Effects Of D-valine On Periodontal Or Peri-implantal Pathogens-Porphyromonas Gingivalis Biofilm

Backgrounds:Implant denture has become the preferred treatment for patients with dentition defect and dentition defect.The 10-year survival rate of implant denture has reached 97.1%,but its biological complication has not been significantly reduced.More than 30%of the total number of failed implants caused by peri-implant mucositis and peri-implantitis.Therefore,we must prevent the occurrence of peri-implant mucositis and further development of peri-implantitis to increase the survival rate of implant dentures.Similar to natural teeth,The initiating factor of peri-implant mucositis is also the formation of biofilm plaque.when the peri-implant is inflamed,its major flora is transformed from G~+to G~-bacteria.And Porphyromonas gingivalis is considered to be the closest bacteria which is associated with the development of peri-implantitis or even the final shedding of the implant.Therefore,to prevent the occurrence of peri-implantitis and prevent its further development,the key is to effectively control the occurrence of mucositis around the implant.Therefore,how to effectively remove the plaque biofilm on the implant surface is the key to prevent and treat peri-implantitis.Recent studies have found that microorganisms contain multiple D-amino acids,but the contents of D-amino acids only begins to increase after the bacteria have entered the plateau stage.The D-tyrosine,D-leucine,D-methionine and D-tryptophan synthesized by Bacillus subtilis during the biofilm dispersion phase could inhibit the formation of biofilm by the free bacteria and trigger the matured biofilms scattered.The inhibition of biofilms by these D-amino acids could be inhibited by enantiomeric L-amino acids,respectively.In addition,the sensitivity of Staphylococcus aureus to D-amino acids in different individuals was obviously different.The combination of D-amino acids could achieve the best destruction effect.The relationship between D-amino acids and biofilms has attracted the attention of various universities.However,the effects of D-amino acids on Porphyromonas gingivalis biofilms remains unclear.Objectives:This study was to investigate the effect of D-valine on the biofilm ofPorphyromonas gingivalis ATCC33277 in vitro.The research results may provide a theoretical basis for the clinical development of new drugs to prevent and treat peri-implantitis.Methods:In this experiment,Porphyromonas gingivalis(P.g ATCC33277)was cultured in Brain Heart Infusion Medium(BHI)and enriched by Brain Heart Infusion Liquid Medium.And draw the growth curve of P.g ATCC33277,select bacteria solution in exponential growth phase for next experiment.Through observing biofilms formed under different bacterial concentrations by fluorescence microscopy,the best biofilm formation concentration was determined.The effect of D-valine on the biofilm biomass of P.g ATCC33277 was detected by crystal violet staining.The effect of D-valine on extracellular polysaccharides produced by P.g ATCC33277 biofilm was detected by phenol-sulfuric acid method.Effect of D-valine on morphology of P.g ATCC33277biofilms were observed by scanning electron microscopy.Results:1.The growth curve of P.g ATCC33277 grew exponentially at the beginning of 48 h,and reached a stable period at the beginning of 60 h.2.The P.g ATCC33277 biofilm formed by the concentration of 10~8CFU/mL showed a grid-like structure,it was homogeneous,and connected tightly,similar to the water channel of the biofilm.And The structure of biofilm formed by the concentration of bacteria 10~7CFU/mL,showed a heterogeneous grid-like structure,the connection was sparse.However,the biofilm formed by 10~9 CFU/mL was an irregular bacterial aggregation without specific structures.3.D-valine at concentrations?20mM reduced the biofilm biomass of P.g ATCC33277and had significant statistical differences compared to the control group(P?0.05).The inhibition of D-valine showed concentration dependence.The higher of the concentration,the more obvious inhibitory effect presented.Compared with the control group,D-valine showed no significant reduction on P.g ATCC33277 biofilm biomass at concentrations from 0.1 mM to 10 mM(P>0.05).4.D-valine at concentrations?50mM reduced the amount of P.g ATCC33277exopolysaccharidesandhadsignificantstatistical differences compared to the control group(P?0.05).At the low concentrations from 0.1mM to 40mM,the effect of D-valine on P.g ATCC33277 has no significant difference compared with the control group.5.Scanning electron microscopy showed that the biofilms of P.g ATCC33277 did not differ significantly between the control group(0 mM D-valine group)and the 0.1 mM D-valine group,both groups of biofilms consist of multilayer bacteria.The biofilms of P.g ATCC33277 did not differ significantly from 1 mM D-valine group to 40 mM D-valine group and consisted of monolayer bacteria.From 50 mM D-valine group to 150mM D-valine group,bacterial biofilm accumulation is significantly reduced and cannot be connected to the membrane.More interestingly,high concentrations from 100 mM to 150 mM of D-valine turned bacteria morphology.Conclusions:1.D-valine could reduce the biofilm biomass of P.g ATCC33277 at the concentrations?20 mM.2.D-valine could reduce the production of extracellular polysaccharides of P.g ATCC33277 biofilm at the concentrations?50mM.3.D-valine at the concentrations?1 mM,the number of P.g ATCC33277 biofilm bacteria was significantly reduced and the biofilm was discontinuous.The morphology of P.g ATCC33277 was changed at high concentrations from 100 mM to 150 mM.