Research ArticleOpen Access

Effect of antibacterial photodynamic therapy on Streptococcus mutans plaque biofilm in vitro

Xiaoyue Liang

Department of Endodontics, Tianjin Medical University Stomatology Hospital, Tianjin 300070, P. R. China

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Zhaohui Zou

http://orcid.org/0000-0001-9195-6988

Department of Endodontics, Tianjin Medical University Stomatology Hospital, Tianjin 300070, P. R. China

E-mail Address: zouzhaohui2005@sina.com

Corresponding author.

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Zheng Zou

Department of Endodontics, Tianjin Medical University Stomatology Hospital, Tianjin 300070, P. R. China

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Changyi Li

Department of Prosthodontics, Tianjin Medical University Stomatology Hospital, Tianjin 300070, P. R. China

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Xiaoxi Dong

Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, P. R. China

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Huijuan Yin

Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, P. R. China

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, and

Guohui Yan

Department of Endodontics, Tianjin Medical University Stomatology Hospital, Tianjin 300070, P. R. China

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https://doi.org/10.1142/S1793545820500224Cited by:9 (Source: Crossref)

Abstract

The main objective of this study is to evaluate the antibacterial effect of antibacterial photodynamic therapy (aPDT) on Streptococcus mutans (S. mutans) biofilm model in vitro. The selection of photosensitizers is the key step for the efficacy of photodynamic therapy (PDT). However, no studies have been conducted in the oral field to compare the functional characteristics and application effects of PDT mediated by various photosensitizers. In this research, the antibacterial effect of Methylene blue (MB)/650nm laser and Hematoporphyrin monomethyl ether (HMME)/532nm laser on S. mutans biofilm was compared under different energy densities to provide experimental reference for the clinical application of the two PDT. The yield of lactic acid was analyzed by Colony forming unit (CFU) and spectrophotometry, and the complete biofilm activity was measured by Confocal Laser Scanning Microscopy (CLSM) to evaluate the bactericidal effect on each group. Based on the results of CFU, the bacterial colonies formed by 30.4J/cm² 532nm MB-aPDT group and 30.4J/cm² 532nm HMME-aPDT group were significantly less than those in other groups, and the bacterial colonies in HMME-aPDT group were less than those in HMME-aPDT group. Lactic acid production in all treatment groups except the photosensitizer group was statistically lower than that in the normal saline control group. The activity of bacterial plaque biofilm was significantly decreased in the two groups treated with 30.4J/cm² aPDT. Therefore, aPDT suitable for energy measurement can kill S. mutans plaque biofilm, and MB-aPDT is better than HMME-aPDT.

Keywords:

1. Introduction

Caries and its complications can cause tooth pain and even tooth loss, seriously affecting the oral and systemic health of patients. Therefore, the prevention and treatment of caries has been the focus of many scholars. The presence of Streptococcus mutans (S. mutans) in the oral cavity is mainly in the form of plaque biofilm settled on the surface of the teeth which is main cause of caries. Plaque biofilm is composed of cells and extracellular matrix composing of proteins and extracellular polysaccharides, which enhances the adhesion and cohesion of the biofilm, protects the attached bacteria, and promotes the local Pondus Hydrogenii (PH) reduction to form an acidic environment.^1,2

In clinical methods, in addition to physical removal of biofilms, chemical methods to kill bacteria in biofilms have been the focus of people’s research. General methods include the use of antibiotics and chemical oxidants, but both have their drawbacks. Plenty of studies have proved that compared with the planktonic bacteria, the bacteria in the biofilm are in different physiological states and are not easily affected by antibacterial agents,³ resulting in poor efficacy of antibacterial agents and fungicides. However, if antibiotics are used for a long time, the bacteria are prone to developing drug resistance, reducing their antibacterial effect and creating a vicious circle. While chemical oxidants such as high concentration of chlorhexidine and sodium hypochlorite have good efficacy in killing bacteria and are not easy to cause bacteria to develop tolerance, however, due to the difficulty in controlling their application range, the drugs can spread from the local oral environment to other tissues, corroding the body and causing tissue inflammation and damage.^4,5 Therefore, a new method is urgently needed in clinic, which can not only achieve good antibacterial effect, but also be safe and nontoxic to the body. With the development of the study of photochemotherapy, photodynamic therapy (PDT) has gradually come into people’s field of vision.

