Modulation of alignment and differentiation of skeletal myoblasts by biomimetic materials

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. We developed a novel cell culture technique that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering and more micro-patterning techniques and surface engineering have been tested by switching differentiating myotubes from growth medium (GM) to differentiative media (DM). One of the goals of tissue engineering is to develop tools allowing in vitro construction and mimicking of the final tissue architectures. The fabrication of polyelectrolyte multilayers (PEMs) may represent a promising approach for recreating physiological nanometer-sized cell environments in vitro. In this study we describe a method for generating biomimetic microstructured surfaces that promote cell adhesion and differentiation of parallel arrays of mature C2C12 myotubes continuously maintained in GM for 7 days. The structure consists of a 'double-sheet' PDMS structure that provides 'compliant' or 'stiff' microdomains to guide the cell self-patterning, coupled to layer-by-layer (LbL) self-assembled multilayers of biocompatible polyelectrolytes that promote C2C12 myoblasts alignment and differentiation. Our findings have relevance to the interpretation of in vitro data as well as to the study of cellular interactions with biomaterials.