Study Of MTAN To Inhibit The Pathogenic Bacteria And Biofilm Of Peri-implantitis In Vitro

Objective:To detect the MIC and MBC values of MTAN,MTAN+EDTA against Porphyromonas gingivalis,Streptococcus sanguis and Fusobacterium nucleatum,and then study the effect of MTAN,MTAN+EDTA on the biofilm of Fusobacterium nucleatum on the surface of cementum tablets.Methods:1.The detection of MIC and MBC valuesAccording to the double dilution method,MTAN and MTAN+EDTA were diluted into 96-well plates,the concentration of nisin in each hole was 1000μg/ml,500μg/ml,250μg/ml,125μg/ml,62.5μg/ml,31.3μg/ml,15.6μg/ml,7.8μg/ml,3.9μg/ml,1.95μg/ml.Positive control group: 0.12% chlorhexidine solution;Negative control group: sterile water.1.1 Micro broth dilution method96-well plates were cultured in each culture condition for 48 h,and the minimum concentration of clear liquid in dark background was MIC.10μl was suctioned from all the wells in which the liquid was clear and was diluted by ten-times-dilution method,and 10μl of each well was suctioned and coated on BHI Agar medium,for aseptic growth was measured as MBC,the MIC was tested for three times.1.2 OD value methodThe original OD value of the diluted mixture was measured by a microplate micrograph(630nm)and recorded,the original number of each bacteria was got from the negative control group.After 24 hours of anaerobic culture in the mold incubator,the OD values of each pore were measured and recorded.The minimum concentration with the change of OD value less than 0.05 was denoted as theminimum inhibitory concentration(MIC).Count the number of bacteria in each concentration larger than the MIC,the minimum concentration was the minimum bactericidal concentration(MBC)when the survival number was reduced by more than 99.9%.Repeat three times to average.The mean value ±standard deviation of OD value was expressed after 24 hours.The single factor variance analysis(ANOVA analysis)was carried out by SPSS20.0 software,and the intra-group pairwise test was carried out by LDS-Test(test level α = 0.05).2.Effects of MTAN,MTAN+EDTA on biofilm formation of Fusobacterium nucleatumTwenty-four dental cemen slices with the size of 4mm×4mm were prepared and wrapped with aseptic saliva in a 37℃ thermostat for 24 h.The obtained membranes wereformed,then the slices were randomly divided into a 24-well plate and added with the same amount of bacterial solution and culture medium.Fusobacterium nucleatum biofilm was formed by anaerobic culture at 37℃ for 48 h.The biofilm was treated with MTAN and MTAN+EDTA with nisin concentration of 125μg/ml,250μg/ml,500μg/ml for 24 h.The negative control was aseptic water,and the positive control was 0.12% chlorhexidine.The morphology of biofilm on the surface of cementum was observed under scanning electron microscope(SEM).Results:1.The detection of MIC and MBC valuesBoth MTAN and MTAN+EDTA showed strong inhibitory effect on the growth and reproduction of three kinds of bacteria.The MIC and MBC values of MTAN and MTAN+EDTA measured by the two methods were consistent with each other.The results of statistical analysis showed that there was a significant difference in OD values between the groups with minimal inhibitory concentration above and the negative control group(P<0.05).The MIC of MTAN to Porphyromonas gingivalis,Streptococcus sanguis and Fusobacterium nucleatum were 125μg/ml,31.3μg/ml and 62.5μg/ml,the MBC was 500μg/ml,125μg/ml and 250μg/ml respectively.While the MIC of MTAN+EDTA to Porphyromonas gingivalis,Streptococcus sanguis and Fusobacterium nucleatum was 62.5μg/ml,31.3μg/ml and31.3μg/ml,the MBC was 250μg/ml,125μg/ml and 62.5μg/ml.2.Effects of MTAN,MTAN+EDTA on biofilm formation of Fusobacterium nucleatumUnder SEM,the biofilm of negative control group was dense,the growth of Clostridium nucleosus was crowed together and the bacteria was full.The number of biofilm bacteria in the positive control group was significantly decreased and distributed in a scattered manner.The number of Fusobacterium nucleatum biofilm bacteria exposed to MTAN and MTAN+EDTA with nisin concentrations of125μg/ml,250μg/ml and 500μg/ml decreased significantly,and with the increase of nisin concentration,the number of Fusobacterium nucleatum in the biofilm decreased significantly with the increase of nisin concentration.Under the condition of the same concentration of nisin,the fragmentation of Fusobacterium nucleatum was observed in MTAN+EDTA group,with the nisin tconcentration increasing,even the cell ablation occurred.Conclusion:1.Under the condition of this experiment,both MTAN and MTAN+EDTA had strong inhibitory effects on Porphyromonas gingivalis,Streptococcus sanguis and Fusobacterium nucleatum.Furthermore,the effect of MTAN+EDTA on inhibiting the growth of Fusobacterium nucleatum and Porphyromonas gingivalis in vitro was much stronger.2.With the increasing of the concentration of nisin,the inhibitory effect of MTAN on the formation of the biofilm of Fusobacterium nucleatum was stronger.When reaching a certain concentration,the structure and morphology of the biofilm were destroyed,and the destruction effect of MTAN+ EDTA was stronger.