Preferential adhesion to and survival on patterned laminin organizes myogenesis in vitro

We have examined a potential role for differential adhesiveness in muscle development using an in vitro model which employed the culture of myoblasts and myotubes, (conditionally immortal myogenic cells, H2k(b)-tsA58), on micropatterned surfaces. These surfaces are made up of multiple alternating tracks of hydrophobic organosilane-treated glass and untreated glass (track width ranging from 5 to 100 microm). We found that myoblasts were aligned on patterns in the presence of serum, by adhering to the tracks of untreated glass, which had preferentially adsorbed serum attachment factors. However, as serum attachment factors are not sufficient for maintenance of adhesion of mature myotubes, we determined whether precoating patterns with laminin, which maintains adhesion, could still provide a differential adhesive cue. Laminin preferentially adsorbs to the hydrophobic regions resulting in alternating tracks that have adsorbed laminin or serum attachment factors. Myoblasts were less well aligned on these patterns as they could adhere both to the untreated glass and to laminin on the previously hydrophobic tracks, but did show a preference for laminin. However, cell alignment increased upon differentiation into myotubes and continued to increase as the myotubes matured. We found that the alignment of myoblasts and myotubes on patterns increased as track width increased. In addition, adhesion to laminin was required for long term survival of the myotubes. Myotubes that had formed on nonlaminin surfaces began to detach after 2 days of differentiation. Although we found that myoblasts preferentially clustered on laminin tracks, this arrangement did not influence the diameter of the myotubes formed, upon differentiation. Instead, the number of myotubes per track increased with track width, while the myotube diameter remained constant. This uniformity of myotube diameter suggests that a mechanism exists which restricts the ability of myoblasts to undergo lateral fusion. Overall, these findings suggest that differential adhesiveness could be an important mechanism for formation and survival of myotubes, and by using these patterns we have demonstrated a mechanism controlling the formation of linear myotubes by restricting the geometry of cell-cell adhesion.