Saliva collection and processing
A sterilized saliva solution was prepared using a previously described method . Unstimulated saliva was collected from a healthy person (one of the authors) who had not eaten, drank, or brushed for at least 2 hours prior to saliva collection. Saliva samples were diluted (1:10) with sterilized Ringer’s solution containing 0.05 % cysteine (Sigma-Aldrich, St. Louis, MO). Diluted solutions were centrifuged at 2,000 g for 10 minutes to remove any debris, and the supernatants were filter sterilized. The saliva solution was used to coat discs with salivary pellicles [18, 23]. All study protocols were approved by the Ethics Committee for Clinical Research of Niigata University (approval no. 2019-0002).
Preparation of the biofilm structure
S. mutans ATCC 25175, which was originally isolated from carious dentin, was purchased from the American Type Culture Collection and was cultured anaerobically at 37°C on brain heart infusion (BHI) (Difco Laboratories, Detroit, MI) agar plates. Single colonies were selected, inoculated in BHI broth without sucrose, and incubated overnight at 37°C under anaerobic conditions. The preculture was transferred to 10 ml of fresh BHI broth containing 0.5 % sucrose under anaerobic conditions and cultured for 4 hours at 37°C under aerobic conditions. The absorbance of all bacterial suspensions at 600 nm was adjusted to 0.05 prior to inoculation.
Composite resin materials (Premise Flowable, Kerr, Orange, CA) were used as the attachment site for the biofilm structure. Standardized discs, 6 mm in diameter and 1.5 mm in thickness, were prepared and polished with 4000 grit waterproof silicon carbide paper; then, they were subjected to ethylene oxide gas sterilization for 4 hours. The discs were coated with 10 % conditioned sterile saliva for 2 hours at room temperature. Biofilm structures were prepared as previously described . S. mutans biofilms were allowed to form on the discs using a rotating-disc reactor (RDR) (Biosurface Technologies Corp., Bozeman, MT). This system is depicted in Fig. 1 and has been previously described in detail .
The discs were incubated for 90 minutes at 37°C in the BHI broth without sucrose, containing the S. mutans cell suspension while stirring at 75 rpm to achieve initial adhesion. Following the adhesion phase, the stir disc was gently rinsed with 100 ml of phosphate buffer (5.0 g l-1 NaCl and 2.5 g l-1 Na2HPO4, pH 7.4), and was aseptically transferred to a sterile reactor vessel filled with 300 ml of diluted BHI broth (1:10) containing 0.05 % sucrose. The biofilm was allowed to form for 72 hours while the solution was stirred at 50 rpm under continuous flow aerobic conditions at a rate of 4.6 ml min-1 during an incubation at 37°C. The medium was changed every 12 hours. After the fixed incubation period, the rotating wheel was aseptically removed, and the specimens were washed three times with phosphate buffer.
A pair of resin composite discs containing biofilms was inserted into recesses located between plastic teeth (Fig. 2A). These plastic teeth were then placed into a typodont model (Nissin Dental Products, Inc., Kyoto, Japan) located inside an exposure chamber containing either phosphate buffer or mouth rinses. The specimens were divided into six groups. Two independent experiments were performed to obtain n = 7 specimens per group. Each experiment consisted of an immersion group and a combination group. The immersion groups were treated with phosphate buffer (group C), a chlorhexidine digluconate Peridex™ (CHX) alcohol-containing (11.6%) mouth rinse (3M ESPE, USA) (group G), or a Listerine® Fresh Mint (EO) alcohol-containing (21.6%) mouth rinse (Johnson & Johnson, USA) (group L). The combination groups received the test mouth rinses and treatment with a sonic toothbrush (designated in the groups as ST) (Philips Sonicare Flexcare HX6930; ST). The combination groups were designated as the C + ST group, the G + ST group, and the L + ST group.
