Effect Of Matrix Metalloproteinases Inhibitors On The Acid Resistance Of Dentin

Objective:?1?To evaluate the effect of various matrix metalloproteinase?MMPs?inhibitors on the acid resistance of human dentin and select the best protocol.?2?To evaluate the effect of the MMPs inhibitor selected based on the in-situ experiment.?3?To explore the application mechanism of MMPs inhibitors by measuring thickness of the demineralized organic matrix?DOM?and content of cross-linked carboxyterminal telopeptide of type I collagen?ICTP?.Methods:?1?A total of 104 sound dentin samples?2 mm?2 mm?2 mm?were collected and prepared.The specimens were randomly divided into 8groups:,deionized water and ethanol?solvent of quercetin?as negative control groups,1.23?10⁴?g/ml sodium fluoride as positive control group,120?g/ml chlorhexidine,183.2?g/ml epigallocatechin gallate?EGCG?,75?g/ml,150?g/ml,and 300?g/ml quercetin?Q75,Q150,Q300?.Two h before daily acid challenge,the samples of each group were immersed in the respective solutions for 2 min and rinsed with deionized water,the samples were then subjected to 4 cycles of acid challenges.This protocol was applied for 7 consecutive days.The surface microhardness?SMH?and surface profile were measured before and after erosion by using the surface microhardness tester and contact profilometry.Scanning electron microscope?SEM?images were taken to observe the surface morphology of the samples.MMPs inhibitor with the best potential of improving the acid resistance of human dentin were further screened using biosafety analysis.The data were analyzed by one way ANOVA and t test.The best type of MMPs inhibitor was selected according to the result of application effect combined with biological safety analysis.?2?The MMPs inhibitor selected was evaluated,against 1.23?10⁴?g/ml sodium fluoride,using an in-situ study model.In vitro acid challenge and in situ remineralization protocol was applied.Ten volunteers?aged from 18 to 30 y?were recruited to make removable jaw pads respectively.Dentin samples?3 mm?4 mm?2.2 mm?were placed in the removable jaw pad,and a window was opened on the dentin surface of the sample.The samples were subjected to 4 cyclic acid challenges every day for 7 consecutive days.The measurements were performed as mentioned above.The independent sample t-test was used for the statistical analysis to evaluate the data.?3?For the first part,dentin specimens were prepared as mentioned above and randomly divided into the following groups:the MMPs inhibitor protocols identified by the in vitro and in situ studies as the experimental groups,the solvent of the MMPs inhibitor as the control group.In vitro erosion cycles were performed as described above.The surface profile after erosion was recorded as P₁.The specimens were then immersed in the artificial saliva containing 100 U/ml type I collagen enzyme at 37°C for 5 d.The surface profile was measured again as P₂.The difference between P₂ and P₁ was obtained and used to calculate the DOM thickness of dentin specimens.For the second part,specimens were treated with 10%phosphoric acid and desiccated,immersed in the respective solutions for 2 min,rinsed and immersed in the artificial saliva at 37°C for 7 d.he content of ICTP in artificial saliva was measured.One-way ANOVA and t test were used to explore the correlation between the effects of MMPs inhibitor and thickness of DOM.Results:?1?After in vitro erosion cycles,SMH%and surface loss of samples in all the experimental groups were significantly reduced than the control groups?P<0.05?.Based on these results,quercetin exhibited significantly better potential against erosion compared with other MMPs inhibitors and the effect was concentration dependent.The relative growth rates of Q75,Q150 and Q300 groups were?108.48±4.85?%,?81.15±2.82?%,and?75.67±0.2?%,respectively,indicating the good biosafety.?2?In the in situ experiment,the SMH%and surface loss in the positive control group and experimental group?Q300?were less significant than the results of in vitro experiment.The SMH%and surface loss of dentin specimens in Q300 group were significantly lower than those in sodium fluoride group?P<0.05?.?3?The DOM thickness in quercetin groups was significantly greater than that of the control group?P all<0.01?.The content of ICTP in the MMPs inhibitor groups was significantly lower than that in the control groups?P all<0.01?.Conclusion:?1?Based on the in vitro study,SMH%and surface loss of samples in each experimental group were significantly reduced compared with the control group?P<0.05?,in which quercetin had greater effect than other MMPs inhibitors.Combined with the cell toxicity analysis,quercetin showed good potential against dentin erosion.?2?Based on the in situ experiment,quercetin showed greater effect on acid resistance of human dentin compared with sodium fluoride.?3?The mechanism of the MMPs inhibitor is related to the preservation of DOM,and MMPs inhibitors could blocked the release of ICTP during erosion.