Integrin α2β1 plays a critical role in osteoblast response to micron-scale surface structure and surface energy of titanium substrates

Efforts to improve bone response to biomaterials have focused on ligands that bind alpha5beta1 integrins. However, antibodies to alpha5beta1 reduce osteoblast proliferation but do not affect differentiation when cells are grown on titanium (Ti). beta1-silencing blocks the differentiation stimulus of Ti microtopography, suggesting that other beta1 partners are important. Stably alpha2-silenced MG63 human osteoblast-like cells were used to test whether alpha2beta1 specifically mediates osteoblast response to Ti surface micron-scale structure and energy. WT and alpha2-silenced MG63 cells were cultured on tissue culture polystyrene (TCPS) and Ti disks with different surface microtopographies: machined pretreatment (PT) surfaces [mean peak to valley roughness (R(a)) < 0.02 microm], PT surfaces that were grit-blasted and acid-etched (SLA; R(a) = 4 microm), and SLA with high surface energy (modSLA). Alkaline phosphatase (ALP), alpha2 and beta1 mRNA, but not alpha5, alpha v, beta3, type-I collagen, or osteocalcin, increased on SLA and modSLA at 6 days. Alpha2 increased at 8 days on TCPS and PT, but remained unchanged on SLA and modSLA. Alpha2-protein was reduced 70% in alpha2-siRNA cells, whereas alpha5-mRNA and protein were unaffected. Alpha2-knockdown blocked surface-dependent increases in beta1 and osteocalcin and decreases in cell number and increases in ALP and local factors typical of MG63 cells grown on SLA and modSLA [e.g., prostaglandin E(2), osteoprotegerin, latent and active TGF-beta1, and stimulatory effects of 1alpha,25(OH)(2)D(3) on these parameters]. This finding indicates that alpha2beta1 signaling is required for osteoblastic differentiation caused by Ti microstructure and surface energy, suggesting that conclusions based on cell behavior on TCPS are not predictive of behavior on other substrates or the mechanisms involved.