Effects Of Dental Alloys With Different Manufacturing Methods On Apoptosis And Autophagy Based On 3D OMM

Objective: Based on the establishment of 3D tissue engineered oral mucosa tissue model,the purpose of this study was to study the expression of apoptosis and autophagy related markers in human oral mucosa under the influence of digital manufacturing or traditional casting dental alloys.It provides experimental basis and guidance for the selection of dental alloys with different manufacturing methods in clinical work.Methods:(1)In this study,Seven commonly used dental alloys with different manufacturing methods,such as cast gold-platinum alloy,cast Co-Cr alloy,computer-aided design / computer-aided fabrication(CAD/CAM)milling Co-Cr alloy,selective laser melting(SLM)Co-Cr alloy,cast titanium,CAD/CAM milling titanium and SLM titanium,are immersed in the cell culture medium for 72 hours,The extract was prepared as the experimental group and the cell culture medium as the blank control group.Firstly,the types and concentrations of metal ions in the extract were detected by inductively coupled plasma mass spectrometry(ICP-MS).(2)Then using the extract to continuously stimulate 3D tissue engineered oral mucosa tissue model(constructed by 3D layered co-culture of human oral keratinocytes and human gingival fibroblasts mixed with matrix)for 72 hours,the stimulated tissue is then fixed and paraffin embedded.There comes the detection of four markers in tissues by immunofluorescence double staining: apoptosis-related proteins Caspase-3,TUNEL(TdT-mediated dUTP nick-end labeling)and autophagy associated proteins p62 and LC3 B.By comparing the positive cell rate of markers between the control group and each experimental group,the effects of different manufacturing methods(digital manufacturing and traditional casting)on the apoptosis and autophagy of oral mucosa cells were analyzed,and the correlation between the apoptosis and autophagy of dental alloys and the concentration of metal ions in the extract was analyzed.Results:(1)ICP-MS showed that metal ions could be detected in the extract of dental alloys in each group.Except for CAD/CAM Ti and SLM Ti,the total concentration of metal ions was significantly higher than that in the blank control group.Compared with the traditional wax loss casting method,digital manufacturing can significantly reduce the total concentration of metal ions precipitated by Co-Cr and CP-Ti in the extract;(2)Immunofluorescence double staining showed that Co-Cr can induce oral mucosal cells to early apoptosis and autophagy.The effect of CAST Co-Cr on early apoptosis and autophagy was the most obvious,and Cp-Ti could increase the level of autophagy in oral mucosa.The CAST Au-Pt can also cause the increase of autophagy level of oral mucosa cells.However,the increase degree of CAST Au-Pt was not as significant as that of CAST Co-Cr;Seven kinds of dental alloys with different manufacturing processes had no significant effect on the late apoptosis of oral mucosal cells;(3)Correlation analysis showed that metal ions had no significant effect on the late apoptosis of oral mucosal cells.The total concentration was highly positively correlated with the degree of early apoptosis in tissues.There was a low or moderate positive correlation between autophagy and autophagy.Conclusions: Compared with Co-Cr,Au-Pt and CP-Ti have less biological effect on oral mucosal cells and show better biocompatibility,which provides a reference for the selection of prosthesis materials in clinical work.Compared with the traditional wax-loss-casting method,the use of new digital manufacturing methods,such as CAD/CAM or SLM,can significantly improve the chemical stability of dental alloys and reduce the precipitation of metal ions.So as to reduce the biological effects of dental alloys on oral mucosal cells,which also laid a theoretical foundation for the popularization of digital manufacturing technology.