Use of CRISPR-Cas9 to Investigate the Role of Integrins on Osteoblast Differentiation of Mesenchymal Stem Cells Induced by Titanium Surfaces

Abstract

Interactions between cells and titanium (Ti) are crucial for dental implant osseointegration. The integrins mediate adhesion of cells to extracellular matrix proteins and some members are involved in the process of osteoblast differentiation. Results of the project entitled "Role of Integrins on Osteoblast Differentiation of Mesenchymal Stem Cells Induced by Titanium Surfaces with Micro and Nanotopography" have shown that Ti with nanotopography (Ti-Nano) and Ti with microtopography (Ti-Micro) modulated the expression of several genes involved in the integrin signaling pathways compared with Ti machined (Ti-Machined), with more pronounced effects of Ti-Nano. The integrins beta 3, alpha 10 and alpha V are among the genes regulates by Ti-Nano. Thus, the aim this study is investigate, through the gene silencing by the clustered regularly interspaced short palindromic repeats / associated nuclease 9 (CRISPR-Cas9), the role of these integrins in osteoblast differentiation induced by Ti surfaces. For this, rat bone marrow mesenchymal stem cells will be transduced with CRISPR-Cas9 and the silencing efficacy will be evaluated by real-time PCR and Western blot. The transduced cells will be cultured on Ti discs with the three different surfaces, Ti-Nano, Ti-Micro and Ti-Machined and the following parameters will be evaluated: (1) gene expression of integrin beta 3, integrin alpha 10 and integrin alpha V and of the osteoblastic markers RUNX2, osterix, bone sialoprotein and alkaline phosphatase (ALP), by real-time PCR at day 3, (2) protein expression of integrin beta 3, integrin alpha 10, integrin alpha V and RUNX2, by Western blot at day 3, and (3) ALP activity, by Fast Red at day 10. The quantitative data will be obtained in triplicate (n=3) and analyzed using One-Way ANOVA with significance level of 5%.