Toxicity Of Porcelain Fused To Metal Substrate To Zebrafish(Danio Rerio) Embryo And Larva

Objective: Porcelain-fused-to-metal(PFM)restorations are the classic standard and most commonly used restoration technique for dentistry,but the mechanism of PFM metal substrate toxicity in vivo is not yet clear.To evaluate its safety and provide a reference for the choice of clinical prosthetic material,in this study,the metal shell of PFM crowns were chosen to detect and compare the effects on the development of zebrafish embryos and larvae.Materials and Methods: The metal substrates of Au-Pd alloy,Ag-Pd alloy,Ni-Cr alloy,Co-Cr alloy and Ti alloy porcelain crown were immersed in artificial saliva for 1,4,7 weeks respectively,and then the leachate was collected.Zebrafish embryos at 4-144 hours postfertilization(hpf)were exposed to leachate to examine their effects on zebrafish embryo and larva.Toxicity was tested by statistical analysisof mortality,spontaneous movement,heart rate,hatchability,malformation andswimming behavior of zebrafish embryo and larvae.The normal 4hpf embryos were pretreated with the extract and the reactive oxygen species in the 24 hpf embryos were detected using the DCFH-DA fluorescence probe;at 24 hpf,detected using acridine orange(AO)staining and real-time fluorescence quantitative analysis(RT-PCR).The expression of apoptotic cells and apoptosis-related genes in zebrafish embryos of control and experimental groups was examined.Results:All 1-week-leachates of five PFMs showed no toxicity to zebrafish.The rate of mortality and malformation of zebrafish in the Ni-Cr alloy group,for 4W and 7W leachates,were significantly increased,meanwhile,spontaneous movement,heart rate and swimming behavior were significantly decreased.Effects of nickel-chromium alloy extracts on ROS level and SOD activity in zebrafish embryos: At 24 hpf,ROS levels and SOD activity in zebrafish embryos were not different between the 1W group and the control group.Zebrafish embryos in the 4W and 7W groups were not observed.In vivo ROS levels were significantly elevated and SOD activity was significantly reduced(P<0.001).The effect of nickel-chromium alloy extract on apoptosis of zebrafish embryos:AO staining showed that there was no significant difference in the early apoptotic cells ofzebrafish embryos in 1W group compared with the control group,and 4W and 7W groups could significantly increase apoptosis;RT-PCR showed that the expression levels of Bax,Caspase9 and Caspase3 in the 1W experimental group were not statistically different from those in the control group(P<0.05).Compared with the control group,the expression of the anti-apoptotic gene Bcl2 decreased in the experimental group.(P<0.05);Compared with the control group,the expression of proapoptotic gene Bax,Caspase9 and Caspase3 was significantly up-regulated in the 4W and7 W experimental groups,and the anti-apoptosis-related gene Bcl2 was also significantly down-regulated(P<0.05).Conclusion:Among these metal substrates commonly used in clinical practice,Nickel-chromium alloy has the strongest toxicity,followed by cobalt-chromium alloy and silver-palladium alloy.Titanium alloy and gold alloy have good biocompatibility.Thence the most suitable alloy material for patients in clinical work should be chosen in consideration of biotoxicity of the alloy.The nickel-chromium alloy 7W extract on the one hand increases the ROS source due to the direct effect of metal ions,and on the other hand,it inhibits the elimination of ROS by inhibiting SOD activity,causing ROS to accumulate in zebrafish embryos and then through oxidative stress.The reaction caused damage to the zebrafish embryo.The nickel-chromium alloy 7W extract on one hand promotes the apoptosis by promoting the expression of apoptosis-related genes,and on the other hand inhibits the repair of DNA damage by inhibiting the expression of anti-apoptosis-related genes,causing damage to zebrafish embryos.