Effect of bone morphogenetic protein 9 on osteoblast differentiation of cells grown on titanium with nanotopography

Abstract

Among bone morphogenetic proteins (BMPs), BMP-9 has been described as one with higher osteogenic potential. Here, we aimed at evaluating the effect of BMP-9 on the osteoblast differentiation of cells grown on titanium (Ti) with nanotopography, a well-known osseoinductive surface. MC3T3-E1 cells were grown either in absence or presence of BMP-9 (20 nM) on Ti with nanotopography (Ti-Nano) or machined Ti (Ti-Machined) for up to 21 days to evaluate the gene expression of RUNX2, osterix, osteocalcin, bone sialoprotein, SMAD6 and SMAD4, protein expression of SMAD4, ALP activity and extracellular matrix mineralization. As expected BMP-9 increased osteoblast differentiation irrespective of Ti surface topography; however, the cells grown on Ti-Nano were more responsible to BMP-9 compared with cells grown on Ti-machined. This could be, at least in part, due to the fact that Ti-Nano may act on both ways, by increasing the activation (SMAD4) and decreasing the inhibition (SMAD6) of the signaling pathway triggered by BMP-9, while Ti-Machined only decrease the inhibition (SMAD6) of this pathway. In conclusion, the combination of the osteogenic potential of BMP-9 with the osseoinductive capacity of Ti-Nano could be a promising strategy to favor the osseointegration of Ti implants.

Participation of integrin β3 in osteoblast differentiation induced by titanium with nano or microtopography

“…The chemical and physical characteristics of this Ti‐Nano have already been described and here we confirmed its differences in terms of topography and roughness compared with a commercially available surface with microtopography . The osseoinductive capacity makes this nanotopography an appealing model of investigation of the signaling pathways triggered by an osteogenic microenvironment that drive the process of osseointegration . In this context, we have already shown that this surface regulates several cell machinery components, including integrins, in order to induce osteoblast differentiation …”

“…This surface exhibits nanopits with an average diameter of 22 nm, a thicker TiO 2 layer and lower amounts of contaminants such as N and Si compared with an untreated Ti surface . The higher osteogenic potential of this nanotopography has been demonstrated using different cell culture and animal models . In addition, we have shown that the signaling pathway triggered by integrin α1β1 complex has a key role in the osseoinductive capacity of Ti with nanotopography since the use of an antagonist of the heterodimer α1β1, obtustatin, prevented the osteoblast differentiation induced by nanotopography …”

“…The Ti discs (12 × 2 mm) of commercially pure (grade II) were polished and cleaned as previously described . To obtain Ti with nanotopography (Ti‐Nano), discs were conditioned for 4 h with a solution of 10 N H 2 SO 4 and 30% aqueous H 2 O 2 (1:1) at room temperature under agitation and cleaned by sonication.…”

“…To detect dCas9 protein, the MC3T3‐E1 and KRAB‐MC3T3‐E1 cells were treated, and the protein of each cell were transferred to PVDF membrane as previous described . For membrane blocking, it was used Tris‐buffered saline, 0.1% Tween‐20 (Sigma‐Aldrich) with 5% nonfat milk (Bio‐Rad Laboratories), for 1 h at room temperature.…”

“…The surface characteristics of titanium (Ti) implants affect cell adhesion, proliferation and osteoblast differentiation, which ultimately dictate the interfacial tissue formation to achieve implant fixation to the host and determine the success rate of dental implants . Modifications of Ti topography may affect osteoblast differentiation by acting on several cell mechanisms and signaling pathways, including those triggered by integrins …”