Titanium with nanotopography induces osteoblast differentiation mediated by endogenous BMP-2/4

摘要

The nanotopography of titanium (Ti) generated by H_2SO_4/H_2O_2 treatment inhibits the expression of microRNAs associated with the modulation of BMP-2 signaling pathway in osteoblasts. Additionally, cells grown on this surface are more responsible to exogenous BMP-2 than cells grown on machined surface. Such findings indicate the relevance of this pathway in the potential of nanotopography to induce osteogenesis. In this context, the aim of this study was to evaluate the endogenous expression of BMP-2/4 and the role of these proteins on the osteoblast phenotype development in cells grown on Ti with nanotopography. MC3T3-E1 pre-osteoblastic cells were cultured for 7 days on Ti discs either with nanotopography or machined for assaying gene and protein expression of endogenous BMP-2/4 by real-time PCR and ELISA, respectively. To repress BMP-2/4 signaling pathway, BMPR1A (one of the BMP receptors)-silenced cells were generated by shBMPR1A transfection and cultured on both Ti surfaces. Cells transfected with nonspecific, scrambled shRNA, (non-silenced cells) were used as control. At days 3 and 7, the effect of BMPR1A silencing on osteoblast differentiation was evaluated by gene and protein expression of bone markers using real-time PCR and western blot, respectively. In addition, at day 7, in situ alkaline phosphatase (ALP) activity was detected by fast red. The expression of genes related to BMP signaling pathway was assessed by PCR array at days 3 and 7. All experiments were carried out in triplicates and the data were analyzed using either non-parametric or parametric test when appropriated (pS0.05). Nanotopography displayed higher BMP-2 gene and protein expression compared with machined Ti surface. The BMPR1A silencing reduced ALP and osteocalcin gene expression, RUNX2 protein expression and in situ ALP activity, mainly in cells grown on Ti with nanotopography. As expected, a higher modulation in the expression of genes related to BMP signaling pathway after BMPR1A silencing was observed in cells grown on nanotopography compared with machined Ti surface. In conclusion, we have shown a relevant role of endogenous BMP-2 on the osteoblast differentiation of cells grown on Ti surfaces. Also, the modulation of the expression of genes related to the BMP signaling pathway in response to the BMPR1A silencing were more evident in cells grown on nanotopography indicating that the positive effects of this Ti surface on osteogenesis is mediated, at least in part, through this pathway. Together, our results open new windows for developing surface modifications at the nanoscale level to control endogenous BMP production and signaling in order to favor the osseointegration of Ti implants.