Topographic guidance of endothelial cells on silicone surfaces with micro- to nanogrooves: Orientation of actin filaments and focal adhesions

To mimic the uniformly elongated endothelium in natural linear vessels, bovine aortic endothelial cells (BAECs) are cultured on micro- to nanogrooved, model poly(dimethylsiloxane) (PDMS) substrates preadsorbed with about 300 ng/cm² of fibronectin. BAEC alignment, elongation, and projected area were investigated for channel depths of 200 nm, 500 nm, 1 μm, and 5 μm, as well as smooth surfaces. Except for the 5 μm case, the ridge and channel widths were held nearly constant about 3.5 μm. With increasing channel depth, the percentage of aligned BAECs increased by factors of 2, 2, 1.8, and 1.7 for 1, 4, 24, and 48 h. Maximum alignment, about 90%, was observed for 1 μm deep channels at 1 h. The alignment of BAECs on grooved PDMS was maintained at least until cells reached near confluence. F-actin and vinculin at focal adhesions also aligned with channel direction. Analysis of confocal microscopy images showed that focal adhesions localized at corners and along the sidewalls of 1-μm deep channels. In contrast, focal adhesions could not form on the bottom of the 5-μm deep channels. Cell proliferation was similar on grooved and smooth substrates. In summary, PDMS substrates engraved with micro- and nanochannels provide a powerful method for investigating the interplay between topography and cell/cytoskeletal alignment.