Optimization of surface-immobilized peptide/protein presentation for control of the biological activity of substrates for tissue engineering applications
The application of the self-assembled monolayer (SAM) technology to perform model studies of the interaction of proteins and cells with biomaterial substrates has yielded new insights in cellular adhesion and function. In this dissertation, SAMs of alkylsilanes of different functional groups (X = -OH, -epoxide, -COOH, -NH2, -CH3) were used to identify and elucidate both non-specific and specific effects which contribute to protein and peptide-mediated cellular adhesion. By using a spinning disc apparatus, quantitative comparison of the magnitudes of adhesion strength was possible and allowed for the sensitive detection of novel substrate-dependent effects. ^ The electrostatic charge of the substrate, especially positive charges, was shown to have a dominant effect on non-specific adhesion, even when a blocking procedure with heat-denatured albumin was used. When an extracellular matrix (ECM) protein, fibronectin, was adsorbed on the SAMs, hydrophobicity was the dominant surface property in determining the amount adsorbed while both hydrophilicity and electrostatic charge appeared to finely tune the orientation and/or conformation of the adsorbed fibronectin molecules and thereby affect adhesion. More specifically, the apparent affinity (Ka′ ) and the average bond strength of the α5β 1 integrin-fibronectin interactions were modulated by surface property. Optimal α5β1 integrin-fibronectin interactions were supported on hydrophilic, negatively-charged substrates. Nano-scale roughness of the substrate was also shown to affect cellular adhesion by alteration of the density of fibronectin adsorbed. ^ Although previous studies have investigated different aspects of arginine-glycine-aspartic acid (RGD) peptide-mediated adhesion and function, a systematic study of the effect of peptide design in optimizing the bioactivity has not been performed to date. Using a maleimide-based immobilization method to attach a series of modularly designed RGD peptides, the residues flanking the RGD sequence were shown quantitatively to modulate the αv-integrin mediated adhesion of MC3T3-E1 cells. Higher adhesion strength generally correlated with enhancements in the differentiation status of these osteoblast-like cells. Quantitative comparisons of the adhesion strengths through the various mechanisms showed that fibronectin-mediated adhesion and non-specific adhesion were significantly larger in magnitude than that mediated by RGD peptides during the cellular attachment phase. ^
Mark H Lee,
"Optimization of surface-immobilized peptide/protein presentation for control of the biological activity of substrates for tissue engineering applications"
(January 1, 2005).
Dissertations available from ProQuest.