Responses of human keratocytes to micro- and nanostructured substrates

We have previously shown that human corneal epithelial cells respond to synthetic topographic features with dimensions similar to those found in the native human corneal basement membrane. Epithelial cells integrated inputs from substrate topography and soluble factors in the culture medium to generate alignment responses to substrate topographic anisotropies. Human keratocytes are the main cellular components of the stroma, the tissue that underlies the corneal epithelium. Here we report that keratocytes aligned more strongly than epithelial cells along topographic patterns of grooves and ridges. On patterns with pitches of 800 nm and larger approximately 70% of keratocytes were aligned along the patterns compared to 35% for epithelial cells. On 70 nm-wide ridges on a 400-nm pitch, keratocyte alignment dropped to 45%, whereas epithelial cell alignment remained constant. Similarly to epithelial cells, focal adhesions and associated stress fibers in keratocytes were aligned mainly along the substrate topographies, although oblique orientations were also observed. Furthermore, keratocytes cultured on the nanoscale patterns had fewer stress fibers and focal adhesions than cells cultured on microscale patterns or on smooth substrates.