Osteoblasts grown on microstructured Ti surfaces enhance osteointegration by producing local factors that regulate bone formation as well as bone remodeling, including the RANK ligand decoy receptor osteoprotegerin (OPG). The objective of this study was to explore the mechanism by which surface microstructure and surface energy mediate their stimulatory effects on OPG expression. Titanium disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT, Ra<0.2microm), microstructured sandblasted/acid etched surface (SLA, Ra=3-4microm), and a microstructured Ti plasma-sprayed surface (TPS, Ra=4microm). Human osteoblast-like MG63 cells were cultured on these substrates and the regulation of OPG production by TGF-beta1, PKC, and alpha2beta1 integrin signaling determined. Osteoblasts produced increased amounts of OPG as well as active and latent TGF-beta1 and had increased PKC activity when grown on SLA and TPS. Exogenous TGF-beta1 increased OPG production in a dose-dependent manner on all surfaces, and this was prevented by adding blocking antibody to the TGF-beta type II receptor or by reducing TGF-beta1 binding to the receptor by adding exogenous soluble type II receptor. The PKC inhibitor chelerythrine inhibited the production of OPG in a dose-dependent manner, but only in cultures on SLA and TPS. shRNA knockdown of alpha2 or a double knockdown of alpha2beta1 also reduced OPG, as well as production of TGF-beta1. These results indicate that substrate-dependent OPG production is regulated by TGF-beta1, PKC, and alpha2beta1 and suggest a mechanism by which alpha2beta1 signaling increases PKC, resulting in TGF-beta1 production and TGF-beta1 then acts on its receptor to increase transcription of OPG.