Subtle variations in polymer chemistry modulate substrate stiffness and fibronectin activity

A family of polymer substrates which consists of a vinyl backbone chain with the side groups -COO(CH2)(x)CH3, with x = 0, 1, 3, 5 was prepared. Substrates with decreasing stiffness, characterised by the elastic modulus at 37 degrees C, and similar chemical groups were obtained. Firstly, we have investigated whether these minute variations in polymer chemistry lead to differences in fibronectin (FN) adsorption: the same FN density was obtained on every substrate (450 ng cm(-2)) but the supramolecular organisation of the protein at the material interface, as obtained with AFM, was different for x = 0 and the other surfaces (x = 1, 3, 5). Consequently, this allows one to use a set of substrates (x = 1, 3, 5) to investigate the effect of substrate stiffness on cell behavior as the unique physical parameter, i.e. after ruling out any influence of the length of the side group on protein conformation. Moreover, the importance of investigating the intermediate layer of proteins at the cell-material interface is stressed: the effect of x = 0 and x = 1 on cell behavior cannot be ascribed to the different stiffness of the substrate anymore, since the biological activity of the protein on the material surface was also different. Afterwards, initial cellular interaction was investigated using MC3T3-E1 osteoblasts-like cells and focusing on actin cytoskeleton development, focal adhesion formation and the ability of cells to reorganize the adsorbed FN layer on the different substrates. Image analysis was used to quantify the frequency distribution of the focal plaques, which revealed broader distributions on the stiffer substrates, with formation of larger focal plaques revealing that cells exert higher forces on stiffer substrates.