Experimental Study On The Effect Of Magnesium Ion On Osteogenic Differentiation Of Human Periodontal Ligament Cells Through Classical Wnt Signaling Pathway

ObjectiveThis paper is based on the study of the effect of gradient magnesium ion(Magnesium Ions,Mg2+)on the osteogenic differentiation of human periodontal ligament cells(PDLCs),and further detection of magnesium ion-mediated osteoblast-related factors in PDLCs.The law of action,and the preliminary exploration of the possible mechanism(Wnt pathway)from the perspective of pathway,in order to provide theoretical basis and experimental basis for the advancement of clinical application of magnesium ion to periodontal tissue regeneration therapy.MethodP3-P5 generation PDLCs were used for inoculation,and the following tests were performed:(1)The proliferation rate of PDLCs in each group was detected by CCK-8 method for 1,3,5,and 7 days.(2)Quantitative detection of ALP activity in PDLCs induced by osteogenic induction by alkaline phosphatase(ALP)activity assay.(3)Using alizarin red staining to observe the degree of mineralized nodules formed by osteogenic-induced PDLCs for 21 days.(4)Real-time quantitative PCR(Realtime PCR)was used to detect the expression of osteogenic related genes ALP,Runx2,ColI and OPN in PDLCs after osteogenic induction,and compare the Wnt signaling pathway related gene Gsk-3β.Differences in expression of Frizzled 3,LRP5,β-catenin,LEF1,Cyclin D1,C-myc,and DKK1.Statistical analysis:SPSS 20.0 software was used in this study.Student t test was used for comparison between the two groups,One-way ANOVA was used for multiple groups.P value<0.05,the difference was statistically significant.Results(1)The results of CCK-8 showed that the cells in the 0-15 mmol/L Mg2+ treatment group continued to proliferate,and the 20,40,50 mmol/L Mg2+treatment group continued to decrease with increasing concentration of cells at 3 days,and the difference was significant.Statistical significance.The speed of the 25 mmol/L Mg2+treatment group was significantly slowed on the 5th day,and the proliferation rate on the 7th day was not significantly different from the control group.The speed of the 30 mmol/L Mg2+treatment group was significantly slowed on the third day,and the proliferation rate on the fifth day was not significantly different from the control group.Compared with the control group,the cell proliferation rate increased on the 7th day of the 5 mmol/L Mg2+group and the cell proliferation rate increased on the 3rd and 5th day of the 7.5 mmol/L Mg2+group.(2)The results of ALP activity test showed that ALP activity in the 15 mmol/L Mg2+ treatment group was significantly higher than that in the other groups after 3 days of PDLCs replacement,and there was a statistically significant difference compared with the control group;the 50 mmol/L Mg2+treatment group significantly down-regulated ALP activity.The ALP activity of 7.5 and 20 mmol/L Mg2+treatment group was higher than that of the control group,and there was significant statistical difference.The trend of osteogenic induction of PDLCs on day 7 was similar to that of day 3,and the ALP activity of 7.5 mmol/L Mg2+treatment group was significantly higher than that of other groups,and there was significant statistical difference.(3)The results of alizarin red staining showed that after 21 days of osteogenic induction of PDLCs,a large number of red deep-stained mineralized nodules were observed in the control group,and only a small amount of spotted mineralized particles appeared in the 7.5 mmol/L Mg2+treatment group.There were no signs of mineralization in the group,and the difference was statistically significant.(4)Real-time PCR:The expression levels of the control group ALP,Coll and RUNX2 on the 7th day were higher than those on the 3rd and 14th day.OPN maintained the lower expression level on the 3rd and 7th day,and the 14th day was significant.Up.Compared with the control group,the expression of Runx2 and ALP in the 7.5 mmol/L Mg2+treatment group continued to increase on the 3rd,7th,and 14th day and was higher than that in the control group.The expression of OPN continued to be low,and there was a statistically significant difference in Coll.Not statistically significant.In the control group,the expression of the wnk signaling pathway related factor Gsk-3βwas down-regulated on the 7th day,and the expression level was close at 3 and 14 days;β-catenin and Frizzled 3 were up-regulated only at 14 days;LEF1,Cyclin D1 and Cmyc genes were at the 3rd,7th and 14th The expression of day was continuously up-regulated;DKK1 expression was significantly up-regulated on day 14,and the expression level of LRP5 was higher on day 7 than on day 3,and on day 14 was lower than on day 3,there was a statistically significant difference.Compared with the control group,GSK-3β and Fizzled 3 in the 7.5mmol/L Mg2+treatment group did not change significantly at 3 time points;LRP5 continued to down-regulate on the 3rd,7th and 14th day;β-catenin,DKK1,Cyclin D1 The expression level gradually increased gradually with time.LEF1 and Cmyc did not change significantly on the 3rd and 7th day,and were up-regulated on the 14th day.ConclusionsMagnesium ions at 7.5-15mmol/L could increase the expression of ALP and Runx2 in PDLCs and decrease the expression of OPN.The expression of early osteogenic factors and the degree of mineralization decreased by PDLCs after treatment with magnesium ions and the Wnt signaling pathway Activation inhibition is relevant.