Controlling macroscale cell alignment in self-organized cell sheets by tuning the microstructure of adhesion-limiting micromesh scaffolds

In vivo tissues, including the cardiac, skeletal muscles, tendon and ligaments display characteristic alignment property which is important for their mechanical property and functionality. Mimicking this alignment property is critical to the realization of physiologically relevant cell sheets for potential application in the regeneration of aligned in vivo tissues. In this study, we aimed to achieve fabrication of aligned cell sheets by harnessing the ability of cells to sense and respond to geometrical cues in their adhesion microenvironment. We demonstrate that macroscale cell alignment in cell sheets formed by C2C12 cells, a mouse myoblast cell line, on adhesion limiting microstructured mesh scaffolds depends on the shape of the scaffold microstructure. Specifically, while square meshes produced cell sheets with random orientation, diamond meshes yielded anisotropic cell sheets with cells aligned uniaxially along the major axis of the diamond shape. Moreover, alignment intensity was found to increase concomitantly with the acuteness of the diamond shape, illustrating alignment dependency on mesh shape anisotropy. Remarkably, myotubes derived from aligned C2C12 cells also displayed a similar alignment trend, demonstrating the robustness of our approach. Taken together, the present study demonstrates the potential to control macroscale cell alignment in self-organized cell sheets by tuning the shape of the scaffold microstructure. Thus, insights from this study could be relevant to the design of instructive scaffolds for fabricating aligned cell sheets for potential application not only in regenerative medicine, but also in developing muscle constructs for toxicity assays