Through the selection of photosensitizer and light source, PDT can achieve excellent targeting therapy, minimize the side effects on the surrounding normal tissues, and make it more comfortable, convenient and noninvasive for patients which is exactly in line with the precise medical needed now. In the field of antibiotics, studies have shown that bacteria do not produce photodynamic effects after repeated use of PDT.^6,7 so it is considered as a promising alternative way to kill antibiotic-resistant bacteria. Most of the photosensitizers commonly used in medical field fall into the following two categories: (1) organic dyes and aromatic hydrocarbons; (2) porphyrin, phthalocyanine and related macrocyclins. Methylene blue (MB) is a blue dye. It can be excited by the light between 550nm and 700nm to produce photodynamic effect.^8,9,10 It is cheap, easy to produce and widely used in the treatment of periodontal and periimplant inflammation. However, the effect of plaque biofilm is still controversial.^11,12,13 Hematoporphyrin monomethyl ether (HMME) is a photosensitizer developed in China and first applied to wine stain. This porphyrin has the characteristics of high photoactivity and low toxicity. HMME at a certain concentration exhibits four different absorption peaks within the range of 500–800nm,^14,15 which enables the selection of excitation wavelength according to the existing clinical conditions in the practical application process and has a high clinical applicability.

In the previous experimental studies of this group, it has been proved that HMME has positive antibacterial effect on oral bacterial biofilm.^16,17 However, horizontal comparison of its effect with other photosensitizers has not been conducted, and the mechanism of HMME in the field of antibacterial has not been in-depth studied. This study will MB/650nm laser and HMME/532nm laser under different energy density of hydroxyapatite on S. mutans biofilm PDT, through the plate colony count and the measurement of L — lactic acid and CLSM living dead bacteria staining methods, from different angles research each PDT effect on S. mutans biofilm, analysis of causes, mechanism, so as to choose suitable photosensitizer in oral clinical application for reference.

2. Materials and Methods

2.1. Subjects

Hydroxyapatite sterile disc (1.14cm², Hospital of Stomatology, SiChuan University).

2.2. Main reagents and equipment

532nm frequency doubling ND:YAG laser (DEKA, America).

650nm Semiconductor laser (Institute of Biomedical Engineering, Chinese Academy of Medical Sciences).

Zeiss_LSM_780 Inverted laser confocal microscope (Zeiss, Germany).

Electric constant temperature incubator (HH-BLL.360, Tianjin Medical University).

VORTEX Genie 2 Vortex oscillator (Scientific Industries Co. Ltd., America).

Lactic acid detection kit (Beijing regen biotechnology co. ltd).

S. mutans (ATCC25175, Guangdong provincial microbial culture conservation center).

HMME (Shanghai yuanye biological technology co. ltd).

0.1% MB(Solebo technology co.ltd).

Dimethyl sulfoxide (Aibixin biotechnology co., ltd).

2.3. Preparation of S. mutans liquid

S. mutans (ATCC25175) at room temperature after recovery, put in BHI culture medium, $3 7^{\circ}$ $3 7^{\circ}$ C aerobic environment culture micro 24h, then pick a single colony to BHI liquid medium to microaerobic training 48h, using vortex should be scattered to the medium, and through a certain proportion dilution in the spectrophotometer at 600nm absorption peak is 0.4, corresponding to the liquid concentration reached 108CFU/mL. Set aside.

2.4. Preparation of photosensitizer

HMME was dissolved in dimethyl sulfoxide (DMSO) at 5mg/mL and stored at $- 2 0^{\circ}$ $- 2 0^{\circ}$ C away from light. When using, remove it from the PBS buffer and reset its concentration to 50 $μ$ $μ$ g/mL. MB re-diluted 0.1% solution in PBS buffers to 50 $μ$ $μ$ g/mL, and the above solution was mechanically sterilized by 0.2 $μ$ $μ$ m membrane filter before use.

2.5. Groups

In this experiment, 80 Hydroxyapatite (HA) sterile disks were randomly divided into 8 groups with 10 samples in each group according to the treatment method. One to six of the groups used photosensitizer concentrations of 50 $μ$ $μ$ g/mL.

