Muscle differentiation and myotubes alignment is influenced by micropatterned surfaces and exogenous electrical stimulation.

An in vitro muscle-like structure with parallel-oriented contractile myotubes is needed as a model of muscle tissue regeneration. For this purpose, it is necessary to reproduce a controllable microscale environment mimicking the in vivo cues. In this work we focused on the application of topological and electrical stimuli on muscle precursor cell (MPC) culture to influence MPC orientation and induce myotube alignment. The two stimulations were tested both independently and together. A structural and topological template was achieved using micropatterned poly-(L-lactic acid) membranes. Electrical stimulation, consisting of square pulses of 70mV/cm amplitude each 30 s, was applied to the MPC culture. The effect of different pulse durations on cultures was evaluated by galvanotaxis analysis. The highest cell displacement rate toward the cathode was observed for 3 ms pulse stimulation, which was then applied in combination with topological stimuli. Topological and electrical stimuli had an additive effect in enhancing differentiation of cultured MPC, shown by high Troponin I protein production and, in parallel, Myogenin and Desmin genes, down-and upregulation respectively