Figure 4. Cellular organization.

Samples were stained for f-actin (green), nuclei (blue), and either muscle-specific myosin heavy chain (red in panels a, b, and c) or gap-junction-specific connexin-43 (red in d, e, and f) to visualize cellular structures. The top image in each pair shows a merged, three-color fluorescence micrograph obtained at low magnification to show typical staining patterns of cells cultured on TCPS (a, d), isotropic ES-PU microfibers, and aligned ES-PU microfibers. The bottom image in each pair shows a 4x zoomed view of the region indicated by the rectangle in each upper image to allow visualization of sub-cellular structures. Cells seeded on TCPS (a, d) and isotropic ES-PU fibers (b, e) were substantially less oriented than those grown on aligned ES-PU microfibers (c, f). In all cases, muscle cells (black asterisks) and non-muscle cells (white asterisks) were present. Striations from organized sarcomeric proteins in cardiomyocytes (white arrow heads) were prevalent in all samples; however, these were generally more difficult to resolve in ES-PU samples due to apparent lensing effects from the underlying biomaterial. Cx-43 staining of gap junctions was also prevalent (black arrowheads). Scale bars represent 20 μm.