Group A (MB group): MB application group alone;

Group B (15.2J/cm²MB-aPDT group): MB/650nm laser, 100mw continuous light irradiation 120s, spot radius 0.5cm, energy density 15.2J/cm²;

Group C (30.4J/cm²MB-aPDT group): MB/650nm laser, 100mw continuous light irradiation 240s, spot radius 0.5cm, energy density 30.4J/cm²;

Group D (HMME group): HMME application group alone;

Group E (15.2J/cm²HMME-aPDT group): HMME/532nm laser, 100mw continuous light irradiation 120s, spot radius 0.5cm, energy density 15.2J/cm²;

Group F (30.4J/cm² HMME-aPDT group): HMME/532nm laser, 100mw continuous light irradiation 240s, spot radius 0.5cm, energy density 30.4J/cm²;

Group G: 0.2% chlorhexidine (CHX) positive control group;

Group H: 0.9% normal saline negative control group.

2.6. Sampling procedure

2.6.1. Preparation of S. mutans biofilm

In order to better determine the irradiation range of the light spot and limit the treatment area to the light area, the effect is more accurate. After the HA sterile disc was demarcated with a cutting knife and its central radius was 0.5cm, it was sterilized with high temperature and high pressure and placed in a 24-well plate. 1.5mL S. mutans solution was placed into the hole to immerse the HA disc. Culture at $3 7^{\circ}$ $3 7^{\circ}$ C for 72h under anaerobic conditions to form S. mutans biofilm. Replace the medium carefully every 24h to avoid the destruction of biofilm.

2.6.2. Treatment of photodynamic group and control group

Carefully remove each HA disc sample and place it in an empty 24-hole plate. Groups 1–6, respectively, dropped 0.5mL MB and 0.5mL HMME into the hole and incubated them under the light for 5min after passing over the surface of the sample. The 650nm laser and 532nm laser were used to connect the optical fiber, respectively. After adjusting the power to make the output power of the light terminal reach 100mw, place the optical fiber 2cm above the sample surface. Make the spot radius up to 0.5cm, respectively, irradiate the samples and blot the liquid dry, then gently rinse with PBS buffer for three times. Groups 7, 8, 0.5mL 0.2% chlorhexidine and 0.5mL 0.9% normal saline were dropped into the hole and passed over the surface of the disc, respectively. After standing for 2 min, they were gently rinsed with PBS thrice. After treatment, the sample surface was gently rinsed with PBS buffer to remove residual drugs in each group.

2.6.3. Colony count

After the treatment was completed, the colonies in the 1cm circle of surface center of all samples were wiped with sterile cotton balls and transferred to the centrifuge tube containing 5mL normal saline, using a vortex to suspense the bacteria in the tube, after continuous dilution, evenly divided samples of each dilution were applied to the agar medium of brain-heart immersion (BHI). Plate colony count was conducted after 24h of culture in $3 7^{\circ}$ $3 7^{\circ}$ C anaerobic environment.

2.6.4. Determination of l-lactic acid concentration

After the corresponding treatment, the treated samples were put into a 24-well plate and added with 1.5mL BHI medium. The samples were further cultured at $3 7^{\circ}$ $3 7^{\circ}$ C for 3h. After that, the samples were placed at $8 5^{\circ}$ $8 5^{\circ}$ C for 5min to stop the production of lactic acid.

Based on the enzymatic conversion principle of l-lactate pyruvate,²⁵ the concentration of l-lactic acid was determined by enzyme spectrophotometry with the lactic acid detection kit. Absorbance readings were performed using a spectrophotometer at a wavelength of 340nm. The absorbance value was converted to lactic acid concentration according to the parameters established by the standard curve.

2.6.5. Observation on the biofilm activity of S. mutans by CLSM

After the corresponding processing, the fluorescein diacetate/ethidium bromide (FDA/EB) was applied for the staining of live and dead bacteria. Mix 2.5mL of each solvent and set aside. Three samples were randomly selected from each group, and then 200 microns of the prepared fluorescent dye was added to the HA disk of the sample. The sample was incubated at $3 7^{\circ}$ $3 7^{\circ}$ C in dark for 15min, and then gently washed with PBS buffer for 1min. After observation under a CLSM microscope, three representative sites of each glass plate containing biofilm were selected for observation under an inverted CLSM. Excitation light was detected at 488nm, FDA at 520nm, and EB at 650nm. The same laser and observation hole Settings were used for image comparison for each observed sample. Three fields of view were randomly selected from each sample and observed with a 20-fold microscope. The observation results were collected in the form of images through the attached software CLSM.

