Multiple Wnt Pathways Differentially Regulate Osteoblast Differentiation on Microstructured Titanium

Introduction: Wnt/beta-catenin and Wnt/Ca²⁺ pathways are associated with osteoblast differentiation, including expression of osteoblast-specific genes. Wnt activity is tightly regulated by several kinds of secreted antagonists and receptors, including Dickkopf-1 (Dkk1), Dkk2, Frizzled (Fzd), and LDL-related receptor protein-5/6 (LRP5/6). We examined these pathways mediated effects of substrate microstructure and surface energy on osteoblast differentiation.  Methods: MG63 cells were grown on tissue culture polystyrene (TCPS) and titanium microstructured surfaces: pretreatment-Ti (PT, Ra<0.2μm), sand-blasted/acid-etched (SLA, Ra=4μm), and modified-SLA (modSLA, hydrophilic-SLA). mRNA levels for Wnt-canonical and Wnt/Ca²⁺ pathways were measured by quantitative PCR. ShRNAs were designed for human Dkk1, Dkk2 and LRP5, and transduced into MG63 cells via lentivirus-particles. Stably silenced cell lines were compared to wild type MG63 cells. Total cell number and alkaline phosphatase specific activity (ALP), as well as secreted osteocalcin (OCN), osteoprotegerin (OPG), transforming growth factor beta-1 (TGF-beta1), and vascular endothelial growth factor (VEGF) protein levels were analyzed. Statistical significance was determined using ANOVA followed by Bonferroni's t-test. Results: Wnt3a mRNAs were undetectable but Wnt5a and Fzd2, Fzd5, Fzd6, Fzd7, Lrp5 and Lrp6 receptors were up-regulated on SLA/modSLA in wild type cells. The Wnt-inhibitor Dkk1 was down-regulated on these substrates whereas Dkk2 was up-regulated. Dkk1-silenced cells exhibited higher levels of ALP, OCN and OPG than wild type cells and the levels increased with surface microstructure/surface energy (modSLA>SLA>PT>TCPS). Dkk2-silenced cells had reduced levels of ALP, OPG, TGF-beta1, VEGF and OCN on all Ti surfaces compared to wild type cells and levels decreased with microstructure/surface energy (modSLAConclusions: Osteoblast differentiation is a complex process that requires activation and inhibition of different Wnt pathways. The Wnt/Ca²⁺-dependent pathway may induce terminal osteoblast maturation on microstructured surfaces, whereas the canonical Wnt pathway may be important in early stages of osteoblast differentiation. (Supported by NIHIR052182, ITI Foundation, and Institut Straumann AG)