Antimicrobial Effects Of Antiseptics With Photodynamic Therapy On Biofilm On Titanium Surface

ObjectiveThe aim of this in vitro study is to investigate the decontamination efficacy of combined application ofantiseptic agents andantimicrobialphotodynamic therapy（PDT）of titanium surfaces previously infected with a porphyromaonas gingivalis（Pg）or Staphylococcus aureus（Sa）biofilm.Methods（1）Polished and sandblasted large-grit acid-etched（SLA）titanium disks were prepared and their surface properties including roughness and surface free energy（SFE）were measured.（2）Pg and Sabacteria suspension were adjusted to 1×10⁸ cell/mL and then inoculate into polished and SLA titanium disk,respectively.The incubation time of Pgwas set as 72 h whileSawas 48 h.Colony forming units（CFU）was used to asses this two bacteria adhesion on two different titanium surfaces.（3）Pg biofilms were grown on polished（n=24）and SLAtitanium surfaces（n=24）for 72 h.Then thetitanium disks were randomly distributed into four different treatmentgroups as follows:（i）immersed in phosphate-buffered saline（PBS）,（ii）immersed in 0.2%CHX,（iii）application ofPDT,（iv）immersed in 0.2%CHX and subsequent PDT.The decontamination effect of each modality was assessed by microbialculture analysis.Residual bacteria amount and cell structure after different treatments was observed by scanning electron microscopy（SEM）.Confocal laser scanning microscopy（CLSM）was conducted to evaluate live/dead bacteria and biofilm structure on titanium surface.（4）Sa biofilm was incubated on polished andSLA titanium surfaces for 48h.Then 72titanium disks（36 polished,36 SLA）were randomly allocated into six experimental groups.Disks immersed in PBSserved as control.（i）PBS,（ii）0.2%CHX,（iii）3%hydrogen peroxide（3%H₂O₂）,（iv）PDT,（v）0.2%CHX+PDT,and（vi）3%H₂O₂+PDT.After treatments,remaining bacteria were cultured and CFU values were measured to determine antimicrobial effects.Residual bacteria and biofilm structure were evaluated using SEM and CLSM.Results（1）Statistical analysis showed all roughnessparameters of SLA titanium disks were significantly higher than those in polished disks.Polished specimens present significantly higher surface free energy（42.35±2.60 mJ/m²）than SLA ones（31.57±4.08 mJ/m²）.（2）The adhesion of Pg on polished titanium disk was 7.02±0.18×10⁷CFU/mL and on SLA titanium disk was 6.87±0.30×10⁷CFU/mL,statistical analysis showed that there is no significant difference between them.The adhesion of Sa on polished titanium disk was 6.98±0.21 CFU/mL and on SLA titanium disk was 7.03±0.18CFU/mL,which is no significant difference between them according to statistical analysis.（3）There was significant difference in the number of remaining alive Pg between the polished and the SLA titanium disks treated with 0.2%CHX or PDT when compared to PBS group.No significant difference was observed in the remaining bacterial load between 0.2%CHX and PDT group.CFU results showed combination protocol （0.2%CHX+PDT）was the mosteffective in eradicatingPg onpolished and SLA titanium disks（P<0.01）.SEM observation showed combined treatment’s bacteria reductionwas significantlygreater than that in 0.2%CHX or PDT groups.Bacteria structure was damaged and cell wall shrinkage was observed in 0.2%CHX+PDT group.CLSM indicated that biofilm depth,bacteria density and dead/live bacteria ratio were changed in various degrees.The most severe structural destruction was found in the 0.2%CHX+PDT group biofilm which was predominantly composed of dead cells.（4）When compared with control group,CFU counts in all decontamination groups were reduced significantly（P<0.001）.PDT group presented the strongest decontamination ability in eliminating Sa biofilm attached to polished and SLA titanium disks in sole decontamination modality（P<0.05）.Preceding antiseptic administration（0.2%CHX/3%H₂O₂）with PDT was the most effective treatment in reduction of adhered bacteria number（P<0.001）,there is no significant difference between 0.2%CHX+PDT group and3%H₂O₂+PDT group.As a negative control,PBS group showed largely intact Sa biofilm which was predominantly composed of living bacteria.After treatment with either 0.2%CHX,3%H₂O₂,or PDT,damage of biofilm structure and a large number of dead cells were observed in CLSM images.The 0.2%CHX+PDT and 3%H₂O₂+PDT group exhibited the most severe structural destruction and the highest proportion of dead cells.Conclusion（1）Surface properties of polished and SLA titanium disk were approximately consistent with clinical implants,which is suitable for further experiment.（2）Polished and SLA titanium disks exert no obvious effect on bacteria adherence.（3）Combination protocol（0.2%CHX+PDT）was the most effective in eradicating Pg（P<0.05）on both polished and SLA surface.（4）The combination of 0.2%CHX or 3%H₂O₂ administration with PDT was more effective in eradicating S.aureus on both polished and SLA Ti disks in comparison with either treatment alone.