In this study, we have shown that micropatterned substrates can regulate myoblast organization, myotube formation, and F-actin assembly. The parallel microgrooves of PDMS effectively aligned the myotubes within 20° from the direction of the microgrooves, in agreement with previous reports [5
]. Moreover, our data shows that micropatterned topography can control myoblast morphogenesis into aligned myotubes. On micropatterned PDMS, the myoblasts attached and fused into myotubes that aligned in close proximity to the direction of the channels. After two days in differentiation media, the percentage of nuclei that incorporated into myotubes on micropatterned PDMS was significantly higher when compared to non-patterned surfaces, suggesting that micropatterned substrates could enhance myotube formation at early time points. However, the enhancement of myotube incorporation on micropatterned substrates did not appear to be modulated by contractile markers, since the mRNA and protein levels of contractile markers were not significantly different between the two groups.
On the other hand, the differential organization of N-cadherin may account for differences in the fusion process on micropatterned substrates. The extracellular domain of N-cadherin mediates cell-cell adhesion through homophilic interactions, whereas the cytoplasmic domain interacts with catenins to anchor to actin filaments [36
]. In previous studies, the role of N-cadherin in myogenesis was demonstrated by the inhibition of myoblast fusion in the presence of N-cadherin antibodies [38
], whereas forced expression of N-cadherin in fibroblast-like cells stimulated myogenic protein expression [39
]. It is likely that the organization of N-cadherin may influence cell-cell adhesion and actin assembly among myoblasts and thereby regulate the fusion process. In addition to cell-cell adhesion, the increase of p21WAF/Cip1
could arrest cell cycle and facilitate myoblast differentiation and fusion.
The micropatterned substrates were designed to incorporate features that may be important for in vitro
muscle formation. As a flexible elastomer, PDMS can be stretched to simulate the mechanically loaded muscle environment in vivo
]. Furthermore, unlike glass substrates, PDMS is deformable, but have tensile strength to withstand dynamic loading environments in vivo
. The spacing between microgrooves were selected to be 10- to 50- μm wide based on our experience that myoblast alignment decreases with increasing groove width (>100 μm; data not shown), and this finding is supported by observations from other researchers [41
]. Since micropatterned surfaces resemble the in vivo
cellular arrangement better than non-patterned substrates, they can be useful for studying myogenesis or engineering aligned muscle for muscle repair. Furthermore, as inverse templates, the PDMS can be used to deposit pHEMA microgrooves onto Petri dishes or biological substrates such as polymer films.
Besides direct culture on micropatterned PDMS, we showed that pHEMA microgrooves could also efficiently control myoblast orientation and generate aligned myotubes. Since the pHEMA microgrooves were found to detach over time, this approach has useful applications for engineering aligned myotubes with limited dwelling time of the biomaterial. The utilization of microfabrication techniques for temporary spatial guidance has also been demonstrated by others. For example, Lam et al
. formed aligned myotubes on micropatterned PDMS before transferring the aligned myotubes onto fibrin hydrogels, generating aligned skeletal muscle hydrogel constructs [4
In addition to myoblasts, microfabrication techniques have been applied to other cell types. In our previous work using adhesive matrix micropatterning as well as topographical patterning, we showed that vascular smooth muscle cells undergo morphological and phenotypic changes in vitro
]. On collagen-patterned glass slides, as well as on topographically patterned PLGA polymer films, smooth muscle cells decreased in spreading area, proliferation, and actin fiber assembly. On microgrooves, cardiac myocytes aligned in the direction of the microgrooves and expressed connexin-43 and N-cadherin around the cell perimeter [42
]. Embryonic neurons exhibited a differential response to micropatterning such that neuronal processes extended in parallel to the grooves when cultured on deep and wide channels, while on reduced dimensions, neuronal process were directed either in parallel or perpendicular to the axis of the grooves [43
]. These studies provide evidence of an association between surface topography and cellular response among numerous types of cells.
In summary, we have demonstrated that micropatterned PDMS or pHEMA can promote cell alignment and fusion along the direction of the microgrooves, and this platform can be utilized to transfer aligned myotubes on biodegradable hydrogels. The results from this study highlight the importance of spatial cues in creating aligned skeletal muscle for tissue engineering and muscular regeneration applications.