纤维提供血液和氧气的血管， 该提案的长期目标是设计血管化的骨骼肌组织 模仿天然肌肉和血管结构的结构，以恢复肌肉 在VML之后执行函数。 这项研究的目的是生物工程骨骼肌组织组成的 骨骼肌前体细胞与血管内皮细胞平行排列 增强细胞存活、肌纤维形成和血管形成的纳米纤维支架 VML小鼠模型的血流灌注恢复。由于血管的重要性 在血流灌注恢复后，支架还将被设计成释放血管新生生长 以修饰的信使核糖核酸(MmRNA)的形式存在的因子，避免了基因组的改变。 提议的目标旨在促进对如何 与平行排列的纳米纤维支架的细胞间相互作用以及瞬时 传递治疗性的mmRNA，可以促进肌肉和血管的再生。 相应地，具体目标是：(1)工程内皮化对齐 骨骼肌由肌肉前体细胞和内皮细胞排列而成 增强细胞存活、肌管形成和收缩的纳米纤维支架 体外功能；(2)增强血管内皮化和平行化的血管生成能力。 利用支架介导的mmRNA传递的排列的工程骨骼肌；以及(3) 内皮化排列工程骨治疗效果的量化研究 在VML小鼠模型中使用瞬时治疗性mmRNAs的肌肉。这个 拟议的研究具有非常重要的意义，因为它们寻求改善治疗 细胞移植治疗VML的好处，从 脱细胞支架植入预制内皮化肌肉组织的实验研究 具有瞬时基因传递的构建物可改善退伍军人和 其他VML患者。

Volumetric muscle loss (VML) is characterized by the loss of a significant portion of skeletal muscle, leading to permanent damage to muscle structure and function. VML results from major traumatic injury, and it is becoming increasingly more frequent in military Veterans as a result of roadside explosions, gunshot wounds, and motor vehicle crashes. VML contributes to long-term disability and $400 billion in economic burden in the US annually. Traumatic injuries leading to VML are associated with impaired endogenous muscle regeneration and revascularization capacity. Current surgical interventions such as muscle flap grafting or scar tissue debridement are associated with significant donor site morbidity and functional deficiency. Experimental approaches using decellularized extracellular matrix scaffolds show limited benefit in muscle recovery. Accordingly, a tissue engineering system that can restore normal skeletal muscle structure and function remains lacking for treatment of VML. Since skeletal muscle is composed generally of a bundle of parallel-aligned myofibers interspersed with blood vessels that provide blood and oxygen to the myofibers, the long-term goal of this proposal is to engineer vascularized skeletal muscle tissue constructs that mimic the native muscle and vessel structure, in order to restore muscle function after VML. The purpose of this study is to bioengineer skeletal muscle tissue composed of skeletal muscle precursor cells and vascular endothelial cells in a parallel-aligned nanofibrillar scaffold that augments cell survival, myofiber formation, and vascular perfusion recovery in a murine model of VML. Owing to the importance of vascular perfusion recovery, the scaffolds will also be engineered to release angiogenic growth factors in the form of modified mRNA (mmRNA), which obviates genomic alterations. The proposed objectives are designed to advance the understanding of how intercellular interactions with parallel-aligned nanofibrillar scaffolds, along with transient delivery of therapeutic mmRNA, can promote muscle and vascular regeneration. Accordingly, the Specific Aims are: (1) To engineer endothelialized aligned skeletal muscle composed of muscle precursor cells and endothelial cells in an aligned nanofibrillar scaffold that augments cell survival, myotube formation, and contractile function in vitro; (2) To enhance the angiogenic capacity of endothelialized and parallel- aligned engineered skeletal muscle using scaffold-mediated mmRNA delivery; and (3) To quantify the therapeutic efficacy of endothelialized and aligned engineered skeletal muscle with transient therapeutic mmRNA delivery in a murine model of VML. The proposed studies are highly significant because they seek to improve the therapeutic benefit of cell transplantation for treatment of VML, shifting away from the transplantation of acellular scaffolds to pre-formed endothelialized muscle tissue constructs with transient gene delivery for improved clinical outcomes in Veterans and other patients with VML.