Control of protein adsorption, cell morphology and intracellular signal transduction using self-assembled monolayers of alkylthiolates on gold

Abstract

Self-assembled monolayers of terminally-functionaiized alkylthiolates on gold have been used to interrogate cell-biomaterial surface interactions at the extracellular and intracellular level. The goal of this research was to provide molecular level information on the surface determinants necessary to produce predictable, controllable biological responses to implanted materials. Various players in "outside-in" communication between a cell and a material's surface were investigated. Surfaces of a variety of alkylthiol chemistries were used to monitor protein adsorption and subsequent Swiss 3T3 fibroblast adhesion, spreading and growth. Distinct alkylthiol substrate chemistry-dependent differences were observed. Formation of focal contacts and stress fibers, early indicators of effective signaling, was observed to be surface-chemistry dependent. These responses could be correlated with protein dynamics on different surface chemistries. Relative deposition and conformations of fibronectin were shown to be dependent on surface chemistry. Results indicate that observed differential cell adhesion, spreading and growth on derivatized surfaces is mediated through modulations of adsorbed ECM molecules (e.g., fibronectin). The primary regulators of the events investigated above were examined. Well-controlled alkylthiol surface chemistries were used to monitor the activation state of RhoA. Activation states of RhoA were determined and shown to be surface chemistry-dependent RhoGDI levels and intracellular localization were also shown to be surface-chemistry dependent Cells cultured on -CH3 terminated SAMs, which normally exhibit a cultured on -CH3 terminated SAMs, which normally exhibit a low growth phenotype, were transfected with a constitutively active RhoA mutant. Transfected cells exhibited significant increases in cel! length. However, no focal contact formation was observed. These results show that genetic alteration of intracellular regulators is incapable of overcoming the lack of extracellular stimuli, in the form of adsorbed ECM proteins, present on -CH3 terminated SAM surfaces. In summary, extracellular and intracellular information indicates that surface chemistry is capable of modulating communication between a cell and its extracellular environment. These data provide new, valuable molecular level information necessary to develop rational cause and effect relationships between a material's surface chemistry and biological response. This information should prove useful to develop biomaterials capable of producing predictable biological responses.