Reduced Surface Alpha-2 Integrin Prevents Osteoblast Differentation on Microstructured Titanium

Objectives: Beta-1 integrin silencing blocks the differentiation stimulus of Ti microtopography on osteoblasts. The present study used siRNA to knockdown α integrin levels to test the hypothesis that α2,β1 specifically mediates the response of osteoblasts to Ti surface micron-scale structure and energy. Methods: MG63 osteoblast-like cells were stably transfected with α2 siRNA. Cells lacking α2 on their surface were quantified by fluorescence activated cell sorting (FACS). α2 silencing was evaluated by comparing attachment of normal and silenced cells to type I collagen and by their response to tissue culture polystyrene (TCPS) and microstructured Ti substrates: machined pretreatment (PT) surfaces (Ra <0.02 μm) and PT surfaces that were grit blasted and acid etched (Ra =4 μm) and were hydrophobic (SLA) or hydrophilic (modSLA). Results: α2 siRNA reduced α2 mRNA and protein by 70%, but did not affect α1 or α5 protein, or mRNAs for α5, αv, β3 or type I collagen on any of the surfaces. β1 mRNAs were also unaffected on TCPS or PT, but α2-knockdown blocked increases in β1 and osteocalcin mRNA and protein on SLA and modSLA seen in normal cells. α2 siRNA reduced the surface-dependent decreases in cell number and increases in ALP activity as well as local factors typical of MG63 cells on SLA and modSLA, including PGE2, osteoprotegerin, and latent and active TGF-β1, including the stimulatory effects of 1α,25(OH)2D3 on these parameters. Cell surface α2 was absent in 98% of silenced cells and these cells did not attach to collagen-coated TCPS. Conclusions: This suggests that signaling via α2,β1 is reduced in the siRNA transfected cells and that α2,β1 signaling is required for osteoblastic differentiation due to Ti microstructure and Ti surface energy. Conclusions based on behavior of cells on TCPS may not predict their behavior on other substrates or the mechanisms involved. NIH AR052102.