[Special] Feasibility study of photodynamic therapy for periodontitis based on LED light source

1 Introduction

Photodynamic Therapy (PDT) refers to the photochemical reaction of photosensitizer (PS) absorbed by tissue by irradiation of light of a specific wavelength. In an aerobic environment, the excited photosensitizer transmits energy to The surrounding oxygen molecules, which produce reactive oxygen species (ROS), react with adjacent biomacromolecules to produce strong cytotoxicity, which leads to cell damage and even death [1]. In recent years, PDT has been widely used as an adjuvant therapy in the treatment of oral diseases, including dental hard tissue diseases, dental pulp diseases, periodontal inflammation [2], [3] and so on.

In recent years, laser light sources have been widely used in PDT. However, all lasers currently developed have limited their application in biomedical applications due to the production principle and practical value [4]. LED has the advantages of high efficiency, energy saving, long life, easy maintenance, simple structure, small power supply and low price. It has the advantage that traditional lasers can't match in the promotion of household portable PDT.

The biggest difference between the light emitted by LEDs and lasers is the monochromaticity and coherence. Since the photosensitizer used in PDT treatment has a wide absorption band, usually 10-20 nm, or even wider, the half-wavelength bandwidth of currently produced LEDs can reach ±10 nm, so monochromaticity has no significant effect on the therapeutic effect. [5]. Further research suggests that there is no theoretical and data basis for the effects of light coherence on phototherapy [6]. Through experiments, this paper not only proves the feasibility of using LED as a light source for photodynamic therapy of periodontitis, but also further explores the optimal bactericidal treatment plan under different illumination parameters, which provides a way to promote family portable PDT treatment in the future. Theoretical basis.

2 experimental light source

The strong absorption band of the new photosensitizer is generally between 650-850 nm [1]. The experiment uses a 660 nm wavelength red LED with methylene blue (MB) as a photosensitizer to in vitro bactericidal experiments on Prevo periodontal bacteria. The photosensitizer strongly absorbs red light with a peak wavelength of 660 nm and releases a large amount of singlet oxygen, which produces cytotoxin and kills bacteria. To ensure that the photosensitizer can be activated, it is necessary to first determine if the wavelength range of the light emitted by the LED array used in the experiment is sufficient. To this end, this paper first used the spectral analysis system to test the LED spectral power distribution. The results of the test are shown in Figure 1.

Figure 1 Spectral power distribution of experimental LED

As can be seen from the above figure, the peak wavelength of the LED is 657 nm, the half width of the spectrum is 20 nm, and the energy is mainly concentrated on the strong absorption band of the MB. Although the LED array cannot be compared with the laser in terms of monochromaticity, as mentioned above, the absorption band width of the photosensitizer used in PDT is usually 10-20 nm or even wider. Therefore, the LED array used in the experiment can fully satisfy the treatment. Claim.

3 Experimental content

3.1 Preparation

3.1.1 bacterial liquid culture

The resistant wild strain isolated from the periodontal abscess of the patient was resuscitated and secreted on an anaerobic basal medium blood plate (OXID, 5% defibrinated sheep blood). The plate was placed in an anaerobic environment (N2 80%, H2 10%, CO2 10%) at 35-37 ° C for 2 days to obtain mature colonies. A certain amount of mature colonies were taken in a liquid medium and incubated in an anaerobic environment (N2 80%, H2 10%, CO2 10%) at 35-37 ° C for 36 hours to obtain a second-generation bacteria.

3.1.2 Selection of bacterial liquid concentration

The bacterial solution incubated for 36 hours was taken and diluted with the culture solution. Take the diluted sample under the spectrophotometer OD600 turbidity, measure the corresponding OD (OD, optical density) value, and then compare the OD value and CFU value curve that has been done before, you can roughly infer the concentration of the liquid Order of magnitude. In this experiment, a bacterial solution having an OD value of 0.65 to 0.75 was obtained (at this time, the bacterial liquid concentration was approximately 107 CFU/mL).

3.1.3 UV sterilization

The equipment used in the experiment of LED array, drive, 12-well cell culture plate, pipetting gun, etc. was placed on a clean bench for 8 minutes of UV sterilization.

3.2 Light experiment

3.2.1 Experimental grouping

The number of 12-well cell culture plates is shown in Figure 2, and the 6 wells with color markers will be used for the experiment. The light conditions are L+, no light is L-; the photosensitizer condition is M+, and the photosensitizer is M-, then the experimental conditions of 6 holes are shown in Table 1.

Figure 2 Hole cell culture plate number map

Table 1 Experimental conditions of each well of cell culture plate

3.2.2 Experimental parameters

The concentration of the floating bacteria solution was 107 CFU/mL, and the MB concentration was 0.05 μmol/mL. The values ​​of light density and illumination time are shown in Table 2.

Table 2: Light density and illumination time parameters of each group of experiments

3.2.3 Experimental steps

1 first add 5μLMB solution in the A1, A2, D2 hole position, then add 500μL floating bacteria liquid, incubate for 2 minutes;

2 Add 500 μL of the control bacterial solution to B1, B2, and D3;

3 buckle the LED array on the cell culture board, turn off the ultra-clean table work light, turn on the LED power supply, and irradiate the bacterial liquid according to the experimental parameters set in Section 3.2.2, as shown in Figure 3;

4 After the end of the irradiation, the LED is turned off, and the floating bacteria liquid in the culture plate is taken out for post-treatment.

