Small facial skeletal muscles often have no autologous donor source to effect surgical reconstruction. Autologously derived muscles could be engineered for replacement tissue, but must be vascularized and innervated to be functional. As a critical step, engineered muscle must mimic the morphology, protein and gene expression, and function of native muscle. This study utilized a self-assembly process to engineer three-dimensional (3D) muscle from a statically strained muscle cell monolayer. Primary mouse myoblasts (PMMs) and mouse embryonic fibroblasts (MEFs) were separately proliferated and coseeded on a fibrin sheet with anchored sutures. Within 10 days of initiating PMM differentiation, the cell-gel layer contracted, lifted, and rolled into a cylindrical 3D structure around the tendon-like suture anchors; the myotubes longitudinally aligned along the lines of tensile force. The objectives of this study were to characterize these engineered muscles and to elucidate the role of the fibroblasts in the self-assembly process. Fibroblasts maintained myotube viability, mediated fibrin degradation, and assisted in muscle self-assembly. The optimal 1:1 PMM:MEF ratio resulted in tissue morphology remarkably similar to native muscle. Through gene and protein expression assays, the development and maturation of the engineered muscle tissue was demonstrated to recapitulate normal skeletal muscle development.
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