Microcontact printing of fibronectin on a biodegradable polymeric surface for skeletal muscle cell orientation

Background and Objectives: Micropatterning and microfabrication techniques have been widely used to control cell adhesion and proliferation along a preferential direction according to contact guidance theory. One of these techniques is microcontact printing, a soft lithographic technique based on the transfer of a "molecular ink" from an elastomeric stamp to a surface. This method allows the useful attachment of biomolecules in a few seconds on a variety of surfaces with sub-micrometer resolution and control, without modifying the biomolecule properties. The aim of this study is to develop an easy and versatile technique for in vitro production of arrays of skeletal muscle myofibers using microcontact printing technique on biodegradable substrata. Methods: Microcontact printing of fibronectin stripes (10, 25, 50 mu m in width) was performed onto biodegradable L-lactide/trimethylene carbonate copolymer (PLLA-TMC) films. C2C12, a murine myoblast cell line, was used for the production of parallel myofibers. Results: This approach proved to be simple, reliable and effective in obtaining a stable pattern of fibronectin on the PLLA-TMC surface as observed by fluorescence microscopy. C2C12 cells were well aligned along the pattern 24 hours after seeding, especially on fibronectin stripes 10 and 25 mu m in width. Seven days after confluence cells fused and formed aligned multinucleated cells expressing alpha-actinin. Conclusions: Fibronectin patterning seems to be a useful method to induce cell alignment and to improve myotube formation. Further studies will be focused on the possibility of applying external stimuli to these structures to obtain healthy myotubes and to induce myofiber development.