Participation of Wnt signaling pathway on osteogenic potential of titanium surfaces with nanotopography

Author(s)

Rodrigo Paolo Flores Abuná

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-05-03

Examining board members

Márcio Mateus Beloti; Denise Carleto Andia; Quamarul Hassan; Willian Fernando Zambuzzi

• Advisor: Márcio Mateus Beloti

Abstract

The Wnt signal transduction is crucial for the structural organization during embryogenesis, participating in mitogenic stimulation, cell differentiation and homeostasis of adult tissues, including bone. It is well established that the titanium surface with nanotopography (Ti-Nano) favors the osteoblastic differentiation by modulating the signaling pathways of integrins and bone morphogenetic proteins (BMPs). However, few studies have addressed the participation of the Wnt pathway on the effects of nanoscale topographies. Therefore, we hypothesized that the Wnt signaling pathway is, at least in part, involved in the higher osteogenic potential of Ti-Nano. To test this hypothesis, we used Ti discs, which were treated with H2SO4/H2O2 solution to produce nanotopographic surfaces, and discs without treatment (Ti-Machined), being considered as control. Then, MC3T3-E1 cells, subclone 14, were cultured on both surfaces to evaluate the effect of Ti-Nano on the gene expression related to the canonical Wnt/β-catenin and non-canonical Wnt/Ca2+ signaling pathways. Based on the real-time PCR results, the most intensely modulated genes by Ti-Nano, the Wnt/β-catenin pathway-related Fzd4 and Wnt/Ca2+ pathway-related Fzd6, were selected and silenced by CRISPR-dCas9-KRAB and CRISPR-Cas9, respectively. Then, we investigated the effect of the silencing on the osteogenic potential of Ti-Nano. Data were compared by Student\'s t-test or one-way ANOVA (p≤0.05). The results of this study revealed that: 1) Fzd4 gene silencing inhibits the osteoblast genotype and phenotype expression of MC3T3-E1 cells, corresponding to the negative modulation of Wnt/β-catenin targets in cells grown on Ti-Nano surface on day 5; 2) Fzd6 silencing inhibits the osteoblast genotype and phenotype expression of MC3T3-E1 cells; however, Wnt/Ca2+ pathway targets were not modulated, but negative expression of β-catenin protein was detected in cells grown on Ti-Nano on day 3. These results indicate that the higher osteogenic potential of Ti-Nano is, at least in part, due to the activation of the Wnt/β-catenin signaling pathway, since the silencing of the Fdz4 and Fzd6 gene significantly reduced the osteoblast differentiation of MC3T3-E1 cells grown on Ti-Nano, compared to Ti-Machined. Taken together, our results revealed a new mechanism involving the Wnt/β-catenin signaling pathway to explain the higher osteogenic potential of the evaluated nanotopography. (AU)

• FAPESP's process: 16/14711-4 - Role of Wnt Signaling Pathways in the Osteogenic Potential of Titanium Surface with Nanotopography
• Grantee: Rodrigo Paolo Flores Abuna
• Support Opportunities: Scholarships in Brazil - Doctorate