The effect of chlorhexidine on dental calculus formation: an in vitro study

Human saliva that had been stimulated by chewing wax was collected from one healthy male (one of the authors) who had not consumed food for 2 h prior to donation. The subject had no evidence of present caries and had not taken antibiotics for at least 3 months prior to donation. The saliva was centrifuged at 10,000 g for 10 min, and the pellet was re-suspended in brain heart infusion (BHI) broth (Becton, Dickinson, and Company, Sparks, MD, USA). The suspension was adjusted to an OD₆₀₀ of 0.2 and was used as an inoculum for biofilm growth. The supernatant was sterilized as described previously [16] and used for pellicle formation.

Biofilms were generated using an MBEC™ device (Innovotech, Edmonton, Canada) as per the method described by Pesciaroli et al. [17] with some modifications. The device consisted of a plastic lid with 96 hydroxyapatite (HA)-coated pegs and individual wells (Fig. 1). Following pellicle formation for 2 h at 37 °C, the pegs were transferred into 200 μl of the bacterial inoculum described above and incubated for 1 h at 37 °C under aerobic conditions, allowing the microorganisms to colonize the peg. The pegs were then transferred into BHI broth containing 0.5% sucrose and incubated under anaerobic conditions at 37 °C for 3 days. The medium was changed every 12 h until the day of harvesting.

The biofilms that formed on the pegs were washed to remove unbound cells by placing the lid into the rinse plate with ion exchanged water (IEW) for 10 s. They were then exposed for 1 min to distilled water (DW) or 0.12% CHG (Sigma-Aldrich, St. Louis, MO, USA), which is the concentration involved in a conventional mouthwash product [18, 19]. After washing with IEW for 1 min, the biofilms were transferred into calcifying solution and incubated for up to 48 h at a maximum of 37 °C under anaerobic conditions (N₂ 85%, CO₂ 10%, O₂ 5%). The calcifying solution was composed of Solution A (NaCl 2.1 g, KCL 1.56 g, Na₂HPO₄·12H₂O 0.9 g, NaH₂PO₄· 2H₂O 0.39 g, KSCN 0.87 g, Urea 0.2 g, 3,3-dimethylglutaric acid 0.8 g, NaOH 0.36 g, and BHI medium 900 ml) and Solution B (CaCl₂·6H₂O 0.33 g and distilled water 100 ml) [20]. Solutions A and B were sterilized separately for 20 min at 121 °C and mixed together just before use. Biofilms were exposed to CHG for 1 min every 12 h and transferred into a new calcifying solution. Treatment with DW served as the control. Biofilms were also immersed in BHI instead of the calcifying solution to determine the amount of mineral uptake from the media. A summary of the experimental design is displayed in Fig. 2. During the experimental process, bacterial viability was checked by assessing the turbidity of the media.

Mineral analysis was performed using a modification of the method described by Wong and Sissons [21]. Following assigned treatment (0, 12, 24, and 48 h in mineral uptake phase; Fig. 2), biofilms were extracted with 1 ml of 0.5 mol/l perchloric acid (Nacalai Tesque, Inc., Kyoto, Japan) and centrifuged at 15,000 g for 15 min. Calcium (Ca) in the supernatant was measured by inductively coupled plasma-atomic emission spectrometry (SPS1500; Seiko Instruments, Inc., Tokyo, Japan), and phosphate (Pi) was measured using the Malachite Green Phosphate Assay Kit (Bioassay Systems, Hayward, CA, USA), according to the manufacturer’s instructions. The pellet was re-suspended in 1 ml of distilled water and the optical density (OD) at 620 nm was recorded. Calculated Ca and Pi concentrations (ppm) were converted to values that corresponded to the same bacterial volume (OD = 1.0) [22]. This assay was performed with a total of four replicates per treatment.

Pegs were broken from the lid and placed into an empty receiver vial, and specimens were desiccated in a dry box. Thereafter, biofilms were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer for 1 h at room temperature. All specimens were washed twice with phosphate buffered saline and dehydrated in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100% for 10 min each). Specimens were then mounted on carbon stubs and sputter-coated with a 300-Å-thick gold layer using an ion coater (IC-50; Shimadzu, Kyoto, Japan). Morphology and elemental composition were analyzed using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer with an image observation function (SEM-EPMA, EPMA1601; Shimadzu). The limit of detection of the elements was approximately 1 μm in depth [23]. This assay was performed with a total of four replicates per treatment. For image analysis, three sample locations on the biofilm surface were randomly selected on each image, and the Ca: Pi ratio at each location was calculated.

The calcifying solution promoted the uptake of both Ca and Pi in biofilms regardless of CHG exposure (Fig. 3a and b). The prolonged incubation time alone, without CHG exposure, did not influence mineral uptake. Incubation for 12 h following a single exposure to CHG significantly increased the amount of minerals in biofilms compared with the control (p < 0.05). Repeatedly exposing biofilms to CHG dose-dependently increased Ca deposition, and the amount of Ca was five times as much as that of the control (Fig. 3a). Pi levels in CHG-treated biofilms were significantly higher than those from the control group (p < 0.05); however, the number of exposures did not influence results significantly (Fig. 3b, p > 0.05). Although the Ca: Pi molar ratio of the total mineral content in CHG-treated biofilms was almost the same as control biofilms between 0 and 24 h, a significant increase was observed in CHG-treated biofilms at 48 h (Fig. 3c, p < 0.05).

An SEM-EPMA enabled morphological observation and quantitative elemental analyses of Ca and Pi from the same field of view. We have presented representative images in Fig. 4. Analysis of SEM data showed that cocci- and bacilli-like bacteria were densely packed within extracellular polysaccharide (EPS)-like structures on the surface of pegs after 3 days of incubation (Fig. 4a). In the control group (Fig. 4b-d), biofilms developed three-dimensionally during further incubation for two days. Microorganisms (white arrow head) were observed to be embedded in EPS-like structures (white arrow). In the CHG treatment group, biofilm structures remained on the peg even after repeated exposure to CHG (Fig. 4e-g). Biofilms in the shape of clusters were observed on the surface, and these clusters were relatively small in comparison with the control. Bacterial growth was achieved in the media until 24 h incubation following exposure to CHG, and it was not detected after 36 h, meaning that microorganisms in the biofilm were completely disinfected after 48 h. Many small particles, which were not microorganisms, were observed on the surface of the biofilm, especially in the CHG treatment group (black arrow heads in the inset of Fig. 4g).

We performed EPMA analysis to determine whether these particles were of mineral composition. Figure 5 showed the profiles of Ca and Pi within a region determined by a rectangle in Fig. 4. The red line graph indicates Ca, and the yellow line graph indicates Pi. The Y axis represents the count, which indicates the X-ray intensity of the spectral peak. The mean Ca: Pi ratio in the control group was 1.06 ± 0.12, 1.12 ± 0.18, and 1.16 ± 0.22 (mean ± standard deviation) for 12, 24, and 48 h incubation, respectively. The mean Ca: Pi ratio in the CHG treatment group was 1.18 ± 0.18, 1.20 ± 0.20, and 1.40 ± 0.26 (mean ± standard deviation) for 12, 24, and 48 h incubation, respectively. There was a significant difference between the control group and the CHG treatment group after incubation for 48 h (p < 0.05).