Effect of Wnt-3a protein on osteoblastic differentiation induced by titanium surface with nanotopography

Abstract

Titanium (Ti) surfaces with nanotopography may favor osteoblast differentiation by modulating several cell signaling pathways. Results from our research group have shown that one of the mechanisms by which the nanotopography, generated by H2SO4/H2O2 treatment, exerts its osseoinductor potential involves the regulation of BMP (bone morphogenetic protein) signaling pathway. This mechanism makes cells grown on this surface to synthesize higher amount of BMP-2 and to be more responsive to exogenous BMP-2. In addition, we have observed that Wnt (wingless-Type mouse mammary tumor virus MMTV Integration Site) signaling pathway is also modulated by Ti surface with nanotopography. Based on these results and considering the participation of the Wnt signaling pathway on osteogenesis, the aim of this study is to evaluate if osteoblastic cells grown on Ti with nanotopography (Nano) are more responsive to exogenous Wnt-3a, a potent Wnt agonist, compared with cells grown on machined Ti surface (Control). In order to test our hypothesis, MC3T3-E1, subclone 14, osteoblastic cells will be cultured on machined Ti and Ti with nanotopography, and on polystyrene. Between days 3 and 5, cells will be exposed to the Wnt-3a recombinant protein (10 ng/mL) and cellular response will be evaluated for periods up to 10 days. At days 7 and 10, the gene expression of the bone markers alkaline phosphatase (ALP), bone sialoprotein, RUNX2, osterix, osteocalcin and osteopontin, and Wnt signaling markers, WNT-3A, Frizzled-4 and Dickkopf will be evaluated by real-time PCR, and the detection and quantification of in situ ALP will be evaluated by Fast Red. The data will be submitted to the Kolgomorov-Smirnov test to evaluate the normal curve adherence and subsequently will be analyzed using parametric or non-parametric tests to detect differences between the evaluated samples. The significance level will be set at 5% (pd0.05). (AU)