Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.


Introduction
Excellent mechanical and biological properties render titanium (Ti) to be the most frequently used biomaterial for manufacturing dental implants. 1

Selection of FAK inhibitor concentration
A potent and specific FAK inhibitor, PF-573228

Effect of FAK inhibition in osteoblastic cells grown on Ti surfaces
The cells were plated on the Ti surfaces at a density of 2×10 4 cells/disc in 24-well polystyrene plates (Corning Incorporated) for up to 10 days, using OM containing PF-573228 0.1 µM, which was selected in a previous experiment. The parameters described below were then evaluated.

Scanning electron microscopy
Exactly 24 h after culture on the Ti surface, SEM was carried out to observe cells grown with or without PF-573228. The samples were fixed in was used to detect their ALP activity as previously described. 25 The absorbance (n=4) was evaluated at 590 nm by using the plate reader μQuant (Bio-Tek Instruments Inc., Winooski, VT, USA), and ALP activity was calculated and expressed as μmol of thymolphthalein/h/mg protein as previously described.

Gene
Gene name Identification

Statistical analysis
The data of cell counting were analyzed by a oneway analysis of variance, followed by the Student− Newman−Keuls post hoc test. Student's t-test was used to analyze the data of gene expression and ALP activity. The significance level used was set to 5% (p≤0.05).     Ti surface modification can improve implant wettability and increase the available surface for bone growth and fixation and blood clotting. 22 Osseointegration of Ti depends mostly on the interactions between the material surface and cells, in which cell signaling pathways play an important role. FAK is involved in several signaling pathways due to its ability to bind to several proteins involved in these pathways; its function is related to many cellular processes such as migration, growth factor signaling, cell cycle progression, and cell survival. 26,27 The present study focused on the role of an FAK inhibitor in the responses of osteoblastic cells to Ti.

Selection of FAK inhibitor concentration
We selected an FAK inhibitor concentration of 0.1 μM because this value is the lowest concentration that did  Focal adhesion complexes are key structures participating in the interactions between cells and surfaces of biomaterials and may affect cell morphology, proliferation, differentiation, and apoptosis. 35 Vinculin detection has been conducted to identify these complexes, but distinct data have been described [36][37][38] . In the present work, we evaluated vinculin expression by immunofluorescence but we did not find a correspondence between the topography and vinculin expression of cells grown with or without the FAK inhibitor (data not shown). This result may be due to the effects of the trial periods chosen for the evaluation or the methodology used. 39

Conclusion
Our results demonstrated the relevance of FAK to the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. We also observed that nanotopography upregulates