After collecting the images, imageJ was used to analyze and process the information to determine the intensity values of green signals and red signals in the images, and the vitality of biofilm was determined by analyzing the ratio of green signals to the sum of green and red signals.

2.7. Statistical analysis

SPSS version 21.0 (IBM@SPSS@Statistics, New York, USA) was used for statistical analysis. The log transformation of CFU. Kruskal-wallis H and Dunn’s tests was used to detect differences between groups. $p < 0.05$ $p < 0.05$ was considered significant.

3. Results

3.1. The results of colony count

As shown in Table 1 and Fig. 1, compared with the negative control group (normal saline group), all the PDT treatment groups (15.2J/cm²MB-aPDT,30.4J/cm²MB-aPDT,15.2J/cm²HMME-aPDT,30.4J/cm²HMME-aPDT) showed a decrease in the number of bacterial colonies, and the difference was statistically significant $(p < 0.05)$ $(p < 0.05)$ . There was no significant difference between the single MB group and the single HMME group and the normal saline group ( $p > 0.05$ $p > 0.05$ ).

**Table 1. Colony count results of each group (log10CFU mL⁻¹) (n = 10, ±s).**
Group	Processing method	$n$ $n$	Bacterial count M ± SD
1	MB	10	4.642 ± 0.211°
2	MB/650 nm 15.2 J/cm²	10	3.738 ± 0.349*
3	MB/650 nm 30.4 J/cm²	10	2.807 ± 0.167*°
4	HMME	10	4.859 ± 0.158°
5	HMME/532 nm 15.2 J/cm²	10	4.147 ± 0.124*
6	HMME/532 nm 30.4 J/cm²	10	3.149 ± 0.160*°
7	0.2% chlorhexidine	10	3.646 ± 0.326*
8	0.9% normal saline	10	4.733 ± 0.350°

Notes: * indicates that p < 0.05 is statistically different from 0.9% normal saline group. ° indicated that p < 0.05 was statistically different from that of the 0.2% chlorhexidine group.

Fig. 1. Results of colony forming count (CFU) in each group.

Compared with the positive control group (sodium hypochlorite group), the colony numbers of the 15.2J/cm²MB-aPDT, 15.2J/cm²HMME-aPDT, MB and HMME groups were significantly higher than that of the sodium hypochlorite group, with statistically significant differences ( $p < 0.05$ $p < 0.05$ ); 30.4J/cm²MB-aPDT and 30.4J/cm²HMME-aPDT groups were superior to 0.2% sodium hypochlorite group, with statistically significant difference ( $p < 0.05$ $p < 0.05$ ).

Comparing different photosensitizers at the same energy density, the sterilization effect of different photosensitizers in the 15.2J/cm²MB-aPDT group was better than that in the 15.2J/cm²HMME-aPDT group, and the difference was statistically significant ( $p < 0.05$ $p < 0.05$ ); The sterilization effect of 30.4J/cm²MB-aPDT was better than that of the group with 30.4J/cm²HMME-aPDT, and the difference was statistically significant ( $p < 0.05$ $p < 0.05$ ).

Compared with the same photosensitizer and different energy densities, the sterilization effect of group 30.4J/cm²MB-aPDT was better than that of group 15.2J/cm²MB-aPDT, and the difference was statistically significant ( $p < 0.05$ $p < 0.05$ ); The sterilization effect of group 30.4J/cm²HMME-aPDT was better than that of group 15.2J/cm²HMME-aPDT, and the difference was statistically significant ( $p < 0.05$ $p < 0.05$ ).

3.2. The results of lactic acid concentration test

As shown in Table 2 and Fig. 2, compared with the negative control group (normal saline), all the PDT treatment groups (15.2J/cm²MB-aPDT, 30.4J/cm²MB-aPDT, 15.2J/cm²HMME-aPDT, 30.4J/cm²HMME-aPDT) showed reduced lactic acid concentration with significant difference ( $p < 0.05$ $p < 0.05$ ). There was no significant difference between the single MB group and the single HMME group and the normal saline group ( $p > 0.05$ $p > 0.05$ ).

