Research Of Growth Inhibition Effect Of Antimicrobial Peptides On Peri-implantitis Pathogenic Bacterium And Biofilm

Objective: Select three kinds of synthetic antimicrobial peptides，research theireffects on inhibiting the three kinds of peri-implantitis pathogenic bacteria experimentstrains. Meanwhile testing different concentrations of antimicrobial peptidescytotoxicity for human osteoblast cell line MG63cells, then select one antimicrobialpeptide to research its effects on porphyromonas gingialis biofilm on smooth puretitanium discs. It can be used to fix and assemble on the surface of pure titaniumimplant materials to endow the implant antimicrobial property. To explore a new type ofimplant antimicrobial coating, so this research can provide the scientific basis for it.Methods:1.Effects of antimicrobial peptides Pac-525、KSL-W、KSL on peri-implantitispathogenic bacteriumAntimicrobial peptides Pac-525、KSL-W、KSL according to their amino acidsequence, and study their purity and integrity through high performance liquidchromatogram(HPLC) and mass spectrographic (MS). Antibacterial SusceptibilitoryTesting procedure is based on the Standards broth microdilution method to research theminimal inhibitory concentration(MIC) and minimal bactericidal concentration (MBC)of antimicrobial peptides for three kinds of experiment strains Streptococcussanguis(S.sanguis), Fusobacterium nucleatum(F.nucleatum), porphyromonasgingialis(P.gingivalis). Evaluate the bacteriostatic effects of the three syntheticantimicrobial peptides.2.Cytotoxicity experiments of antimicrobial peptides Pac-525、KSL-W、KSLCytotoxicity experiments is based on MTT assay to evaluate the effects of the threes antimicrobial peptides on MG-63cells3.Effect of Pac-525on P.gingialis biofilm on smooth pure titanium discs. Titanium discs preparation: The test specimens in the form of discs10mm indiameter and1mm thick were ground down and polished one side as a mirror finish andthen they were sterilized in ethylene oxide, air-dried and stored in a desiccator.Saliva collection for biofilm inoculums: Unstimulated human saliva was collectedfrom four healthy,non-smoking donors, Saliva was centrifuged (30min,4500rpm,4℃),and the supernatant was sterile-filtered, and stored at4℃.Model of P.gingialis biofilm on smooth pure titanium discs: Titanium discs weresoaked under37℃with saliva for24hours, for the formation of acquired film on thesurfaces. Titanium discs were transferred to the sterile24well plates. Bacteriumsuspension was added500ml per well, then add1500ml medium. After anaerobiccultured48hours, the samples were observed under scanning electron microscopy(SEM).Effects of Pac-525on P.gingialis biofilm on smooth pure titanium discs: Selectthree concentrations which are greater than the MIC of Pac-525vs. P. gingialis. CLSMwas used to observe activity of three concentrations of Pac-525treated or untreatedbacterial biofilm.Then calculate the biofilm thinckness and percentage of livingbacterium using microsofts and SPSS17.0.Results:1.Effects of antimicrobial peptides Pac-525、KSL-W、KSL on peri-implantitispathogenic bacteriumThree kinds of antimicrobial peptides were synthesized successfully, We can acceptthat the Pac-525MIC and MBC to S. sanguis is0.125mg/ml and0.500mg/ml; MIC andMBC to F.nucleatum is0.0078mg/ml and0.0156mg/ml; MIC and MBC to P.gingivalisis0.0625mg/ml and0.125mg/ml. The KSL-W MIC and MBC to S. sanguis is notmeasured; MIC and MBC to F.nucleatum is0.0156mg/ml and0.0313mg/ml; MIC andMBC to P.gingivalis is0.125mg/ml and0.500mg/ml. The KSL MIC and MBC to S.sanguis is not measured too; MIC and MBC to F.nucleatum is0.0156mg/ml and0.0313mg/ml; MIC and MBC to P.gingivalis is0.125mg/ml and0.500mg/ml.2.Cytotoxicity experiments of antimicrobial peptides Pac-525、KSL-W、KSLUnder the effects of three kinds of antimicrobial peptides, the viability of the MG63cells was not affected compared with the DMEM negative control group, with nosignificant difference (P＞0.05).Viability of MG63cell is more than93%. 3.Effect of Pac-525on P.gingialis biofilm on smooth pure titanium discs.After anaerobic cultured48hours,Trough the SEM it shows that the model ofbacterial biofilm formed well on smooth pure titanium discs. Observed under theconfocal laser scanning microscope（CLSM）, in the chlorhexidine group,0.25mg/mlgroup and0.5mg/ml group,there are large area of red fluorescence, dead bacteriaincreased, biofilm thickness and percentage of living bacterium was decreasedobviously with significant difference (P＜0.05).Conclusions:1.Pac-525, KSL-W, KSL with high purity were synthesized by artificial solid-phasepeptide synthesis. The Pac-525groups have visible bacteriostasis effect to all bacterium,the MIC is0.0078mg/ml～0.125mg/ml, the MBC is0.0156mg/ml～0.500mg/ml. Butthe KSL-W and the KSL MIC and MBC to S. sanguis have not been measured under thetest concentration ranges. It suggested that the bacteriostasis efficiency of the Pac-525isbetter than the KSL-W and the KSL.2.The antimicrobial peptides Pac-525, KSL-W, KSL have no cytotoxicity on MG63cell.3.Pac-525has significant destructive effect on the biofilm formed by P.gingialissingle experimental strain in vitro which demonstrates that Pac-525has effectiveness ofanti-peri-implantitis, and the inhibition effect of0.25mg/ml and0.500mg/ml Pac-525group are significant.