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EFFECT OF PHOTODYNAMIC THERAPY ON CARIOGENIC BACTERIAL GROWTH AND BIOFILM FORMATION : AN IN VITRO STUDY

Dental caries, the third chronic non-infectious disease recommended by WHO after cancer and angiocardiopathy, is a common disease affecting the oral and systemic health. Bacteria and their metabolite are major factors involving in the development and progress of dental caries. They form biofilms on the tooth surfaces and destroy the hard tissue structure of he tooth.
Plaque control is the key preventive method for dental caries. At present mechanical and antibiotic methods are widely used in the clinics to control dental plaque formation. Although both are effective, mechanical method is difficult to maintain the ideal result of plaque control for a long time. Meanwhile , it is difficult to achieve the satisfactory effect because some covert areas, such as the deep fossa, groove and proximal surface, can not be reached by tools. Long-term antibiotics application results inbacterial drug resistance and dysbacteriosis, which increases the possibility of opportunistic infection.
Photodynamic therapy (PDT), also known as photoradiation therapy,or phototherapy, refers to the use of a photoactive dye (photosensitizer) that is activated by exposure to light of a specific wavelength in the presence of oxygen. It involves three components: light, a photosensitizer,and oxygen. Many researches indicate neither independent photosensitizer nor laser has effect on oral pathgen. However, photodynamic effect resulted from union application of appropriate photosensitizer and the light can kill oral pathogenic bacteria without inducing drug tolerance, indicating its superiority compared with antibacterial drugs. Besides, it’s easy to utilize syringe to spread photosensitizer on teeth surface or carious lesions and is deliver the proper light through optical fiber.The clinical operation is simple and convenient.
In summary, PDT is a promising method for caries prevention and plaque control. There are little domestic papers or reports about caries prevention with photodynamic therapy, while massive overseas research are restricted to floating bacteria or single bacterium biomembrane, few about multi-bacteria biofilm. Accordingly, further researches are needed to establishing the appropriate PDT system for dental caries prevention.
Objectives:
The study is to observe the cariogenic bacteria growth in vitro after PDT sensitized by toluidine bule to explore the proper parameters for PDT , SEM is used to observe the morphology of the artifical multi-bacteria biofilm to see the anti-carious effect of PDT.

Methods:
1. Measurement of absorption spectrum of photosensitizer toluidine blue(TB). Absorption spectrum of TB solutions with different concentrations were measured under the light with the wavelength from 500nm to 700nm. The maximum absorption peak values were record. Based on the result we hoped to find the appropriate wave length to achieves the desired effect of photodynamic therapy.
2. Experiment of in vitro growth inhibition of oral cariogenic bacteria by TB. Four cariogenic bacteria were chosen as experimental subject for measuring minimal bactericidal concentration(MBC) of TB.
3.Effect of in vitro photoinactivation of Photodynamic therapy on Streptococcus mutans before and after PDT, the colonies of Streptococcus mutans illuminated by light for different time or cultured in TB with different concentrations were counted, the antibacterial ratio of PDT was calculated. Cell morphologic changes of streptococcus mutans was observed under scanning electron microscope.
4. Four cariogenic bacteria were chosen to form artificial biofilm on enamel surface in vitro. The biofilms were treated by photodynamic therapy in which the photosensitizer concentration was 100μg/ml and light intensity was 94.5J/cm2. Samples were observed under Scan Electron Microscope on the first, third and seventh days respectively to investigate the effect of photodynamic therapy on plaque formation.
Result:
1. The absorbance values of toluidine blue increased along with its concentration increasing. When its concentration was for 25μg/ml, 50μg/ml and 100μg/ml respectively, The wavelength values, at which the TB curves reached the peaks were 635nm, 635nm and 620nm. The result suggested concentration of TB would influence the absorption value.
2. Toluidine blue can resist bacteria growth in high concentration.None of four bacteria had a growth when concentration of the solution was for 1000μg/ml, The minimal bactericidal concentration of toluidine blue to Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus and Actinomyces viscosus respectively was 500-1000 μg/ml, 500-1000μg/ml, 250-500 μg/ml and 125-250 μg/ml.
3. Neither light nor toluidine blue had ideal bacteriostasis action.There was no showed difference (P﹥0.05)between the light-only of TB group. There was significant statistics difference (P<0.05) between experimental group and control group by PDT. Photosensitizer concentration and the light dosage existenced correlation actions .
4. Result of scanning electron microscope picture showed that compared with the control group bacterial morphology in PDT group changed obviously. Under a magnification of 20,000 times, the cell outline of the Strep. mutans in the control group was clear and the cell surfaces were smooth and intact, while the cell contour of the experimental group was clear, the majority of bacterial cell surfaces became rough , with blister shaped bulge and amorphic substance was surrounded the cells.
5. In the control group, bacteria, mainly the streptococci, adhered to the acquired pellicle on coverslip on the first day. Number of bacteria obviously increased on the third day, Lactobacillus acidophilus and Actinomyces viscosus were wrapped by the Streptococci forming corn cob structure. Amorphic substance existed surrounding the cells. On the 7th day, both the cell numbers and stack thickness of the biofilms were increased, a more complex three dimensional structure of the biofilms was formed. In the bioflim ,the bacteria were connected by massive matrix.
Compared with the control group, Photodynamic therapy inhibited the formation of biofilm and altered the biofilm morphology. On the 1st,3rd,and 7th days , thickness of biofilm in experimental group were obviously thinner than that in the control group, and the bacterial adherence was relatively looser ,with less matrix and amorphic substance.
Conclusion:
1.The absorbance value of toluidine blue solution increases along with its concentration. When its concentration is for 25μg/ml, 50μg/ml and 100μg/ml respectively, The wavelength values, at which the TB curves reached the peaks are 635nm, 635nm and 620nm.
2. Toluidine blue can resist bacteria growth in high concentration. The minimal bactericidal concentration of toluidine blue to Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus and Actinomyces viscosus respectively is 500-1000 μg/ml, 500-1000 μg/ml,250-500 μg/ml, 125-250 μg/ml.
3. Neither light nor toluidine blue has ideal bacteriostasis action.Photosensitizer concentration and the light dosage existences correlation actions .
4. Bacterial morphology in PDT group changes obviously compared with the control group.
5. On the 1st, 3rd, and 7th days , thickness of biofilm in experimental group is obviously thinner than that in the control group, and the bacterialadherence is relatively looser ,with less matrix and amorphic substance.
Key words: photodynamic therapy; toluidine blue O; dental caries;oral cariogenic bacteria; dental plaque biofilm