**Table 2. Determination results of l-lactic acid concentration in each group (mmol/L) $(n = 10, ¯ x \pm s)$ $(n = 10, \bar{x} \pm s)$ .**
Group	Processing method	$n$ $n$	Results of lactic acid concentration M ± SD
1	MB	10	1.29 ± 0.27
2	MB/650 nm 15.2 J/cm²	10	0.76 ± 0.18*
3	MB/650 nm 30.4 J/cm²	10	0.71 ±0.19*
4	HMME	10	1.33 ± 0.26
5	HMME/532 nm 15.2 J/cm²	10	0.75 ± 0.16*
6	HMME/532 nm 30.4 J/cm²	10	0.69 ± 0.17*
7	0.2% chlorhexidine	10	0.84 ± 0.16*
8	0.9% normal saline	10	1.41 ± 0.31

Notes: * indicates that compared with the 0.9% saline group, p < 0.05, there was a statistical difference.

Fig. 2. Lactic acid concentration in each group.

Compared with the positive control group (sodium hypochlorite group), the concentration of lactic acid in MB group and HMME group was higher, and the difference was statistically significant ( $p < 0.05$ $p < 0.05$ ). The lactic acid concentration of 15.2J/cm²MB-aPDT, 30.4J/cm²MB-aPDT, 15.2J/cm²HMME-aPDT, and 30.4J/cm²HMME-aPDT groups was not significantly different from that of sodium hypochlorite group ( $p > 0.05$ $p > 0.05$ ); There was no significant difference between the single MB and the single HMME group and the normal saline group ( $p > 0.05$ $p > 0.05$ ).

Comparing different photosensitizers at the same energy density, there was no statistically significant difference between the15.2J/cm²MB-aPDT and the 15.2J/cm²HMME-aPDT group ( $p > 0.05$ $p > 0.05$ ); There was no significant difference between the 30.4J/cm²MB-aPDT and the 30.4J/cm²HMME-aPDT group ( $p > 0.05$ $p > 0.05$ ).

Comparing between the same photosensitizer and different energy densities, there was no significant difference between the 15.2J/cm²MB-aPDT and the 30.4J/cm²MB-aPDT group ( $p > 0.05$ $p > 0.05$ ).There was no significant difference between the 15.2J/cm²HMME-aPDT group and the 30.4J/cm²HMME-aPDT group ( $p > 0.05$ $p > 0.05$ ).

3.3. The analysis of CLSM

As shown in Table 3 and Fig. 3, compared with the negative control group (normal saline group), all the aPDT treatment groups (15.2J/cm²MB-aPDT, 30.4J/cm²MB-aPDT, 15.2J/cm²HMME-aPDT, 30.4J/cm²HMME-aPDT) showed a decrease in the proportion of live bacteria ( $p < 0.05$ $p < 0.05$ ). There was no significant difference between MB group and normal saline group ( $p > 0.05$ $p > 0.05$ ). The difference between HMME group and normal saline group was statistically significant ( $p < 0.05$ $p < 0.05$ ).

**Table 3. The ratio of green and red signal intensity in each group ( $n = 9, ¯ x \pm s$ $n = 9, \bar{x} \pm s$ ).**
Group	Processing method	$n$ $n$	Green signal intensity ratio M ± SD
1	MB	9	77.53 ± 6.46
2	MB/650 nm 15.2 J/cm²	9	65.96 ± 8.09*
3	MB/650 nm 30.4 J/cm²	9	31.18 ± 6.12*°
4	HMME	9	73.65 ± 12.34*
5	HMME/532 nm 15.2 J/cm²	9	61.24 ± 28.52*
6	HMME/532 nm 30.4 J/cm²	9	36.51 ± 9.93*°
7	0.2% chlorhexidine	10	68.29 ± 18.37*
8	0.9% normal saline	9	81.87 ± 5.34

Notes: * indicates that compared with the 0.9% saline group, p < 0.05, there was a statistical difference. ° indicated that p < 0.05 was statistically different from that of the 0.2% chlorhexidine group.

Fig. 3. Confocal laser images of each group. The green signal represents live bacteria. The red signal indicates that the cell membrane is damaged and dead. The yellow image is the result of the superposition of red and green signals.