In the immersion groups, the biofilm constructs were immersed in the tested solution for 5 seconds. In the ST combined groups, the biofilms were treated with a sonic toothbrush for 5 seconds at a position located 2 mm away from the disc (Fig. 2B-C). Thereafter, the discs with biofilms were gently rinsed with sterile phosphate buffer three times to remove excess treatments, and the residual biofilm structure was observed with a scanning electron microscope (SEM). In addition, the biofilm was recovered by an ultrasonic treatment, the viable cell count was estimated using the colony counting method, and the total number of bacteria was calculated using the PCR-Invader method.
After treatment, the biofilm structure was observed with a SEM (EPMA-1610, Shimadzu, Kyoto, Japan). Specimens were washed with phosphate buffer and fixed with 2.5 % glutaraldehyde for 2 hours. After fixation, the fixed specimens were dehydrated using a series of ethanol solutions (10 minutes each in 60, 70, 80, 90, 95, and 100 % ethanol) and then air dried. The samples were sputtered with gold-palladium and examined using the SEM.
Cryo-embedding, cryo-sectioning, and measurement of the thickness of the S. mutans biofilms
The treated biofilm samples were embedded in a protective medium (Tissue-Tek O.C.T. compound, Sakura Finetek, Tokyo, Japan) as previously described . The resin discs were placed on dry ice, and the medium was gently poured on them from above and allowed to freeze. The resin discs were peeled away from the embedded medium to ensure that the biofilm remained attached to the medium side. Then, the biofilm was placed on dry ice with the embedded side down. The embedding medium was used to cover the bottom surface of the exposed biofilm. The embedded biofilm samples were sectioned into 8 μm cross sections using a cryostat (CM 3050 S; Leica, Nussloch, Germany). The thickness of the biofilm was measured and partitioned into 10 section intervals. Twenty-five sections from each embedded sample were analyzed.
Quantitative analysis of viable and total cells
Colony counts and the PCR-Invader assay were used to quantify the numbers of viable and total bacteria in the test samples, respectively. Samples were washed three times with phosphate buffer and immersed in 3 ml of phosphate buffer. Biofilms were collected by vortex mixing for 3 minutes, followed by ultrasonication for 5 minutes. Samples were serially diluted in autoclaved distilled water, and 100 ml of each dilution were plated on BHI agar. The plates were incubated anaerobically for 48 hours at 37°C, after which the number of viable colonies were counted.
The total number of bacteria was determined using the modified Invader PLUS method developed by BML Inc. (Saitama, Japan). The details of the PCR-Invader assay have been previously reported . The PCR-Invader assay combines polymerase chain reaction amplification and invader detection, and can calculate the total number of bacteria with high sensitivity and speed. The primers for S. mutans were based on a region of the 16S ribosomal RNA sequences. Bacterial DNA was extracted using Pure LC (Roche, Tokyo, Japan) and a MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche). The template DNA (3 ml) was added to 12 ml of a reaction mixture containing 20 mM primers, 2.5 mM dNTP, 2.5 U of AmpliTaq gold, 3.5 mM primary probe, 0.35 mM Invader oligo, and the Invader core reagent kit, which consisted of FRST mix and enzyme/MgCl2 solution (F-primer, 5’-GGATTCGCTAGTAATCG-3’; R-primer, 5’-TACCTTGTTACGACTT-3’; Tb-Primary probe, 5’-CGCGCCGAGGCCGGGAACGTATTCACC-3’; and Tb-Invader oligo, 5’-TGACGGGCGGTGTGTACAAGGCA-3’). Reaction mixtures were preheated at 95°C for 20 minutes, and then a two-step PCR was performed for 35 cycles (95°C for 1 second and 63°C for 1 minute) using the ABI PRISM 7900 sequence detection system (Applied Biosystems, Foster City, CA). Fluorescence values for carboxyfluorescein (wavelength/bandwidth: excitation 485/20 nm; emission 530/25 nm) were measured at the end of the incubation/extension step at 63°C for each cycle. Each assay was performed in triplicate and the mean values from the six independent samples were determined.
Statistical analyses were performed using the SPSS 11.0 (IBM, Armonk, NY) program. When applicable, data are presented as means ± standard deviations (SD). The significance of measurement biofilm thickness, viable and total cell counts were determined using Kruskal-Wallis test and the post hoc Steel-Dwass test.