Effect Of HMME-PDT On The Activity And Structure Of The Dental Plaque Biofilms

Objective:Dental Caries is an endogenous infectious diseases caused by bacteria in oral ecosystems which damages the dental hard tissue. The third national oral health epidemiological survey results show that the most prominent oral health problem is dental caries:5-year-old children’s caries prevalence is66%, and the caries prevalence of35to44-year-old middle-aged is88.1%,65to74-year-old up to98.4%. Moreover, dental caries and its concurrent endodontics or periapical diseases are the dental diseases with the highest rate. In view of the frequency and severity of dental caries, the prevention of dental caries is much more important than passive treatment.The incidence and development process of dental caries can be mediated and completed which only rely on the dental plaque. Therefore, the appropriate method that affect the growth and metabolism of bacteria in the plaque biofilms, interfere with plaque formation or promote plaque dissociation can achieve the purpose of preventing dental caries. In this study, using photodynamic therapy (PDT) mediated by the semiconductor laser with the wavelength of532nm as light source and hematoporphyrin monomethyl ether (HMME) as photosensitizer treated the mixed bacterial suspension of four main cariogenic bacteria to investigate the killing effects of PDT and filter out the best parameters; Establishing mixed bacterial plaque biofilms in vitro for PDT, to explore the impact of PDT on the structure of plaque biofilms, bacterial activity and acid production, and verify the role of singlet oxygen in the sterile process of HMME-PDT.Methods:1. S.mutans, S.sangius, L.acidophilus, A.viscosus were recovered in the standing-temperature cultivator at37℃and anaerobic cultured for48h, and prepared concentration of standard bacteria solution with1×108CFU/mL and set aside.2. The mixed strains of bacteria suspension incubated with different concentrations (10μg/ml,20μg/ml,40μg/ml,60μg/ml) of HMME for five minutes, then irradiated by the semiconductor laser whose wavelength is532nm and exposure time was30s,60s,90s,120s. Finally, the viable counts of bacterial suspension recorded by counting colony forming unit were compared with the control groups. Chose the best parameters of PDT effects, to provide the basis for the following plaque biofilms experiments.3. Four main cariogenic bacteria had been chosen to establish dental plaque biofilms model on the enamel surface in vitro. Separated them into five groups:group of HMME-PDT, group of HMME-PDT+sodium azide, group of sodium azide, group of cetylpyridinium chloride(positive control), group of normal saline (negative control). Observed the activity of the cariogenic bacteria in dental plaque biofilms according to the plate colony count; Observed their acid production by PH records in24h; observed the structure of plaque biofilms by confocal laser scanning microscope (CLSM).Results:1. The plate colony counts of mixed strains of bacteria suspension experiment showed that, under the experimental conditions, the best experimental parameters of PDT for the best effect of cariogenic bacteria:photosensitizer concentration40μg/ml of HMME, the semiconductor laser of wavelength532nm and power density0.14W/cm2irradiated90s.2. Plate colony counts of the elimination of HMME-PDT on the cariogenic bacteria in plaque biofilms showed that:compared to normal saline group, separate azide sodium group had almost no effect on the growth of cariogenic bacteria in plaque biofilms (P>0.05); cetylpyridinium chloride made the number of cariogenic bacteria(CFU/mL) in plaque biofilms significantly reduced (P<0.05), with the bactericidal rate51.86%; after HMME-PDT treatment against the plaque biofilms model, the number of cariogenic bacteria significantly reduced (P<0.05), the bactericidal rate up to94.92%; The number of cariogenic bacteria in plaque biofilms of HMME+sodium azide group was not significant different (P>0.05), with the bactericidal rate only9.47%.3. The pH results of the supernatant by the different groups at different time within24hours showed that:compared to normal saline group, HMME-PDT and cetylpyridinium chloride treatment could prevent the acid production of the cariogenic bacteria in plaque biofilms, and⊿pH (range of variation) significantly reduced (P<0.05); after HMME-PDT treatment24h, the pH value of culture supernatant was6.01which is higher than the cariogenic critical pH values (5.0to5.5); HMME-PDT+sodium azide group and separate sodium azide group had no significant effect on the acid production of the cariogenic bacteria in plaque biofilms, and⊿pH had no statistical significance either(P>0.05).4. The CLSM tomography image showed that:in the inner and outer layers of saline treatment group’s dental plaque biofilms, there were a small amount of sparse survival bacteria; the bacteria of the middle layer increased significantly and accumulated such as cotton wool; the three-dimensional reconstruction image displayed that the structure of the dental plaque biofilms is dense and thickness, and there were a lot of dark piping systems. After HMME-PDT treatment, The structure of dental plaque biofilms was sparse and did not form the typical three-dimensional; there were almost no presence of bacteria in the inner layers and the outer layers; and a few bacteria survive in the middle layer, spread star-liked.Conclusions:1. The elimination of HMME-PDT on the mixed strains of bacteria suspension in vitro was significant. The killing effect was closely related to the photosensitizer concentration and irradiation time.2. HMME-PDT had significantly reduced the activity of cariogenic bacteria in dental plaque biofilms and inhibited the metabolic acid production by them.3. Singlet oxygen played an extremely important role in the sterile process of HMME-PDT.4. HMME-PDT could destroy the structural integrity of dental plaque biofilms and promote its dissociation.