The Role of Fibroblasts in Self-Assembled Skeletal Muscle

Abstract

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.

Implanted Scaffold-Free Prevascularized Constructs Promote Tissue Repair

“…This hypothesis is supported by the inherent regenerative capacity of skeletal muscle conferred by satellite cells 9,11–13 ; the observed inverse relationship between muscle wound size and vascularization/degree of wound healing; and the fact that extracellular matrix proteins synthesized by our constructs are present in the satellite cell microenvironment and implicated in their activation 9,10,13,14 .…”

“…Arranged into bundles of multinucleated myofibers, skeletal muscle has an inherent regenerative capacity due to the presence of resident mononuclear stem cells, called satellite cells. Satellite cells reside between the basal lamina – a region rich in laminin 9,10 – and the sarcolemma (myofiber plasma membrane) 11 . In addition to a role in regulating muscle turnover during homeostasis, activated satellite cells have been shown to be required for restoration of skeletal muscle tissue function following injury 12 .…”

Cell Density and Joint microRNA-133a and microRNA-696 Inhibition Enhance Differentiation and Contractile Function of Engineered Human Skeletal Muscle Tissues

“…Fibroblasts help stabilize engineered muscle tissue and produce important ECM. 35 While Duisters et al reported that levels of miR-133 were 15-fold greater in rat cardiac myocytes compared to rat fibroblasts, 36 the passive force in skeletal muscle myobundles was not sensitive to the anti-miR treatment (Fig. 5B) and transfection of fibroblasts with the anti-miRs did not affect compaction of bundles prepared only with fibroblasts.…”

Factors That Affect Tissue-Engineered Skeletal Muscle Function and Physiology

“…The first reported mouse constructs were created using the myoid technique and resulted in greater force production than rat MPCs (19.2 vs. 2.9 kN/m 2 ) [Dennis et al, 2001]. Using fibrin casting, Li et al [2011] showed that coculture with mouse embryonic fibroblasts was required for more complete remodeling and degradation of fibrin, suggesting that mouse myoblasts show a decreased ability to remodel and secrete sufficient ECM. In contrast to Li et al [2011] [Lee and Vandenburgh, 2013], approximately 3 orders of magnitude higher than we have reported in fibrin/Matrigel hydrogels.…”

“…Using fibrin casting, Li et al [2011] showed that coculture with mouse embryonic fibroblasts was required for more complete remodeling and degradation of fibrin, suggesting that mouse myoblasts show a decreased ability to remodel and secrete sufficient ECM. In contrast to Li et al [2011] [Lee and Vandenburgh, 2013], approximately 3 orders of magnitude higher than we have reported in fibrin/Matrigel hydrogels. However, when engineering muscle from the same cell isolation using collagen/Matrigel and fibrin/ Matrigel hydrogels, we have found that fibrin/Matrigel constructs produce approximately 5 times higher force ( table 1 ).…”