Dental caries is thirdly common diseases in the world followed cardiovascular diseases and cancer．Streptococ—ells mutans(S．mutans)，the primary etiological agent of human dental caries，has developed multiple mechanisms to colonize and form biofilms on the tooth surface．
1t is important to control the growth of S。mutans of the oral biofilms for preventing dental caries。Ano tibacteriat agents has been widely used，but problems with general efficacy due to access of topical agents tonplaque[1]and the possibility of development of bacterialnresistance[2j mean alternative strategies are desirable to control plaque and treat caries。
Photodynamic therapy fPDT)may emerge a8 a suit—able process to combat both biofilm and antimicrobial．related resistance．Since dental caries are localized infections．such plaque-related diseases would be well suited to PDT。The photodynamic approach to kill bacterian i8 clearly a rapidly emerging alternative to current antimicrobialbregimens[31。SignificantlY,it is unlikely thebbacteria could develop resistance to the photodynamic action of cytotoxic singlet oxygen oree radicals．as has been reported with conventional antimicrobiaIs and antibioticsl4]．
The aim of this study was to carry out a preliminary assessment of hematoporphyrin monomethyl ether fHMME)一mediated PDT on biofilms of S．mutans in vitro—generated and the cell killing efficacy of PDT with HMME compared with other antimicrobial methods．25 Freshly extracted human permanent premolars for othodontic reason，free of defects and／or cracks when trans-illuminated，were selected for this study．The roots were resected from enamel-cementum joint and the crowns were splited with handpiece from mesialdistal direction．Aher ultrasonic cleanout for 5 min to remove debris，each sample was covered with nail polish only exposed 4 x 4(mm)on the surface of enamel．50 Enamel pieces were sterilized with 75％ethanol for 72 h。Unstimulated saliva of human free of dent8l carries and periodontal disease was collected．Enamel pieces were Dreconditioned in the filter—sterilized saliva for 37。C and 48 h to form the achievement membrane．
，I、he microorganism used in this study was S．mutans NCTC 10449(Capital Medical University School of StomatoloKy,Beijing。China)。To prepare the inoculum，S．mutans was first grown anaerobically on Trypticase Soy Agar(TSA)plates for 48 h．Subsequently,single colonies were inoculated into 10 mL of Trypticase Soy Broth(TSBl and incubated anaerobically at 37。C for 24h．The nutrient source in all experiments were TSA and TS8．
Later。50 enamel pieces were took out om the saliva and immerged into lmlinoculum of S．mutans(1×108)respectively and anaerobically at 37。C for 48 h．HMME (Shanghai nldan， Zhangjiang Bio-Pharmaceutical Co．，Ltd．China)was dissolved to obtain a final concentration of 10 mg／mL and was subsequently kept in the dark。The light sources used were a diode laser fLaser Medical Lab of Tianjin Medical University,China)．which produces 1ight with the wavelength of 635 nm．50 enamel pieces were divided into 5 groups with each group of 10 random samples。Each group was disposed as following：1 1 HMME group：20#g／mL HMME added as photosensitizer alone．Incubation time ws．s 2 h．2)Laser group：diode laser irradiation alone．3)PDT group：after incubated with 20 lzg／mL HMME for 2 h and then irradiated with diode laser．The power output of laser was 10 mW．The irradiation times was 90 s and the energy density was 12．74 J／cm2 fpower density was 0．14W／cm21．41 Positive control group：treatment with l mL0．05％chlorhexidine for 90 s served as a positive contr01．5)Negative control group：treatment with 1 mL 0．9％of sterile physiological saline solution for 90 s served 8．8negative controls．After antimicrobial treatment．each enamel piece was immerged into 1 mL inoculum of S．mutans f 1 x 108 1 respectively and anaerobically culti—vated at 37。C for another 48 h．
To harvest S．mutans of biofilm．each enamel pieces was transferred to a sterile 5一mL polypropylene tube containing sterile physiological saline in sterile physiological saline and inoculated on TSA incubated anaerobically at 37。C．After 48 h，the number of colony—forming units(CFUs)was counted with the aid of a stereomicroscope．
