Spatial control of ligand presentation on biomaterial surfaces

Abstract

Adhesion of many cell types to the extracellular matrix or to synthetic bioactive surfaces is mediated by transmembrane integrin receptors. Integrin clustering is believed to be closely associated with focal contact formation and signaling, as assessed by the behavior of cells on surfaces presenting relatively uniform ligand distributions. It has therefore been hypothesized that controlled clustering of 2, 3.....n integrins might be achieved by controlling the spatial distribution of adhesion ligands on biomaterial surfaces. Substrates were prepared on which cell-surface interactions are controlled by modifying non-adhesive poly(ethylene oxide) (PEO) hydrogels with the minimal cell-adhesion peptide sequence GRGDY (RGD). The peptide is tethered to the hydrogel surfaces via star PEO molecules, producing surfaces on which the ligands are presented to cells in "clusters", or domains of high concentration. The substrates are compared with others on which the RGD peptide is uniformly distributed. Control of the RGD cluster size was achieved by varying the relative concentrations of reactants in solution. The binding of RGD-modified stars to surfaces was found to be a non-linear function of its concentration in solution and degree of modification, and is reasonably explained by a Langmuir model of competitive adsorption. Quantitative techniques for visualizing the ligand distribution on the surface were developed, and indicated that surfaces to which ligands had been tethered via star molecules showed a significant deviation from normal, random distribution. Thus, control of the ligand spatial distribution was achieved. In addition, preliminary biological testing suggests that substrates on which adhesion ligands are presented to cells in a clustered format produces more physiological behaviour than those on which ligands are uniformly distributed at the same average ligand density. Thus, we have fabricated surfaces which, because of their resistance to non-specific cell interactions and the control of specific interactions at the molecular level, can serve as a model for artificial matrix development and can be used for fundamental in vitro studies.