The Effect Of Artificial Synthesis Antimicrobial Peptides Pac525on The Formation Of Peri-implantitis Pathogenic Bacteria Biofilms In Vitro

Objective： This experiment selects artificial synthesis antimirobial peptidespac525which has been proved in our preliminary experiments that has differentinhibiting effects on three pathogenic bacteria of peri-implantitis includingPorphyromonas gingivalis (P.Gingivalis), Fusobacterium nucleatum(F.nucleatum)andStreptooccus sanguis(S.sanguis) for further research.We draw the kill-time curve ofthese three bacteria through living bacteria counting,in order to find inhibiting functionsof Pac525under the condition of different time and different drugconcentration.Meanwhile,we build bacteria biofilm of these three bacteria, usingscanuining electron microscopy (SEM) to observe the growth.Also we use crystal violetspectrometry to test the minimum biofilm inhibition concentration(MBIC) and theminimum biofilm reduction concentration(MBRC) of pac525.Methods:1.the kill-time curve of three pathogenic bacteria of peri-implantitisAccording to the amino acid sequence of pac525, we compound the syntheticantimicrobial peptide. We draw the kill-time curve of these three bacteria through livingbacteria counting,in order to find inhibiting functions of Pac525under the condition ofdifferent time and different drug concentration.2.three pathogenic bacteria biofilms built on pure titanium surface To simulate oral environment, we immerse the smooth titanium discs with saliva aspretreatment (37℃,24hours), then,the surface could form acquired biofilm. Movetitanium discs into24well plates, wash their surfaces slightly with PBS, then add1500ml BHI liquid medium and500ml bacteria suspension per well, each will be cultivatedat their condition at for24to48hours, then observe the form of the biofilm usingscanning electron microscopy (SEM).3.Effect of pac525on three pathogenic bacteria biofilmsThe measurement of MBIC (minimum biofilm inhibition concentration)：we setup three kinds of bacteria on96-well plates respectively, add200m l Pac525liquidculture medium and20m l1x107CFU/ml bacteria liquid, cultivate at37℃for24to48hours, wash with PBS, fix with maldehyde, crystal violet stain, sterile deionized waterflushing, ethanol is relapsed, measure absorbance with enzyme standard instrument.According to the formula we calculate the MBIC. Use the SPSS17.0statistical softwarefor statistical analysis.The measurement of MBIC (minimum biofilm reduction concentration): add200ml BHI liquid medium into96-well plates and20ml1x107CFU/ml bacteriasuspension, then cultivate at37.C for24to48hours, suck out planktonic bacteria,washwith PBS, add200μ l pac525liquid medium into wells,cultivate for24to48hours,suck out planktonic bacteria,wash with PBS, fix with maldehyde, crystal violetstain, sterile deionized water flushing, ethanol is relapsed, measure absorbance withenzyme standard instrument. According to the formula we calculate the MBIC. Use theSPSS17.0statistical software for statistical analysis.Result:1.The kill-time curve of three pathogenic bacteria of peri-implantitisThrough the kill-time curve we can see that with the change of time and differentconcentrations of Pac525,all results showed a certain effection of anti-bacteria.ForS.sanguis, at0.5h,different concentrations of Pac525showed different degrees ofanti-bacteria, especially on2mic and4mic;at1h,4mic has killed all the S.sanguis;at3.5h,we don’t find obvious change; at1h,2MIC’s result showed a stead change, all maintained at a higher level. For F.nucleatum, at4h Pac525which shows differentdegrees of different concentration of anti-bacteria, at8h2MIC has killed all theF.nucleatum, MIC of anti-bacteria effect tend to be stable at this time. For P. Gingivalis,at4h2MIC has killed all the bacteria, at8h MIC also has killed all the bacteria.Results showed that pac525effect on bacteria is positively correlated with theconcentration of the drug and time.2.Three pathogenic bacteria biofilms built on pure titanium surfaceBy one-way anova analysis(P <0.05),pac525of S.sanguis MBIC50is1mg/ml,8times of the MIC. Pac525to F.nucleatum MBIC50is0.125mg/ml,16times of the MIC.Pac525to P. Gingivalis MBIC500.5mg/ml,8times of the MIC. But MBRC has notbeen measured.Conclusions:1.Pac525anti-bacteria effect on peri-implantitis pathogenic bacteria can bechanged with time and the concentration of the drug.2.Pac525can damage bacteria plaque biofilm. MBIC is8~16times of the MIC.3.MBRC50cannot be measured, it is likely to be related to its early bacteriostasis.