Integrin subunits responsible for adhesion of human osteoblast-like cells to biomimetic peptide surfaces

We have identified the integrin subunits responsible for the initial adhesi on of human osteoblast-like cells to peptide-modified surfaces. Biomimetic peptide surfaces containing homogenous RGD (Arg-Gly-Asp), homogenous FHRRIK A (Phe His-Arg-Arg-Ile-Lys-Ala), and a mixed ratio of FHRRIKA:RGD (25:75) w ere used to assess integrin-mediated adhesion. The RGD and FHRRIKA peptides were selected from the cell-binding and putative heparin-binding domains o f bone sialoprotein. A panel of monoclonal antibodies against human alpha(1 ), alpha(2), alpha(3), alpha(4), alpha(5), beta(1), alpha(v), and alpha(v)b eta(3) was used to identify the subunits most dominant in mediating short-t erm (10 or 30 minutes) and long-term (4 hours) cell adhesion to the peptide surfaces. Anti-alpha(2), anti-beta(1), and anti-alpha(v), significantly (p < 0.05) diminished cell attachment to homogenous RGD surfaces following 30 minutes of incubation. After 4 hours of incubation on RGD-grafted surfaces , immunostaining of these integrin subunits revealed discrete localization of the alpha(v) subunit at the periphery of the cell (similar to focal cont act points), whereas the alpha(2) and beta(1) subunits stained very diffuse ly throughout the cell. A radial-flow apparatus was used to determine the e ffect of anti-integrin antibodies on strength of cell detachment following 10 minutes of incubation on peptide grafted surfaces. The strength of detac hment from surfaces containing RGD was significantly reduced (p < 0.5) in t he presence of anti-alpha(2), anti-alpha(v), or anti-beta(1) compared with controls (presence of preimmune mouse IgG). None of the antibodies signific antly influenced cell attachment to homogenous FHRRIKA-grafted surfaces. Th ese results demonstrate that initial (30 minutes) attachment of human osteo blast-like cells to homogenous RGD surfaces was mediated by the collagen re ceptor alpha(2)beta(1) and the vitronectin receptor alpha(v)beta(3), wherea s only the vitronectin receptor governed longer term (longer than 30 minute s) adhesion (localization to focal contacts). The importance of distinct in tegrins in mediating the attachment of bone cells to RGD-immobilized surfac es indicates a strategy for engineering orthopaedic implants with a built-i n surface specificity for cell adhesion.