Microstructured Titanium Surfaces Promote Terminal Osteoblast Differentiation via DKK2

Objective: Surface topography promotes osteoblast differentiation and increases osteogenic local factors. Studies examining osteoblast differentiation on tissue culture polystyrene (TCPS) surfaces show that the process requires an initial activation of Wnt/beta-catenin-signaling, which up-regulates osteoblast-specific genes, including Wnt inhibitors like Dickkopf (Dkk2). Expression of Dkk proteins upstream of Wnt-signaling inhibits osteogenesis, but Dkk expression after Wnt-signaling induces a terminal osteoblast phenotype. In this study we hypothesized that surface topography and hydrophilicity modulate Wnt-Dkk2 signaling and Dkk2 is necessary for surface-dependent osteoblast differentiation. Methods: MG63 cells were grown for 6 days on TCPS (control) and titanium microstructured surfaces (PT [Ra<0.2µm], SLA [Ra=4µm], modSLA [hydrophilic-SLA]). Changes in mRNA levels were measured by an RNA-microarray and confirmed by real time PCR. Surface effects on Dkk2 protein were measured by ELISA. MG63 cells were transduced with lentivirus particles containing the Dkk2-siRNA template and selected with puromicyn. MG63 and Dkk2-silenced cells were cultured on the test surfaces +/- exogenous Dkk1 and Dkk2 protein. Alkaline phosphatase specific activity (ALP), OCN, OPG, TGFb, VEGF and Dkk2 protein levels were analyzed. Results: SLA and modSLA surfaces up-regulated Dkk2 expression and protein content based on RNA-microarray, real time PCR, and direct measurement of Dkk2 protein. Dkk2 was reduced in siRNA-Dkk2 cells by more than 70%. These cells had significantly lower levels of ALP, OCN, OPG, VEGF and TGFb than MG63 cells on each test surface. Effects of Dkk2 knock-down were comparable on SLA and modSLA. Dkk2 but not Dkk1 protein treatment partially rescued the osteoblast differentiation phenotype in Dkk2-silenced cells, and this effect was dose-dependent. Conclusions: These results indicate a major role for Dkk2 in late-stage osteoblastic differentiation on microstructured and hydrophilic surfaces, and suggest the importance of Wnt/beta-catenin-signaling and Dkk2 during implant osteointegration in vivo. Supported by AR 052102, the ITI Foundation, and Institut Straumann.