In Vitro Assessment of Scaffoldless Tissue‐Engineered Skeletal Muscle for Volumetric Muscle Loss Repair

Abstract

Volumetric muscle loss (VML) is the traumatic loss of skeletal muscle resulting in damage that overwhelms the body's capacity for self‐repair, leading to functional impairment. Thus, the need for technologies that promote regeneration of skeletal muscle fibers to integrate with remaining muscle architecture is imperative. Our lab has developed scaffoldless, tissue‐engineered skeletal muscle units (SMUs) for VML treatment in sheep. Additionally, in order to ensure proper innervation of the SMU myofibers, we developed an engineered neural conduit (ENC) to bridge the SMU and the damaged nerve. This study aims to develop a fabrication method for SMUs and ENCs that restore function following an acute VML injury in a more clinically relevant, load‐bearing model in sheep. Ovine bone marrow stromal cells (BMSCs) were harvested and used to fabricate ENCs. Before 3‐D formation, silicone tubing was pinned in the tissue culture dish to allow the delaminating monolayer to roll around the tubing, creating a lumen (Fig 1A.). For SMU fabrication, semimembranosus muscle was harvested from female lambs and the cell isolation mixture was plated onto tissue culture plastic. When elongating myotubes began to form a network, the plates were shifted to differentiation media until spontaneous delamination of the monolayer occurred. The monolayers were then pinned into 3‐D cylindrical constructs and 2–3 single constructs were placed side‐by‐side and allowed to fuse (Fig 1C.). 2–3 fused SMUs were then sutured together, just prior to implantation (Fig. 1B). A subset of SMUs, fabricated on 60mm dishes, were used to measure contractile and structural properties. All animal procedures were conducted in accordance with The Guide for Care and Use of Laboratory Animals. The biomechanical properties of the SMUs indicated that on average the isometric tetanic force was 657 ± 667 μN. Structural maturation of the constructs was evaluated histologically with H&E, myosin heavy chain, and laminin. Images of developing monolayers were taken 10 days after initial plating and showed a highly aligned and dense myotube network without fibroblast overgrowth. Immunohistochemistry for myosin heavy chain (MF‐20) and laminin showed that the construct is largely composed of aligned muscle. Picrosirius red staining revealed that the ENC's are mostly composed of collagen. We evaluated the development, structure, and function of our SMUs and ENCs throughout the fabrication process. We were successfully able to fabricate and implant 60 SMUs, all 13cm long and 5–10mm in diameter. The biomechanical data showed that we were able to consistently fabricate constructs that met our release criteria for force production. It is important to note that improper alignment of the fibers along the longitudinal axis may have reduced potential force production of the constructs and account for the variability in force data. Histology revealed that the overall structure of the constructs is linear and that the core is not necrotic, indicated by the presence of positively stained‐DAPI...