Large Scale Analisys of MicroRNAs in Osteoblastic Cells Grown on Titanium with Nanotopography

Abstract

MicroRNAs (miRs) are small noncoding RNAs ( ~ 22 nucleotides) that significantly regulate the translation of protein coding genes in higher organisms and are involved in a plethora of biological processes, including in vitro osteoblast differentiation and in vivo bone formation, key events in the titanium (Ti) osseointegration process. It is well known that surfaces with nanotopography favor the extracellular matrix mineralization, despite the cellular mechanisms are still unclear. In this way, it is possible to speculate that miRs are part of the cellular machinery mediating cell/Ti surface interactions. Thus, the aim of our study is to evaluate the large-scale expression of miRs in osteoblastic cells grown on nanostructured Ti surface compared with Ti surface without nanotopography (control). Human mesenchymal stem cells will be cultured under osteogenic conditions and the following parameters will be assessed: cell proliferation and viability by counting the number of cells; gene expression (messenger RNA - mRNA), using Real-time PCR, of apoptotic markers, Bax and survivin, and of osteoblast phenotype markers, runt related transcription factor 2 (Runx2), type I collagen, osteopontin, alkaline phosphatase, bone sialoprotein, osteocalcin and BMP-2; protein expression of Runx2 by Western blot; and miRs expression using Microarray. Extracellular matrix mineralization will be evaluated with Alizarin red stain at 21 days.