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The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time-point, miR-sequencing analysis revealed that 20 miRs were upregulated (>2 fold) while 20 miRs were downregulated (>3 fold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR-4448, -4708 and -4773, which were significantly downregulated (>5 fold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs that directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP-2) osteogenic signal, were upregulated by Ti with nanotopography. Overexpression of miR-4448, -4708 and 4773 in MC3T3-E1 pre-osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP-2 displayed a higher osteogenic effect on MC3T3-E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP-2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR-SMAD-BMP-2 circuit governs the osteoblast differentiation induced by Ti with nanotopography.

We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP-2 and BMP-4 and the relevance of this process to the nanotopography-induced osteoblast differentiation. MC3T3-E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP-2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF-β/BMP signaling were evaluated. Nano supported higher BMP-2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF-β/BMP signaling was deeply affected by BMPR1A-silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP-2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718-1726, 2016. © 2015 Wiley Periodicals, Inc.