Roles Of YAP And TAZ In Osteoblasts Differentiation On SLA Titanium

Background:Currently,Sandblasted,Large-grit,Acid-etched(SLA)is recognized as the primary modification of dental implants.The hierarchy microtopography of SLA implants enhances surface roughness,which is in favor of protein adsorption and cell adhesion,subsequently induces osteogenic differentiation,finally promotes bone formation and implant osseointegration.YAP/TAZ are transferred from cytoplasm to cell nuclear,which may be mediated by specific extracellular environment.YAP and TAZ both play vital roles in osteogenic differentiation by regulating Runx2 transcriptional activity.ECM hardness and surface topography are regarded as critical factors in YAP/TAZ-dependent regulation of osteogenic differentiation in BMSCs and osteoblasts,which is promising in tissue engineering.Purposes:Polished and SLA titanium plates were fabricated for experiments in vitro.The expression and distribution of YAP/TAZ in osteoblasts were observed in polished Ti and SLA Ti.Furthermore,YAP and TAZ knockdown in SLA Ti were performed,respectively,following by detecting the expressions of osteogenic-related genes.Accordingly,the effect of SLA Ti in osteoblast differentiation associated with YAP/TAZ was studied.Methods:1.Ti6A14V titanium disks were ground and polished(Polished Ti).Sandblast and acid-etching were subsequently implemented to fabricate SLA Ti.The surface topography of the two titanium plates was observed with a scanning electron microscope.The surface roughness was measured with an Optical Profiler.2.MC3T3-E1 cells were cultivated on Polished Ti and SLA Ti.Cells were divided into Polish Ti group and SLA Ti group.Immunofluorescence staining was performed to observe the intracelluar localization on Polished Ti and SLA Ti.Quantitative RT-PCR and Western blot were implemented to analyze the expressions of YAP/TAZ on the two titanium plates.3.MC3T3-E1 cells were cultivated on SLA Ti.SiRNA were transfected into cells to reduce the expression of YAP and TAZ,respectively.Cells were divided into three groups:non-specific siRNA group,siYAP group and siTAZ group.Quantitative RT-PCR and Western blot were performed to analyze the expressions of Runx2 and BSP in the three different groups.Results:1.Polished Ti appears as a sliver-white and lustrous metal.,while SLA Ti appears as a gray,rough and lackluster metal.Under a scanning electron microscope,Polished Ti possessed a flat surface with a few scratches.And SLA Ti had small micro pits,overlapping each other.Under a higher magnification,SLA Ti showed s with a diameter of 30 ?m and pits with a diameter of 3-8 ?m.The arithmetical mean deviation of the profile(Ra)of Polished Ti and SLA Ti was 0.24?m and 2.6?m,respectively.2.Immunofluorescence staining showed that YAP and TAZ were both observed in the cytoplasm and cellular nuclear on Polished Ti.And the fluorescent spreading in the cytoplasm appeared large.On SLA Ti,the fluorescence of YAP aggregated mainly in the nuclear.In the cytoplasm only a little fluorescence were observed around the nuclear.Nine tenths of the fluorescence of TAZ was in the nuclear.3.Quantitative RT-PCR results showed that the expressions of YAP and TAZ on SLA Ti were significantly higher than that on Polished Ti.The similar results were obtained from the western blot analysis.4.Quantitative RT-PCR results showed that silencing YAP and TAZ could significantly attenuate the expression of Runx2 and BSP,which were consistent with that of western blot analysis.Conclusion:1.SLA microtopography promotes the gene expression of YAP and TAZ in osteoblasts,as well as the protein expression.2.SLA micromorphology induces YAP and TAZ from cytoplasm to cell nucleus.3.YAP and TAZ both facilitates osteoblasts differentiation on SLA titanium.