Mammalian cells are sensitive to the physical properties of their micro-environment such as the stiffness and geometry of the substrate. It is known that the stiffness of the substrate plays a key role in the process of mammalian myogenesis. However, the effect of geometrical constraints on the process of myogenic differentiation needs to be explored further. Here, we show that the geometrical cues of substrates can significantly influence the differentiation process of C2C12 skeletal myoblasts. Three different geometries including lines of different widths, tori of different inner diameters, and hybrid structures (linear and circular features with different arc degrees) were created by micro-contact printing of fibronectin on the surface of Petri dishes. The differentiation of C2C12 cells was studied over a period of seven days and was quantified; we report the differentiation parameters of (1) fusion index, (2) degree of maturation, (3) alignment, and (4) response to electrical pulse stimulation (EPS). Hybrid structures with the smallest arc degree (hybrid 30°) showed the best results for all four differentiation parameters. The hybrid 30° pattern exhibits an ~2-fold increase in the fusion index when compared to the line patterns and an ~3-fold increase when compared to the toroid patterns. The hybrid 30° also showed a higher maturation index compared to the line or the toroid patterns. In response to electrical stimulation (20 V, 50 ms pulse, 1 Hz), mature myotubes on hybrid 30° patterns showed an ~2-fold increase in cellular displacement when compared to myotubes on the line and torus patterns. We tested the influence of C2C12 cell density on fusion and maturation indices, and the results suggest that density does not exert significant influence on cellular differentiation under these conditions. Our results can have important implications in engineering skeletal muscle tissues and designing muscle cell bio-actuators.
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