Figure 3 Illuminating the floating bacteria solution with an LED array

3.3 Post-processing

3.3.1 dilution of bacterial solution

The bacterial liquids in A1, A2, B1, B2, D2, and D3 and the bacterial stock solution (7 samples in total) were subjected to several 10-fold dilutions for plate colony counting. In order to make the count value fall within the confidence interval, six different concentrations of bacterial liquid were inoculated for each sample, and the expected treatment effect was considered, and the dilution ratio of each sample is shown in Table 3.

Table 3 dilution ratio of each sample

3.3.2 Plate colony count

The diluted bacterial solution was inoculated on an anaerobic basal medium blood plate, one plate per sample. The plate is divided into six equally sized areas, each inoculated with a concentration of bacterial fluid. The plate inoculated was placed in an anaerobic environment at 35-37 ° C, and after 2 days, the plate was taken out for colony counting. The number of colonies counted, the CFU value of each bacterial liquid sample was converted according to the dilution factor and the sampled inoculum amount, thereby obtaining the sterilization rate of the sample to determine whether the PDT treatment was effective.

4 Experimental data and analysis

4.1 Experimental data

In order to accurately measure the concentration of each sample after treatment, for each experimental sample, the inoculation sample with the number of colonies falling within the confidence interval after incubation was selected, and the concentration of the original sample was converted according to the dilution factor. Table 4 records the original bacterial concentration of each set of experiments and the concentration of the experimental sample bacteria that were treated and incubated.

Table 4 The concentration of floating bacteria in the original bacterial solution and the treated experimental groups

4.2 Data Processing and Analysis

4.2.1 PDT treatment feasibility analysis

According to Table 4, the concentration of the original bacterial solution is the denominator, and the concentration of the experimental sample is liquid, and the survival rate of the periodontal pathogen after treatment of each sample can be calculated. 4(a), (b), and (c) show the pathogenic bacteria survival rate of each experimental sample at different irradiation times when the light density is 10, 20, or 40 mW/cm2, respectively. It can be seen from the figure that although the parameters of the illumination are different, in each of the separate experimental groups, the bacterial survival rate of the experimental sample to which the photosensitizer is added and received light is lower than that of the other experimental samples.

Figure 4: (a) Bacterial viability of each experimental group at 10 mW/cm2 light density; (b) Bacterial viability at 20 mW/cm2 light density; (c) Fine survival at 40 mW/cm2 light density

Considering the influence of the incubation environment and the incubation time on the concentration of the bacteria solution, the concentration of the bacterial solution of the double negative control sample (LM-) was used as the denominator, and the concentration of the other sample liquid was used as the molecule to obtain the survival of the periodontal pathogen. rate. Figure 5 (a), (b) and (c) show the periodontal results of each experimental sample after considering the time factor at different illumination times when the illumination densities are 10, 20, 40 mW/cm2, respectively. Pathogen survival rate. It can be more clearly seen from the figure that the bacterial survival rate of the experimental sample group to which the photosensitizer was added and irradiated was much lower than that of the other sample groups. The experimental samples with light alone or with photosensitizer showed a large dispersion of bacterial survival with changes in light density and illumination time. Thus, PDT using LED as a light source is effective for the treatment of periodontitis, and the use of a photosensitizer while illuminating the bacteria can produce a better bactericidal effect than using a photosensitizer alone or merely receiving light.

Figure 5: (a) Bacterial survival rate of each experimental group at 10 mW/cm2 light density after considering the time factor; (b) Bacterial survival rate at 20 mW/cm2 light density; (c) 40 mW/cm2 light density Fine survival rate

4.2.2 Optimal illumination parameters for PDT treatment

Figure 6 shows the bacterial survival rate of experimental samples with simultaneous addition of photosensitizer and illumination, under different combinations of illumination parameters. It can be seen from the figure that when the light density is low (10-20mW/cm2), the survival rate of pathogenic bacteria mainly depends on the illumination time - as the illumination time increases, the bacterial survival rate decreases significantly; when the illumination time After more than 16 minutes, the bacterial survival rate stabilized and remained at a very low level (less than 3%). When the light density is high (40mW/cm2), the bacterial survival rate increases greatly with the increase of illumination time. After 4 minutes of irradiation, the survival rate of pathogenic bacteria is as low as 3% or less; similarly, after irradiation for 16 minutes. After that, the bacterial survival rate tends to be stable and remains at a lower level (less than 0.1%). Because in the actual treatment process, it is very inconvenient to irradiate the patient's affected area for a long time, and the survival rate of bacteria is still maintained at about 3% after the treatment with low light density. Therefore, this paper believes that in the 18 different light parameters of the experiment, it is a suitable treatment to irradiate the affected area with light intensity of 40mW/cm2 for 8min.

Figure 6 Periodontal pathogen survival rate under different illumination parameters

5 Conclusion

The experimental data showed that the survival rate of periodontal pathogens in planktonic bacteria was significantly lower than that of the other control groups in the experimental group using photosensitizer and receiving light, that is, the bactericidal effect of the experimental group was much better than other groups. The application of LED light source to PDT treatment of extracorporeal pathogenic bacteria has proven effective. At the same time, it can be seen from the experimental data that the longer the illumination time, the higher the sterilization rate of PDT treatment; when the illumination density is higher, the sterilization rate increases with the increase of illumination time. Among the 18 different illumination parameters of the experiment, the light density of 40mW/cm2 combined with the irradiation time of 8min is a suitable treatment plan. The study in this paper only focuses on the effect of PDT treatment with LED as a light source on the survival rate of periodontal pathogens in vitro. As to whether the clinical therapeutic effect of PDT is consistent with in vitro experiments, further research is needed in the future. If clinical trials are also proven to be effective, PDT therapeutic devices for portable LED light sources are expected to enter thousands of households in the near future, and PDT-assisted therapy will become a routine means of periodontal treatment.

Edit: Cedar