Collagen nanofibre anisotropy induces myotube differentiation and acetylcholine receptor clustering.

To create musculoskeletal tissue scaffolds for functional integration into host tissue, myotubes must be properly aligned with native tissue and spur the formation of neuromuscular junctions. However, our understanding of myoblast differentiation in response to structural alignment is incomplete. To examine how substrate anisotropy mediates myotube differentiation, we studied C2C12 myoblasts grown on aligned collagen substrates in the presence or absence of agrin. Myoblasts grown on microfluidically patterned collagen substrates demonstrated increased multinucleated myotubes and nicotinic acetylcholine receptor (AChR) clusters. However, agrin treatment did not synergistically increase differentiation of myoblasts seeded on these patterned collagen substrates. Myoblasts grown on aligned electrospun collagen nanofibres also demonstrated increased formation of multinucleated myotubes and AChR clusters, and agrin treatment did not increase differentiation of these cells. Using fluorescently labelled collagen nanofibres, we found that AChR clustered in cells grown on nanofibres with significantly higher anisotropy and that this clustering was eliminated with agrin treatment. Interestingly, anisotropy of substrate had no effect on the localization of AChRs along the myotube, suggesting that additional signalling pathways determine the specific location of AChRs along individual myotubes. Taken together, our results suggest a novel role for fibre anisotropy in myotube differentiation, specifically AChR clustering, and that anisotropy may guide differentiation by activating similar pathways to agrin. Our data suggest that agrin treatment is not necessary for differentiation and maturation of myoblasts into myotubes when myoblasts are grown on aligned collagen substrates.