Compared with the positive control group (sodium hypochlorite group), the difference between the 15.2J/cm²MB-aPDT, 15.2J/cm²HMME-aPDT and the 0.2% chlorhexidine group was not statistically significant ( $p > 0.05$ $p > 0.05$ ). The proportion of live bacteria in the group of 30.4J/cm²MB-aPDT and 30.4J/cm²HMME-aPDT was lower than that in the group of 0.2% chlorhexidine, and the difference was statistically significant ( $p < 0.05$ $p < 0.05$ ). The difference between MB group and sodium hypochlorite group was statistically significant ( $p < 0.05$ $p < 0.05$ ). There was no significant difference between HMME group and sodium hypochlorite group ( $p > 0.05$ $p > 0.05$ ).

Comparing different photosensitizers at the same energy density, there was no significant difference in the proportion of live bacteria between the 15.2J/cm²MB-aPDT and the 15.2J/cm²HMME-aPDT group ( $p > 0.05$ $p > 0.05$ ). There was no significant difference between the 30.4J/cm²MB-aPDT and the 30.4J/cm²HMME-aPDT groups ( $p > 0.05$ $p > 0.05$ ).

Among the same photosensitive agents and under different energy densities, the proportion of live bacteria in the group of 30.4J/cm²MB-aPDT was lower than that in the group of 15.2J/cm²MB-aPDT, and the difference was statistically significant ( $p < 0.05$ $p < 0.05$ ). The proportion of live bacteria in group 30.4J/cm²HMME-aPDT was lower than that in group 15.2J/cm²HMME-aPDT, and the difference was statistically significant ( $p < 0.05$ $p < 0.05$ ).

4. Discussion

In PDT, the more energy provided before the quenching of the photosensitizer itself, the more singlet oxygen produced, as shown in Fig. 4.^31,32 Therefore, increasing the energy density can effectively improve the effect of aPDT. This is consistent with the results of this experiment. Through aPDT guided by LED light source, some scholars believe that in the antibacterial field, in order to achieve effective sterilization effect, the energy density of aPDT should be above 30J/cm² when LED light is used for irradiation.¹⁸ Some scholars also use incandescent lamp as the light source and believe that PDT has a significant sterilization effect only when the energy density is above 500J/cm².^19,20 The energy density of different light sources is mainly due to the different absorption efficiency of photosensitizer to different wavelengths of light. In this experiment, a more stable and more efficient laser was used as the light source, so the 15.2J/cm² group and the 30.4J/cm² group were set up to compare the effect of aPDT therapy under different energy densities. According to the results, the sterilization rate of the two groups of 30.4J/cm² was higher than that of the two groups of 15.2J/cm², while aPDT therapy still had sterilization effect under the energy density of 15.2J/cm², similar to that of chlorhexidine of 0.2%.This suggests that the use of a more efficient light source can improve energy efficiency in aPDT. Increasing energy density can obviously improve the effectiveness of PDT, however, energy density cannot be increased indefinitely. Although with the deepening of the research on photosensitizer, the quenching time of the newly developed photosensitizer itself is far longer than the treatment time of PDT, however, the energy density depends on the power density and exposure time. Especially, in clinical applications, the power density selected in aPDT is usually lower (most of which are less than 100mw/cm²) for safety reasons. Therefore, prolonged exposure time is often the main choice to improve aPDT energy supply. In the treatment of clinical tumors and other diseases, the treatment duration of aPDT is usually 15–30min under the premise of low power density.^21,22 In the treatment process of oral clinical diseases, due to the limitation of treatment time, the treatment time cannot be too long. Therefore, finding the best combination of power density and time is also one of the main goals of aPDT research in the field of stomatology.

The selection of excitation light of photosensitizer should be based on its own spectral characteristics. The absorption peak of MB in the visible light range appears at 550–700nm,²³ and the absorption peak of HMME to blue light (wavelength around 450nm) is the highest in addition to ultraviolet light, as shown in Fig. 5.^24,25 However, in clinical application, due to the low popularity of blue laser, the green light (512nm) laser with wider application is selected. Its wavelength is the second highest in the visible light absorption peak of HMME, and it is also one of the laser wavelengths commonly selected in clinical research. Compared with red light, green light has insufficient soft tissue penetration, so PDT mostly chooses red light as excitation light in the field of tumor therapy. However, infectious diseases commonly seen in oral clinic, such as caries, periodontal disease, wisdom tooth pericoronitis and other diseases, can be directly irradiated to the infected lesions by instruments and optical fiber in the treatment process, avoiding the problem of insufficient penetration ability of soft tissue under green light.

