Early Osteoblast Attachment, Spreading, and Focal Adhesions on RGD Coated Surfaces

Abstract

Cells recognize and interact with the extracellular matrix (ECM) through heterodimeric receptors known as integrins. The objective of our work is to immobilize integrin-stimulating peptides to bone-contacting implants in order to control cellular activity and response.We have previously demonstrated that cysteine (C) containing peptides self-assemble onto gold-coated substrates. Investigations have focused on the RGD (Arg-Gly-Asp) peptide sequence since it is found in several bone ECM proteins. Gold was first coated onto glass coverslips by evaporation and the peptide was applied in a 0.22 mM solution. Contact angle and surface plasmon resonance verified RGDC peptide attachment and formation of a monolayer.Rat calvarial osteoblasts isolated from six-day-old rat pups were used from passages one to three. Cell attachment at 20 minutes is 100% greater on RGDC than on CG (control peptides) or plain gold surfaces. Cells on RGDC also stain positively for vinculin, a protein which is present in focal adhesions (functional structures into which integrins assemble) whereas surfaces without integrin stimulating peptides do not. Scanning electron micrographs show cells to be more spread and have more processes at 20 minutes, 1, 3, and 24 hours on RGDC. Live video images of these surfaces from zero to three hours after plating confirmed earlier and greater cell spreading on RGDC.Ongoing in vitro experiments are investigating the long-term response of osteoblasts to RGDC and other immobilized peptides in terms of differentiation, matrix production, and integrin expression.