Manufacturing substrate nano-grooves for studying cell alignment and adhesion

Nano-scale pattern templates have been manufactured in order to study the differences in cell behaviour between fibroblasts cultured on smooth and on grooved substrata. The pattern templates were made on silicon wafers using electron beam lithography in hydrogen silsesquioxane (HSQ) and subsequent reactive ion etching (RIE). These masters were replicated in polystyrene cell culture material using solvent casting. The replicas were assessed with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. It appears that both groove depth and width determine the cellular alignment on patterns with a ridge/groove ratio of 1:1. On smooth substrata, cells always spread out in a random fashion. There appears to be a threshold groove barrier size of around 70-80 nm, above which random cell spreading is not possible anymore and contact guidance occurs. It is speculated that this threshold size may be associated with the size of contact molecules at the cell extensions, which grow and find anchoring spots preceding cell spread out and cell alignment.