Fig. 5. MB and HMME absorption spectrum.

The concentration of sensitizer also has a great influence on the effect of PDT. When the concentration is low, there is not enough photosensitizer in the target cells or tissues to participate in photochemical reactions, so the efficiency of PDT is reduced and no obvious curative effect can be achieved. At high concentration, photosensitizer will change its aggregation form and affect the absorption peak of the solution. For example, when the concentration is greater than 0.0003mg/mL, MB becomes dimer due to self-aggregation, and its peak excitation wavelength becomes 610nm.²⁶ The absorption peak of HMME also changes with the concentration. Due to the change of absorption peak, the absorption rate of the same wavelength laser at different concentrations is different, thus affecting the efficiency of producing singlet oxygen by photosensitizer.^27,28 Due to the enrichment of photosensitizer by bacteria and their surrounding proteins, what form of photosensitizer exists in the microscopic environment and whether this aggregation form will affect the selection of excitation light still needs further experimental clarification.

The lactate concentration assay was based on lactate dehydrogenase colorimetry. The results showed that the acid production capacity of plaque biofilm was decreased after aPDT, but there was no significant difference between the groups. This may be due to the lack of sensitivity of the method itself or the fact that the alkaline environment required to prepare photosensitizer solution affected the presence of lactic acid and thus interfered with the experimental results. However, the results still proved to a certain extent that aPDT could reduce the acid production capacity of biofilm and was consistent with the bactericidal efficiency represented by colony count results.

In this experiment, FDA/EB dye was selected for fluorescent staining. The emission light wavelength of EB combined with DNA was about 600nm, and the emission light signal received above 660nm was weakened. However, as a porphyrin, HMME has the property of fluorescence induced by excitation. According to the literature, the fluorescence peak of HMME is located at about 620nm.^29,30 In order to minimize the influence of HMME autofluorescence on this experiment, this experiment chose to use 650nm wavelength signal to detect EB fluorescence. However, HMME still has a weak fluorescence signal, so it may lead to a higher intensity of red signal in HMME photosensitive group than the fluorescence intensity emitted by the actual EB dye, which will interfere with the results. Different from colony count, laser confocal microscopy can observe the activity of plaque biofilm immediately after treatment, which can be used to analyze the mechanism of action of treatment steps on biofilm. However, since the staining principle of various fluorescent dyes is mostly based on the integrity of cell biofilms to distinguish between living and dead bacteria, therefore, the observation of CLSM is quite limited and easy to be interfered by other nonfluorescent dyes introduced in the experimental process. So, the fluorescence characteristics of each substance should be taken into account in the design of experiments and analysis results to minimize the experimental errors.

5. Conclusion

APDT therapy has a sterilizing effect on S. mutans in bacterial plaque biofilm, among which, the sterilizing effect of high energy density aPDT is better than that of low energy density aPDT, and the sterilizing effect of MB-aPDT is better than that of HMME-aPDT. aPDT can reduce acid production capacity of S. mutans biofilm. aPDT reduces the proportion of live bacteria in plaque biofilms.

List of abbreviations

S. mutans Streptococcus mutans

PDT Photodynamic therapy

HMME Hematoporphyrin monomethyl ether

MB Methylene blue

CHX Chlorhexidine

ROS Reactive oxygen species

HpD Hematoporphyrinderivative

DMSO Dimethyl sulfoxide

CFU Colony forming unit

BHI Brain Heart Infusion Medium

PBS Phosphate Buffer Saline

CLSM Confocal Laser Scanning Microscopy

PH Pondus Hydrogenii

HA Hydroxyapatite

FDA Fluorescein diacetate

EB Ethidium bromide

Conflicts of Interest

The authors declare that they have no conflict of interest.

Acknowledgments

This study was supported by the Construction Plan of the Tianjin Characteristic Subject Group, Oral Medical Engineering. We thank the Institute of Biomedical Engineering of the Chinese Academy of Medical Sciences for providing the site and technical support for our project. Xiaoyue Liang, Zhaohui Zou and Zheng Zou contributed equally to this paper.

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Vol. 13, No. 06

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Received 19 December 2019

Accepted 7 June 2020

Published: 8 August 2020

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This is an Open Access article. It is distributed under the terms of the Creative Commons Attribution 4.0 (CC-BY) License. Further distribution of this work is permitted, provided the original work is properly cited.

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