Participation of Hedgehog and Notch signaling pathways in the osteoblastic differentiation induced by titanium with nanotopography

Abstract

Osteogenesis is a biological event characterized by steps highly dependent oo cell signaling pathways, including the Hedgehog and Notch pathways that act in osteoblastic differentiation. Thus, the modulation of these signaling pathways through the use of agonist and antagonist drugs may generate relevant data for therapies related to bone tissue in dentistry, such as the use of osseointegrated titanium (Ti) implants. It is known that Ti surfaces with nanotopography favor osteoblastic differentiation and extracellular matrix mineralization; however, the cellular mechanisms involved in these effects are not entirely understood. In this context, we hypothesized that the Hedgehog and Notch pathways are involved in the osteogenic potential of nanostructured Ti surfaces. Thus, the aim of this study is to investigate the participation of Hedgehog and Notch signaling pathways in the osteoblastic differentiation induced by Ti with nanotopography (Ti-Nano) compared to Ti-Machined. Rat calvaria-derived osteoblastic cells will be cultured on polystyrene in presence of three concentrations of the agonist and antagonist drugs for the Hedgehog and Notch pathways, purmorphamine and cyclopamine, bexarotene and DAPT, respectively. The drug concentrations with more pronounced effects on cells will be selected based on the following evaluations: (1) gene expression of bone markers by real-time PCR, (2) alkaline phosphatase (ALP) activity and (3) extracellular matrix mineralization. Then, osteoblastic cells will be cultured on Ti-Nano and Ti-Machined to evaluate: (1) gene expression of bone markers and components involved in Hedgehog and Notch signaling by real-time PCR, (2) expression of proteins involved in Hedgehog and Notch signaling, and bone markers by Western blot, (3) ALP activity and (4) extracellular matrix mineralization. Finally, osteoblastic cells will be cultured on both Ti surfaces in presence of agonists and antagonists, at the previously selected concentrations, to investigate the effect of activation and inhibition of Hedgehog and Notch signaling pathways in the osteogenic potential of Ti-Nano compared to Ti-Machined by evaluating the same cellular parameters mentioned above. The results of this study may contribute to the understanding of the cellular mechanisms involved in the interaction between bone tissue and nanostructured Ti implants and consequently to the development of new strategies combining signaling pathways and surface topographies that favor the events related to the process of osseointegration. (AU)