To determine the significance of the presence of sensitizer alone．the laser irradiation alone and the combination of sensitizer and laser．the data was analysed by a variance analysis(ANOVA)model using the factorial(2 x 2)design．The r11ukey test was chosen for evaluating the significance of all pairwise comparisons with a significance limit of 5％．Inoculum of S．mutans incubated with HMME for 2 h were placed into a Petri dish(5 am in diameter)，and examined with a BX51 fluorescence microscopy(Olympus Jappen．INCl．
To determine the antimicrobial activity of PDT．the number of CFU obtained from different treatment methods groups were compared with negative control group fsterile physiological saline)．The results are summarized in Table 1．
PDT group．with 20#g／mL HMME and diode laser irradiated，effectively eliminated bacteria(P<0．05)．Laser group and 0．05％chlorhexidine group were caused reduction in the viable COUnts of S．mutans significantly different(P<0．05)also，but these two test treatments did not statistically differ from each other．HMME group did not statistically differ with negative control group．
PDT group exhibited a significantly(P<0．051 increased antimicrobial potential compared with 20#g／mL Fig．1．Fluorescence microscope imaging shown uptake of HMME into S．mutans of biofilms．(a)Reflective S．mutans
cellular is located using fluorescence microscope reflectionmode imaging and(b)the red areas of HMME localization is detected in fluorescence mode．HMME only．1aser only，0．05％chlorhexidine，and 0．9％sterile physiological saline．which reduced the S．mutans of the biofilm most effectively．
S．mutans biofilm，imaged on the fluorescence microscope in reflected—light mode，is shown in Fig．1(a)．Poorly reflective regions correspond to regions of S．mutans cellular，while highly reflecting areas are the channels and voids typical of biofilm architecture．By viewing the same image in fluorescence mode，the red areas of HMME localization are revealed in Fig．1 fb)．The two images are superimposable．indicating that HMME localizes primarily in the biomass of the biofilm．although whether the Dhotosensitizer is associated with the bacterial cells．the extracellular mattix or a combination of the two is not known at present．PDT is a medical treatment that utilizes 1ight to ac．
tivate a photosensitizing agent n)hotosensitizer)in the presence of oxygen．The exposure of the Dhotosensitizer to light results in the formation of oxygen species．such as singlet oxygen and free radicals．causing localized Phm todamage and cell death．
Several studies have shown that oral bacteria are SUS—ceptible to PDT when they are grown as planktonic cultures．However．the causative agents of caries and other oral diseases are present as organized biofilms．It has been known that biofilm-grown cells differ from their planktonic counterparts in a number of respects including the presence of a extracellular polymeric substances(EPS)，cell wall composition，growth rate，metabolic activity,and gene expression．
Bacteria in biofilms display increased resistance to an— timicrobial agentsiSJ．PDT represents an alternative antibiotic treatment for drug-resistant organisms[9J．It is unlikely that bacteria would develop resistance to the cytotoxic action of singlet oxygen or free radicals．Bacteria that grow in biofilms，implicated ln diseases like dental caries is susceptible to PDTtloj．
The results of this study showed that PDT was effective in significantly reducing the viability of S．mutans biofilms．whereas laser and chlorhexidine caused significant，but limited，reductions in the viable counts．and HMME failed to demonstrate a significant antimicrobial efficacy．Fluorescence microscopy images of biofilms after exposure to diode laser in the presence of HMME showed uptake of HMME into S．mutans of biofilms．
In conclusion，the results of this study showed that S mutans biofilms were susceptible to diode luser in the presence of HMME，suggesting that this approach may be useful in the treatment of dental plaque-related diseases．Wb report the use of the dental plaque-disclosing agent HMME in the PDT of oral biofilm bacteria．Wb have demonstrated HMME to be an effective photosensitizer for the killing of the cariogenic bacterium S．mutans，which highlights the excellent clinical potential of HMMBmediated PDT in the control and treatment of dental plaque biofilm bacteria．Further work is now required to evaluate the clinically effect of antibiotic treat—ment of HMME-mediated PDT．We will further inves． tigate the mechanism of HMME—mediated PDT for the treatment of dental caries．