Effect of the association of BMP-2 and Wnt-3a on osteoblastic cells grown on titanium surfaces 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 the osseoinductor effect of the nanotopography, generated by H2SO4/H2O2 treatment, involves the regulation of, at least, two intracellular mechanisms related to BMP and Wnt signaling pathways. These mechanisms induce cells grown on this surface to synthesize higher amount of BMP-2 and to be more responsive to exogenous BMP-2. In addition, Wnt signaling pathway is also modulated by Ti surface with nanotopography. Based on these results and considering the participation of the BMP and Wnt signaling pathways on osteogenesis, the aim of this study is to evaluate if osteoblastic cells grown on Ti with nanotopography (Ti-Nano) are more responsive to exogenous BMP-2 and Wnt-3a compared with cells grown on machined Ti surface (Ti-Control). To test our hypothesis, MC3T3-E1 osteoblastic cells, subclone 14, will be cultured on Ti-Nano and Ti-Control discs. Recombinant proteins, BMP-2 (100 ng/mL) and/or Wnt-3a (10 ng/mL), will be added to the culture medium and cell responses will be evaluated for periods of up to 21 days. At days 3, 5 and 7, the cell proliferation will be evaluated by MTT assay. At day 7, the gene expression of the bone markers alkaline phosphatase (ALP), RUNX2, osterix and osteocalcin will be evaluated by real-time PCR, and the detection and quantification of in situ ALP will be evaluated by Fast red. At day 21, the extracellular matrix mineralization will be detected by Alizarin assay. The data will be submitted to the Kolgomorov-Smirnov test to evaluate the adherence to the normal curve and subsequently they 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)