Engineering muscle tissues on microstructured polyelectrolyte multilayer films

The use of surface coating on biomaterials can render the original substratum with new functionalities that can improve the chemical, physical, and mechanical properties as well as enhance cellular cues such as attachment, proliferation, and differentiation. In this work, we combined biocompatible polydimethylsiloxane (PDMS) with a biomimetic polyelectrolyte multilayer (PEM) film made of poly(L-lysine) and hyaluronic acid (PLL/HA) for skeletal muscle tissue engineering. By microstructuring PDMS in grooves of a different width (5, 10, 30, and 100 microm) and by modulating the stiffness of the (PLL/HA) films, we guided skeletal muscle cell differentiation into myotubes. We found optimal conditions for both the formation of parallel-oriented myotubes and their maturation. Significantly, the myoblasts were collectively prealigned to the grooves before their differentiation. Before fusion, the highest aspect ratio and orientation of nuclei were observed for the 5 and 10 microm wide micropatterns. The formation of myotubes was observed regardless of the size of the micropatterns, and we found that their typical width was 10-12 microm. Their maturation was characterized by the immunolabeling of type II isomyosin. The amount of myosin striation was not affected by the topography, except for the 5 microm wide micropatterns. We highlighted the spatial constraints that led to an important nuclei deformation and further impairment of maturation within the 5 microm grooves. Altogether, our results show that the PEM film combined with PDMS is a powerful tool that is used for skeletal muscle engineering. This work opens perspectives for the development of skeletal muscle tissue in contact with films containing bioactive peptides or growth factors as well as for the study of pathogenic myotubes.