Preliminary Study For The Efficacy Of Photodynamic Therapy On Microbe And Plaque Biofilm In Vitro

Background:Dental caries is one of the most common and frequent oral diseases,and dental plaque biofilm is main factor of caries. Extracellular polysaccharide especially insoluble polysaccharide is playing a decisive role on biological membrane structure, Such as: promote the adhesion of bacteria,the formation and development of biofilm, and play an important role in the formation of the disease incidence and concentration of symbiotic bacteria. Therefore, it is important to elucidate the amount of exopolysaccharides produced by dental plaque biofilm. Many scholars are looking for new ways to prevent dental caries, of which have greater potential application prospect including photodynamic therapy. Toluidine blue o was applied as photosensitizer to measure the amount of exopolysaccharides when cultivated with 1%sucrose,and treatment with 0.1%chlorhexidine served as a control,for discussing the feasibility of PDT for the prevention of dental caries. This study will refer the results of home and abroad to evaluate the susceptibility of bacteria in the planktonic phase to PDT.Discussing the safety of the periodontal tissues, on the basis to measure the amount of exopolysaccharides when cultivated with 1%sucrose,and treatment with 0.1%chlorhexidine served as a control,for discussing the feasibility of PDT for the prevention of dental caries.Material & Methods:1 The induction of Streptococcus sobrinus and Actinomyces viscosus. Freeze-dried bacteria Streptococcus sobrinus(S.s) and Actinomyces viscosus(A.v) were inoculated into BHI blood agar medium, 37℃micro-aerobic activated cultured 48h (80%N2,10%H2,10%CO₂). After Gram staining and biochemical identification,the same size and shape of colonies were moved to BHI liquid medium,re-inoculated in BHI blood agar medium , after 37℃micro-aerobic activated cultured 48h (80%N2,10%H2,10%CO₂),in accordance with the NCCLS standard method,adjust it by the 0.5McFarland (equivalent to 1.5×108CFU/ml) bacterial suspension and set aside.2 The inhibiting ability of PDT against treptococcus sobrinus(S.s) andActinomyces viscosus(A.v) in vitro. In dark room,each well of a 48 cell hole were placed with different concentrations of TBO 900μl, the two types of bacteria liquid 0.5McFarland standards were diluted 100-fold into 1.5×106CFU/ml, take 100μl bacterialObjective: suspension to each well, after the text, 100μl liquid from each well were diluted 100-fold by PBS, and then take 20μl drops on TSA agar plates, drops 3 times each partition, reading the results of colony counts after 48h. Groups: The first one as control received neither TBO nor light exposure,the second was respectively treated with 50μg/ml,100μg/ml TBO alone,the third respectively treated with 15min,25min LED(105J/cm²,635nm),the fourth were subjected to 50μg/ml,100μg/ml TBO and 15min,25min LED.3 The analysis of extracellular polysaccharides by artificial plaque biofilm treated with PDT.3.1 The preparation of artificial plaque biofilmLiquid sterilized agar were added in both sides of 6 cell holes,after it become hardened,22mm×11mm sterilized cover glasses were plugged in the agar,each hole with 3 cover glasses,A.v bacterial suspensions and BM-5 medium were mixed into 6 cell holes at ratio of 1:9 (V/V),fresh BM-5 medium was changed every 12h. After 4 days,L.a were mixed in the same way,and then S.m and S.s were mixed in the same way after next 4 days. It was needed for observing 4 days when mixed-biofilm formed. The groups as described,and plus thefifth group: 0.1% chlorhexidine 15min.3.2 The analysis of extracellular polysaccharides.The cover glasses as described were removed from the 6 cell holes and placed with 10ml 10g/L sucrose TSA liquid medium in tubes, 37℃micro-aerobic activated cultured 18h, scraped and collected the bacteria on the cover glasses and collected the culture medium,then centrifugated at 4℃750×g 30 min, discarded supernatant,The bacterial sediment was washed with 5ml distilled water, then centrifuged 2 times, combined supernatant, that is soluble polysaccharide; The washed bacteria was washed with 5ml 0.4 mol/L NaOH, centrifuged 3 times, combined supernatant, that is insoluble polysaccharides. Proper amount of supernatant, The anthrone method was applied to measure the amounts of extracellular polysaccharides produced by artificial dental plaque biofilm attached S.sobrinus and planktonic ones measured by anthrone method of extracellular polysaccharide4 The effect of PDT on periodontal tissues of miceFifty-four mice were randomly divided into nine groups; the experimental photodynamic therapy (PDT) group was respectively treated with 50μg/ml,100μg/ml TBO and 15min,25min LED(105 J/cm²,635nm).The other groups were subjected to 15min,25min laser irradiation alone or to 50μg/ml,100μg/ml TBO alone or received neither TBO nor light exposure. All the mice were killed 3 days after they had been subjected to PDT, and periodontal tissue samples were taken for histological examination.Results:1 S.s inhibitory rate: TBO 50μg/ml with 15min, 25min LED illumination inhibitory rate was 89.13%, 91.30%. TBO 100μg/ml with 15min, 25min LED illumination inhibitory rate was 94.02%, 96.20%。After analysised by single-factor of variance F = 2.88 P <0.05,have interaction.A.v inhibitory rate: TBO 50μg/ml with 15min, 25min LED illumination inhibitory rate was 64.71%, 91.18%. TBO 100ug/ml with 15min, 25min LED illumination inhibitory rate was 91.18%, 88.24%。After analysised by single-factor of variance F = 9.95 P <0.05,have interaction.2 After treated, the insoluble polysaccharides: the group and the control group were not significantly different (P> 0.05), chlorhexidine and control groups were significantly different (P<0.05);the soluble polysaccharides: Each group and the control group were not significantly different (P> 0.05), chlorhexidine and control groups were significantly different (P <0.05).3 During the 72h observation period, no mice showed necrotic,ulcer,hyperplasia or inflammatory changes in the gingival, dental pulp or alveolar bone of any of the mice in this study.Conclusion:1 Photodynamic therapy killed more than 90% of bacteria present in Suspension suggests that it is sensitive.2 The combined use of a TBO and LED was not bactericidal to artificial dental plaque biofilm and was unable to achieve total kill, unlike 0.1% chlorhexidine.3 The results suggest that TB-mediated PDT is a safe antimicrobial approach for the prevention of dentine caries without damaging effects to adjacent normal tissues.