Alignment of multi-layered muscle cells within three-dimensional hydrogel macrochannels

This work describes the development and testing of poly(ethylene glycol) (PEG) hydrogels with independently controlled dimensions of wide and deep macrochannels for their ability to promote alignment of skeletal myoblasts and myoblast differentiation. A UV-photopatterned thiol-ene mold was employed to produce long channels, which ranged from similar to 40 to 200 mu m in width and from similar to 100 to 200 mu m in depth, within a PEG-RGD hydrogel. Skeletal myoblasts (C2C12) were successfully cultured multiple cell layers deep within the channels. Decreasing channel width, increasing channel depth and, interestingly, increasing cell layer away from the channel base all contributed to a decreased interquartile range of cell angle relative to the long axis of the channel wall, indicating improved cell alignment. Differentiation of skeletal myoblasts into myotubes was confirmed by gene expression fcr myoD, myogenin and MCH Ilb, and myotube formation for all channel geometries, but was not dependent on channel size. Qualitatively, myotubes were characteristically different, as myotubes were larger and had more nuclei in larger channels. Overall, our findings demonstrate that relatively large features, which do not readily facilitate cell alignment in two dimensions, promote cell alignment when presented in three dimensions, suggesting an important role for three-dimensional spatial cues. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.