Role of Wnt Signaling Pathways in the Osteogenic Potential of Titanium Surface with Nanotopography

Abstract

The titanium surface with nanotopography, generated by H2SO4/H2O2 treatment, exhibits high osteogenic potential thanks to its ability to modulate signaling pathways involved in osteoblast differentiation such as integrin and bone morphogenetic protein (BMP) signals. Considering the relevant participation of Wingless-Type mouse mammary tumor virus [MMTV] Integration Site (Wnt) signaling pathways in osteogenesis, we hypothesized that these pathways are, at least in part, involved in the osteogenic potential of Ti with nanotopography. In order to test our hypothesis, MC3T3-E1 osteoblastic cells will be cultured on discs of Ti with nanotopography and machined (control) and the gene expression of canonical Wnt and non-canonical Wnt/Ca2+ signaling markers will be evaluated by real-time PCR. Based on gene expression findings, one target gene of each signaling pathway, canonical and non-canonical, will be selected for acquisition of the clustered regularly interspaced short palindromic repeats (CRISPR-Cas9/gRNA) to be used for silencing the target genes of interest. The silencing efficacy will be evaluated by real-time PCR and Western blot to determine the gene and protein expression, respectively, of the two target genes. Then, MC3T3-E1 cells will be transduced with the previously selected CRISPR-Cas9/gRNA at the predetermined concentration. Transductions will be carried out as follows: (1) CRISPR-Cas9/gRNA for canonical Wnt signaling, (2) CRISPR-Cas9/gRNA for non-canonical Wnt/Ca2+ signaling, (3) CRISPR-Cas9/gRNA for canonical Wnt and non-canonical Wnt/Ca2+ signaling or (4) control (CRISPR-Cas9 without gRNA). The transduced cells, and consequently silenced for canonical Wnt and/or non-canonical Wnt/Ca2+ signaling, will be cultured on Ti with nanotopography and control surfaces and the following parameters will be evaluated: (1) expression of genes related to canonical and non-canonical Wnt signaling and the osteoblastic markers RUNX2, osterix (OSX), alkaline phosphatase (ALP) and osteocalcin (OC), by real-time PCR, (2) RUNX2 protein expression by Western blot, and (3) ALP activity. The quantitative data will be obtained in quintuplicate (n=5) and submitted to the normal curve adherence and variance homogeneity tests. If data normality is detected, t-test or ANOVA (to compare two groups or multiple comparisons, respectively) will be used or if not, data will be compared by Mann Whitney or Kruskal-Wallis. The significance level will be set at 5%. (AU)