In vitro antibacterial effects of photodynamic therapy against Enterococcus faecalis in root canals of deciduous teeth

Sample collection and preparation

A total of 44 maxillary deciduous anterior teeth obtained from the Department of Pediatric Dentistry, Stomatological Hospital, Southern Medical University were used. The study was approved by the Human Research Ethics Committee of our hospital (No 2019–32).

The inclusion criteria were: i. a complete absence of dental caries or an absence of pulp infection and ii. single-rooted with complete root. To standardize the roots, crowns were removed from the 9 mm roots and the pulps extracted. The working length was established to be 1 mm lower than the apical foramen, so, the root canals were enlarged to #35 K-file (Dentsply-Maillefer, Ballaigues, Switzerland). 1% NaClO (Fujian weizhenyuan medical science and technology Co., Fujian, China) was used as the irrigation solution. To remove the smear layer, the root canals were rinsed in 17% EDTA (Yongan Laboratory, Shandong, China) for 4 min while the removal of residual irrigations was done in 5 ml of normal saline.

An adhesive system (Dentsply, Konstanz, Germany) and a composite resin (Densberg, Milford, USA) were used to seal the apices. The roots were placed in a glass bottle containing distilled water and sterilized in an autoclave (Shanghai Boxun Co., Shanghai, China) at 121 ℃, 1.5 MPA, for 15 min. Two root samples were randomly selected and incubated in brain–heart infusion (BHI) broth (Hukai microorganism Co., Guangzhou, China) for 48 h at 37 °C. Two samples were randomly selected for observation under SEM(Hitachi, Japan) to make sure that the smear layer of the root canal wall was removed.

The root canals were infected with a standard strain of E. faecalis (ATCC29212). E. faecalis was grown in BHI broth at 37 °C overnight. At a wavelength of 600 nm, its concentration was spectrophotometrically adjusted to an absorbance of 0.1 (about 1 × 108 cells/ml).

The root canal samples were placed on the silicone rubber base in a 24-well plate. A 2 ml aliquot of the culture of E. faecalis was inoculated into the root canal of each sample. To promote growth, the E. faecalis cultures were maintained for 21 days. After every 48 h, BHI was replaced.

Disinfection of the root canals

Forty samples were randomly assigned into 4 groups of 10 each.

Group 1(Normal saline group): The root canals were irrigated with 5 ml of saline for 1 min. To rinse the root canals, a 27-G lateral perforated needle was introduced 2 mm short of the working length, pull up and down slightly.

Group 2(1% NaClO group): 5 ml of 1% NaClO was used to irrigate the root canal for 2 min after which they were irrigated with 5 ml of saline for 1 min.

Group 3(PDT group): 0.1 mg/ml methylene blue (Zhengzhou Jiatai Biotechnology Co., Ltd., Zhengzhou, China) was injected into the root canal for 1 min. Next, following the manufacturer’s instruction,diode laser (Zhengzhou Jiatai Biotechnology Co, Ltd, Zhengzhou, China), with 13.2 J/cm2, 200mW and red continuous emission (660 ± 10 nm wavelength), with an intracanal fiber (diameter 0.6 mm) attached, was used to irradiate the apical third for 1 min and this process was conducted twice. Subsequently, 5 ml saline was used to irrigate the root canal for 1 min.

Group 4(1% NaClO + PDT group): Refer to group 2,the PDT procedure was performed and the diode laser irradiate for 1 min twice.Then 5 ml of 1% sodium hypochlorite was used to rinse the root canals for 2 min after-which they were rinsed with 5 ml of normal saline for 1 min.

Microbiological analysis

The samples collected immediately after contamination with E. faecalis and before the decontamination process were referred to as the initial sample (S1), while the samples collected after the decontamination process were referred to as the final sample (S2). A sterile size 35 paper point(Daya Ding Medical Devices Co., Ltd., Beijing, China)was placed in the root canal for 1 min and transferred to a sterile Eppendorf tube containing 1 ml saline. The suspension in the Eppendorf tube was vortexed for 60 s. Tenfold serial dilutions of the suspension was done in saline. 0.1 ml aliquots were spread on BHI agar plates and incubated at 37 °C for 48 h. Colony counts were done after incubation. The operator who is conducting the experiment and the examiners when assessing the results must be provided are different people.

Sample preparation for confocal laser scanning microscopy

Five root canals of deciduous teeth were randomly selected from each group and split using a slow turbine. Each sample was treated with LIVE/DEADTM BacLight Bacterial Viability Kit-L7012 (Invitrogen, Oregon, USA) staining solution for 15 min. Staining was done in the dark at room temperature. Residual fluorescent dyes were removed by rinsing in PBS for 1 min. Samples on the confocal plate were observed by CLSM (LSM 880 with Airyscan, ZEISS,Germany). From each sample, three fields of view were randomly selected and the CLSM image of the surface layer of the biofilm (1024 × 1024 pixels) was observed under a × 20 lens. The green channel represented living bacterial cells while the red channel represented dead bacterial cells. The fluorescence intensity of the image is analyzed using Image J software, and the ratio of red fluorescence to red fluorescence + green fluorescence, that is, the ratio of dead bacteria to total bacteria, was calculated.

SEM preparation and analysis

After treatment, the root canals and the remaining 5 samples in each group were detected by SEM. Samples were stored in glutaraldehyde solution at 4 ℃ for 24 h and were then detected by a scanning electron microscope after gradient dehydration, critical point drying and gold spraying. Magnifications of × 1000, × 5000 and × 20,000 were used focus the residual biofilm on the surface of the root canal.

Data analysis

The statistical software SPSS 25 was used in data analysis. Normal distribution and data homogeneity were tested. Since the data were not normally distributed, the Kruskal–Wallis test and Dunn test with boferroni adjustment were used to analyze the effect of the different treatment techniques on the E. faecalis in root canals. Values with p < 0.05 were considered significantly.

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