Fibronectin activity on substrates with controlled OH density

Abstract

Adhesion of human fibroblast to a family of fibronectin (FN) coated model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios to obtain a controlled surface density of --OH groups was investigated. Cell adhesion and spreading surprisingly decreased as the fraction of --OH groups on the surface increased. AFM studies of FN conformation revealed formation of a protein network on the more hydrophobic surfaces. The density of this network diminished as the fraction of --OH groups in the sample increased, up to a maximal --OH concentration at which, instead of the network, only FN aggregates were observed. The kinetics of network development was followed at different adsorption times. Immunofluorescence for vinculin revealed the formation of well-developed focal adhesion complexes on the more hydrophobic surface (similar to the control glass), which became less defined as the fraction of --OH groups increased. Thus, the efficiency of cell adhesion is enhanced by the formation of FN networks on the substrate, directly revealing the importance of the adsorbed protein conformation for cell adhesion. However, cell-dependent reorganization of substrate-associated FN, which usually takes place on more hydrophilic substrates (as do at the control glass slides), was not observed in this system, suggesting the increased strength of protein-to-substrate interaction. Instead, the late FN matrix formation--after 3 days of culture--was again better pronounced on the more hydrophobic substrates and decreased as the fraction of --OH groups increase, which is in a good agreement with the results for overall cell morphology and focal adhesion formation.