Response Of Human Endothelial Cells To Oxidative Stress On Niobium

Objective:Oral implant has been widely used as prosthodontic method for decades.However,implant failure do occur and this is due to be connected with Complex body fluid environment.Inflammatory process cause oxidative stress,which may accelerate metal corrosion.Metal corrosion products are harmful to surrounding cells.Niobium is a corrosion resistant material and better than Titanium in some respects.Our purpose of this experiment is to study the cytotoxic effect of Niobium under H₂O₂ induced oxidative stress compared to Titanium and polystyrene?PS?.Methods:1.Cells cultureWe purchased EA.hy926 cell line from the Cells Bank of the Chinese Academy of Sciences?Shanghai,China?.EA.hy926 cell was cultured in high-glucose DMEM supplemented with 10%fetal bovine serum.Cells were incubated in a humidified atmosphere at 37°C?5%CO₂?.Cells with amount of 1mL were seeded on surface of three different materials in a density of 50,000 cells/mL in 24 well plate.After 2 days H₂O₂ solution with different concentration in DMEM medium without phenol red was applied to the cells for 1 or 24 h..2.Oxidative stress model Building upEA.hy926 cells were seeded on 24 wells plate as the method described above.After 2 days H₂O₂ solution?0/0.1/0.2/0.5mM?in DMEM medium without phenol red was applied to the cells for 24 h.Cell activity was measured by the conversion rate of cell counting kit-8.3.Titanium and Niobium preparationWe prepared Titanium and Niobium disks with 15 mm diameter and 2 mm thickness using in this study.The metal surfaces were prepared by grinding and polishing to roughness values below 20 nm?Ra?.Before we use,ultrasonic cleaning was done in 1%Triton X-100,acetone,and ethanol for 20 min in each materials.All metal samples were sterilized with autoclave.4.Cytotoxicity assayEA.hy926 cells were seeded on Niobium plates Titanium plates or PS as the method described above..After 2 days H₂O₂ solution?0/0.1/0.2mM?in DMEM basal medium without phenol was applied to the cells for 24 h.Cell activity was measured by the conversion rate of cell counting kit-8.5.Cell apoptosis detectionEA.hy926 cells were seeded on Niobium plates Titanium plates or PS as the method described above.After 2 days H₂O₂ solution?0/0.1/0.2mM?in DMEM basal medium without phenol was applied to the cells for 24 h.Cells in each group were harvested and stained with FITC/PI dye.Apoptotic rates in each group was tested by flow cytometry.6.Immunofluorescence stainingEA.hy926 cells were seeded on Niobium plates Titanium plates or PS as the method described above.After 2 days H₂O₂ solution?0/0.1/0.2mM?in DMEM basal medium without phenol was applied to the cells for 24 h.Cells on each materials were fixed in 4%PFA for 15 min and permeabilized with 0.1%Triton X-100 in PBS for 5min followed by 3 times washing with PBS.Then the cells were staining with rhodamine labelled phalloidin for 30min,the nucleus were stained with DAPI for5min.Cell morphology on each materials were observed by Fluorescence microscope.7.Determination of ROSOxidative stress was assayed utilizing the fluorescent probe DCDHF-DA.EA.hy926 cells were seeded on Niobium plates Titanium plates or PS as the method described above for 2 days.EA.hy926 cells were incubated with DCDHF-DA solution?20?M?in the DMEM cell culture medium for 30 min,then the cells were washed 3times with DMEM medium.H₂O₂ solution?0/0.1/0.2mM?in DMEM basal medium without phenol was applied to the cells for 1h.Fluorescence was measured 1 h after incubation with H₂O₂ using fluorescence microscope at 488 nm excitation filter and525 nm emission filter.8.Determination of GSH amountEA.hy926 cells were seeded on Niobium plates Titanium plates or PS as the method described above..After 2 days H₂O₂ solution?0/0.1/0.2mM?in DMEM basal medium without phenol was applied to the cells for 24 h.Cells on each materials were harvested and washed twice with PBS,then the cell were processed under ultrasonic and centrifuged at 12,000g.The protein amount of supernatants were quantified with BCA Assay.The amount of reduced glutathione?GSH?on each materials were determined by using commercial kits according manufacturer'instructions.9.SOD activity assaysEA.hy926 cells were seeded on Niobium plates Titanium plates or PS as the method described above.After 2 days H₂O₂ solution?0/0.1/0.2mM?in DMEM basal medium without phenol was applied to the cells for 24 h.Cells on each materials were harvested and washed twice with PBS,then the cell were processed under ultrasonic and centrifuged at 12,000g.The protein amount of supernatants were quantified with BCA Assay.The SOD level on each materials were determined by using commercial kits according manufacturer'instructions.Results:1.Oxidative stress model Building upCell activity decrease with increase of H₂O₂ concentration.We set 0mM H₂O₂group cell activity as 100%.From the results we can find that 0.1mM H₂O₂ group maintain nearly the same activity as 0mM group,while 0.2mM H₂O₂ group has nearly50%activity compared with 0mM group which meet IC50?half-maximal-inhibitory concentration?.0.5mM H₂O₂ group has only 0.9%activity as 0mM group.So we chose0,0.1,0.2mM H₂O₂ for following test.2.Cytotoxicity assayIn cell counting kit-8 assay,cell grown on Niobium Titanium and PS displayed no significantly difference?p>0.05?in?0/0.1/0.2mM?H₂O₂ concentration group.0.1mM H₂O₂ treatment did not significantly induce cytotoxicity while 0.2mM H₂O₂ induce half Inhibitory of cell activity.3.Cell apoptosis detectionThe apoptosis rate in cells exposed to Niobium,Titanium and PS surface showed no significant difference?p>0.05?in each group and raised by the elevation of H₂O₂concentration.4.Immunofluorescence stainingThere is no significant difference on Niobium,Titanium and PS that cells shape extended well in 0mM H₂O₂ condition,cells maintained same number and shape in0.1mM H₂O₂ condition while there are some cells become round up or even detached in0.2mM H₂O₂ condition.5.Determination of ROSIn ROS DCF-assay,cell grown on Niobium Titanium and PS displayed no significantly difference?p>0.05?in?0/0.1/0.2mM?H₂O₂ concentration.0.1mM H₂O₂treatment did not significantly increase ROS intensity while 0.2mM H₂O₂ treatment increase ROS intensity almost two times than 0mM H₂O₂ group.6.Determination of GSH amountThe level of GSH in cells exposed to Niobium,Titanium and PS surface showed no significant difference?p>0.05?in each H₂O₂ concentration and decreased by the elevation of H₂O₂ concentration.7.SOD activity assaysThe SOD activity in cells exposed to Niobium,Titanium and PS surface showed no significant difference?p>0.05?in each H₂O₂ concentration and decreased by the elevation of H₂O₂ concentration.Conclusion:Niobium compared to Titanium and PS showed no more cytotoxicity effect to Ea.hy926 cells which grown on their surface under different H₂O₂concentration condition.