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Myoblasts are precursor muscle cells that lie nascent to mature skeletal muscle. Once muscle is damaged, these cells migrate, fuse, and regenerate the muscle tissue. It is known that skeletal muscle can partially regenerate in vivo after muscle tissue damage. However, this regeneration does not always occur, especially in more severe injuries. Cellular therapy using tissue-engineering approaches has been shown to improve organ repair and function. To exploit potential benefits of using cell therapy as an avenue for skeletal muscle repair, it is important to understand the cellular dynamics underlying skeletal myocyte formation and growth. Cardiac fibroblasts have been shown to have a major influence on cardiomyocyte function, repair, and overall spatial distribution. However, little is known regarding fibroblasts' role on skeletal myocyte function. In this study, we utilized a reconfigurable co-culture device to understand the contact and paracrine effects of fibroblasts on skeletal myocyte alignment and differentiation using murine myoblast and fibroblast cell lines. We demonstrate that myotube alignment is increased by direct contact with fibroblasts, while myotube differentiation is reduced both in the gap and contact configurations with fibroblasts after 6 days of co-culture. Furthermore, neutralizing antibodies to FGF-2 can block these effects of fibroblasts on myotube differentiation and alignment. Finally, bi-directional signaling is critical to the observed myoblast-fibroblast interactions, since conditioned media could not reproduce the same effects observed in the gap configuration. These findings could have direct implications on cell therapies for repairing skeletal muscle, which have only utilized skeletal myoblasts or stem cell populations alone.

Fibroblasts influence muscle progenitor differentiation and alignment in contact independent and dependent manners in organized co-culture devices