Abstract
Tissue engineered cell sheets composed of oriented muscle fibers differentiated from myoblasts can be regarded as more suitable for the regeneration of skeletal muscles. A novel method for fabricating monolayer cell sheets by culturing cells on a suspended mesh patterned structure consisting of narrow mesh lines (< 5 μm) and large apertures (> 100 μm) was developed in our laboratory. In this study, we investigated the influence of mesh shape on the orientation of myoblast and myotubes derived from them. To do this, we cultured C2C12 (mouse myogenic cell line) on fibronectin-coated mesh patterned structure and differentiated them to myotubes after confluency was reached. Using a diamond shaped mesh with a 100 nm long minor axis and a minor angle of 70°, we obtained myoblast cell sheets with cells oriented in the longest axis, in accordance with our previous studies. Importantly, myotubes derived from the oriented myoblasts were also similarly oriented, suggesting that lengthwise fusion as opposed to sidewise fusion occurs predominantly in orientated myoblasts. Using Fluo-4 indicator, we monitored calcium dynamics in the oriented myotubes and found that some myotubes displayed a unique calcium wave that travelled from end-to-end at a speed of 10-30 μm/s. This calcium dynamics was clearly distinct from the typical calcium spike displayed by a majority of myotubes. In our future studies, we will investigate the relationship between this calcium dynamics and the beating motion also observed in some oriented myoblast.
