Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the α2β1 integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via α2β1 signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for α2 integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces. Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1α,25(OH)2D3 enhanced levels of VEGF-A on SLA and modSLA. Silencing the α2 integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via α2β1 integrin signaling.

Microstructured and high surface energy titanium substrates increase osseointegration in vivo. In vitro, osteoblast differentiation is increased, but effects of the surface directly on multipotent mesenchymal stem cells (MSCs) and consequences for MSCs in the peri-implant environment are not known. We evaluated responses of human MSCs to substrate surface properties and examined the underlying mechanisms involved. MSCs exhibited osteoblast characteristics (alkaline phosphatase, RUNX2, and osteocalcin) when grown on microstructured Ti; this effect was more robust with increased hydrophilicity. Factors produced by osteoblasts grown on microstructured Ti were sufficient to induce co-cultured MSC differentiation to osteoblasts. Silencing studies showed that this was due to signaling via α2β1 integrins in osteoblasts on the substrate surface and paracrine action of secreted Dkk2. Thus, human MSCs are sensitive to substrate properties that induce osteoblastic differentiation; osteoblasts interact with these surface properties via α2β1 and secrete Dkk2, which acts on distal MSCs.