Bovine Dentin Discs Preparation
In the present study, freshly extracted caries-free bovine incisors from animals killed for commercial reasons kept in 0.01% sodium hypochlorite were used as bacterial biofilm culturing substrates, as described previously [17]. After soft tissues and calculus deposits were removed with a periodontal curette, these bovine teeth were horizontally cut at the cement-enamel junction with a 0.6-mm-thick (Isomet, Buehler Ltd, Lake Bluff, IL, USA) under water cooling. A thin coronal groove was prepared in the middle of each crown using a low-speed handpiece with a bur, which was then fractured with a blade and a hammer into two pieces. To obtain uniform substrate to culture bacterial biofilms, the enamel of each piece was discarded, and the dentin surface was ground by 320-grit, 600-grit and 1500-grit silicon carbide paper in sequence and shaped by a fine carbide bur (Tulsa Dentsply, Tulsa, OK, USA) with a low-speed handpiece to standardize thickness of 1.5 mm and diameter of 12 mm. Using 5.25% NaOCl and 6% citric acid (pH 4.0) in an ultrasonic bath for 5 minutes to remove the smear layer, each dentin disc was irrigated and then rinsed in distilled water for 5 minutes. These samples placed in a glass bottle containing distilled water were autoclaved at 121 oC for 20 minutes, and then kept at 4 oC before any subsequent experiments.
Two specimens were fixed in 4% glutaraldehyde in 0.1 M phosphate-buffered saline (pH 7.2) at 4 oC for 24 hours, dehydrated by increasing concentrations of ethanol solutions (30%, 50%, 70%, 90% and 100%), and dried using a critical point drier. Then the samples were sputter-coated with gold-palladium in a vacuum evaporator. The opening of dentinal tubules in bovine dentin blocks was observed by SEM (JEOL JSM-7500F, JEOL, Japan) at a magnification of 2000×-10000× operating at 10 kV.
E. faecalis Biofilms in Dentin Canal Samples
In the present study, E. faecalis (American Type Culture Collection 29212) stored at -80 oC was used as the test organism and grown overnight on brain-heart infusion (BHI) agar (Becton-Dickinson, sparks, MD, USA) plates at 37 oC in air. A single colony from one BHI agar plate was inoculated onto another sterile BHI agar plate for incubation at 37 oC in air overnight. The pure bacteria were harvested and suspended in BHI broth. Cell density was spectrophotometrically standardized to 3×106 colony-forming units (CFU)/mL in BHI broth.
Each bovine disc was kept in a well of 24-well tissue culture plate (Costar, Corning, NY, USA) in order to make sure the dentin surface of each sample was infected by E. faecalis suspensions. Eighteen hundred µL of sterile BHI broth and 200 µL of bacterial suspension were transferred to the dentin surface of each bovine sample for 1- and 3-week-old incubation at 37 oC in air. Fresh BHI medium was changed once a week for the 3-week-old specimens. The 1- and 3-week-old E. faecalis biofilms on four samples were examined by SEM as described previously.
Disinfection of Dentin Samples with aPDT
After incubation for 1 and 3 weeks, the dentin discs with E. faecalis biofilms were taken out of the wells and rinsed with sterile saline for 1 minute to remove unattached bacteria and culture broth. The 1- and 3-week-old bacterial biofilms on dentin discs were randomly subjected to the following three disinfecting treatments for 3, 12 and 30 minutes with nine specimens in each group: aPDT with 0.01% MB (Sigma, St. Louis, MO, USA), 2 mL of 5% NaOCl (Tianjin Dengke Ltd, Tianjin, China) and 2 mL of 0.85% sterile saline (control).
In the aPDT with 0.01% MB group, each sample was treated with 300 µL of 0.01% MB for 5 minutes in dark incubation, and then with aPDT (LaserHF, Hager & Werken, Tubingen, Germany) with a wavelength of 660 nm for 3, 12 and 30 minutes according to the manufacturer's instructions. The maximum output energy was 50 mW, and the outer diameter of optical fiber in the needle tip of the handpiece was 320 µm. The needle tip of aPDT was placed 1 mm above the top of the biofilms, and moved like letter "Z" at the same level (Fig. 1). In 12- and 30-minute aPDT groups, the LaserHF handpiece was activated every 3 minutes and powered off for 1 minute. Two mL of 5% NaOCl or saline was added every three minutes in the 12- and 30-minute groups. After exposure, each specimen was gently washed with saline for 1 minute and then examined with viability staining and CLSM as described previously [17, 18].
Confocal Laser Scanning Microscopy Examination
The LIVE/DEAD BacLight Bacterial Viability kit L-7012 (Molecular Probes, Eugene, OR, USA) containing SYTO 9 and propidium iodide (PI) was used to stain the E. faecalis biofilm cells in bovine dentin discs according to the manufacturer's instructions. The excitation/emission maxima for the two dyes are approximately 480/500 nm for SYTO 9 and 490/635 nm for PI. Bacteria with intact cell membranes stain fluorescent green by SYTO 9, whereas bacteria with damaged membranes stain red by PI. All of the specimens were rinsed with phosphate-buffered saline for 1 minute, and then viewed immediately by CLSM (Olympus FV1200, Olympus, Japan) using a 40× lens. Five randomly selected areas (0.3 mm × 0.3 mm for each area) on bacterial biofilms were examined by CLSM and 3D reconstruction analysis in each bovine disc per time point.
The CLSM images were captured using Fluoview version 4.3 software (Olympus, Melville, NY, USA) at a resolution of 512 × 512 pixels. A stack of 15 slices (1-µm step size) was scanned at each randomly chosen area on 1-week-old biofilms, and the scanning depth for 3-week-old biofilms was set at 50 µm. The dead cell volume (red fluorescence) and live cell volume (green fluorescence) were reconstructed into a 3D model and the portion of dead cell volume was analyzed by using the Imaris 7.2 software (Bitplane Inc, St Paul, MN).
Visible Changes in Structure of Bovine Dentin Discs
Six uninfected bovine dentin discs were randomly subjected to aPDT with MB for 12 and 30 minutes as described previously. There were three samples in each group at the same exposure time. After 1-day-old incubation, the dentin specimens were washed for 1 minutes using saline. The visible structural changes in three uninfected bovine dentin blocks and six aPDT-treated specimens were observed by SEM.
Statistical Analysis
The proportions of dead cell volume in E. faecalis biofilms were presented as the means and standard deviations (means ± SDs). Differences between the proportions of dead cell volume were subjected to univariate analysis of variance using SPSS 22.0 software (SPSS Inc, Chicago, IL, USA). Post hoc multiple comparisons were used to isolate and compare the results at a significance level of P < 0.05.