Abstract
Objectives
To compare the antimicrobial efficacy and kill penetration of essential oils (EO) mouthrinse versus stannous fluoride, and triclosan dentifrice slurries on saliva-derived biofilms using confocal laser scanning microscopy (CLSM).
Methods
Saliva-derived biofilms were grown for 48 h on hydroxyapatite discs using pooled, homogenized saliva from 8 healthy volunteers as the inoculum. The mean thickness of these biofilms was 84 μm (range, 23–241 μm). CLSM with viability mapping was used to visualize the antimicrobial kill penetration of each treatment regime within a biofilm.
Results
At 30 s treatment durations, CLSM imaging revealed greater antimicrobial activity and kill penetration of EO mouthrinse compared to sodium fluoride-, stannous fluoride-, and triclosan-containing dentifrice slurries. Quantification of biovolume revealed that EO mouthrinse treatment at 30 s resulted in a greater non-viable biovolume proportion (84.6% ± 15.0%) than other treatment groups. Increasing the treatment duration of the triclosan dentifrice (to 60 and 120 s) resulted in better penetration and an increased reduction of viable cells, comparable to EO mouthrinse treatment at 30 s duration. Further, CLSM imaging showed that the combined treatment of a non-antimicrobial dentifrice (45 s) with EO mouthrinse (30 s) showed superior antimicrobial activity (96.2% ± 3.7%) compared to the antimicrobial triclosan-containing dentifrice used without a mouthrinse step (26.0% ± 32.0%).
Conclusions
Within typical exposure times, the EO-containing mouthrinse can penetrate deep into the accumulating plaque biofilm compared to the chemotherapeutic dentifrice slurries, and may provide an efficacious alternative to triclosan, when used as an adjunct with a mechanical oral care regimen.
Clinical significance
Using viability mapping and CLSM, this study demonstrated that EO-containing mouthrinse penetrates and kills microorganisms deeper and more effectively in plaque biofilm in typical exposure times when compared to dentifrice chemotherapeutic agents, providing an efficacious alternative to triclosan or stannous fluoride when used as an adjunct to mechanical oral care.
1
Introduction
Oral biofilms (dental plaque) are aggregates of multiple microbial species that can adhere to enamel and oral soft tissues. They comprise more than 700 identified species of oral microbes held together within an exopolymeric matrix , and are characteristic of commensal oral micro flora. However, uncontrolled oral biofilm formation disrupts the balance of synergistic and antagonistic interactions between bacterial aggregates and often leads to dental caries and periodontitis . Oral biofilms exhibit slower growth rates and tend to be far more virulent compared to planktonic bacteria, and are thus less susceptible to host defenses and show greater antibiotic resistance . Eliminating and preventing dental plaque accumulation is, therefore, imperative in combating dental and periodontal diseases.
Increasingly, clinical studies show that brushing alone may not be completely effective in preventing plaque accumulation . This is attributed to ineffective brushing technique, or the inability to provide complete plaque removal from all surfaces in the typical brushing times dedicated by patients (∼45 s ). It is also hypothesized that antimicrobial agents present in dentifrices cannot effectively penetrate hard to reach areas in the oral cavity, resulting in biofilm-dwelling bacteria accumulation in the interproximal and interdental spaces.
It has been repeatedly demonstrated that the therapeutic benefit of mechanical removal and disruption of accumulating plaque layers is enhanced when used in combination with the antimicrobial action of chemotherapeutic agents present in anti-septic mouthrinses . This is because antimicrobial mouthrinses in combination with brushing not only provide additional antimicrobial effects but also penetrate areas in the oral cavity less accessible to mechanical implements and dentifrices. Mouthrinses containing essential oils (EOs), cetylpyridinium chloride (CPC), or chlorhexidine (CHX), have proven effective in combating plaque formation and gingivitis . The quaternary compound CPC, when used as an adjunct to oral hygiene, provides a significant additional benefit in reducing plaque and gingival inflammation, compared to only brushing or brushing accompanied by a placebo rinse . The bacteriostatic and bactericidal effects of CHX make it most effective against plaque and gingivitis. However, one of the side effects associated with its use is an increase in staining of teeth . The hydrophobicity of EOs enables disruption of bacterial cell membranes and inhibition of bacterial enzymatic activity . EO-containing mouthrinses were found to be significantly effective at reducing bacterial vitality and thickness and density of oral biofilms . When used as an adjunct to a mechanical oral hygiene regimen, EO mouthrinses provide an additional benefit with regard to plaque and gingivitis reduction compared to brushing/flossing alone or brushing/flossing accompanied by use of a control non-EO mouthrinse .
Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) allow direct visualization of the anti-biofilm activity of these antimicrobial agents within the complex architecture of oral biofilms . CLSM is an especially valuable tool as it permits non-invasive spatial examination of the three dimensional (3D) structure of dental plaque. When paired with viability staining, it becomes possible to measure antimicrobial solutions for general effectiveness, speed of activity, and depth of penetration into a bacterial biofilm.
LISTERINE ® Antiseptic (LISTERINE ® Cool Mint ® , Johnson and Johnson Consumer Inc., New Jersey, USA), a mouthrinse containing a fixed combination of EOs, is well documented for its antigingivitis and antiplaque effects by multiple clinical trials . LISTERINE Antiseptic is also classified by the U.S Food and Drug Administration (FDA) as Category I, safe and effective for use against plaque and gingivitis .
In this study, using viability mapping through CLSM, we aimed to demonstrate the comparative antimicrobial efficacy and kill penetration of EO mouthrinse versus several dentifrices on in vitro saliva-derived biofilms. In addition, we tested the effects of a proposed regimen of a non-antimicrobial dentifrice paired with EO mouthrinse versus an antimicrobial triclosan-containing dentifrice used alone.
2
Materials and methods
2.1
Subjects
Healthy adults (aged >18 years) with a minimum of 20 natural teeth; no partial dentures, removable orthodontic appliances, or tongue piercings; and no antibiotic or anti-inflammatory therapy 30 days before saliva donation, dental prophylaxis during the 2 weeks preceding saliva donation, or any dental procedure (surgical/restorative), were included in the study.
Written informed consent was obtained from all subjects prior to saliva collection.
2.2
Saliva collection and processing
Subjects refrained from smoking, eating, drinking, and any oral hygiene procedure for 12 h before saliva collection. Donors chewed a 4″ × 4″ piece of Parafilm™; subsequently, 5–25 mL of saliva was collected in a sterile 50 mL capped centrifuge tube and stored at 4 °C. The saliva from each donor was then pooled into a sterile container and homogenized using a non-aerating mixer (Glass-Col, Indiana, USA). Pooled saliva was only used on the day it was collected.
2.3
Biofilm growth
Autoclaved hydroxyapatite discs (Himed Hitemco Medical Applications Inc., New York, USA) were incubated with homogenized, pooled human saliva statically at 35 °C for 30 min to form a pellicle and provide an initial organism source. Thereafter, further incubation steps were carried out with additional saliva and basic media/phosphate buffer saline mixture for 24 h; media was exchanged 2 times after the initial 24 h with a saliva-free basic media/phosphate buffer saline mixture for an overall incubation period of 48 h. Further details are provided in the Supplementary Methods section.
2.4
Preparation of dentifrice slurries
Three commercially available dentifrices, including Crest ® Pro-Health ® (0.45% stannous fluoride), Crest ® Cavity Protection (0.24% sodium fluoride) (The Procter & Gamble Co., Ohio, USA), and Colgate ® Total ® (0.30% triclosan) (Colgate-Palmolive Company, New York, USA) were used for this study. An equal weight of sterile saliva (Complete Saliva, Northeast laboratory services, Maine, USA) and dentifrice (10 g each) was combined in a 1:1 by weight ratio . Conical tubes were vortexed at maximum speed for 5 min each and then centrifuged at 7000 RPM for 10 min. The liquid supernatant of each dentifrice slurry was subsequently used for biofilm treatment.
2.5
Biofilm treatment
Biofilms were treated with: filtered sterilized water (as a negative control); LISTERINE ® Antiseptic mouthrinse (essential oils: 0.092% eucalyptol, 0.042% menthol, 0.060% methyl salicylate, and 0.064% thymol), the stannous fluoride dentifrice slurry, and the sodium fluoride dentifrice slurry for 30 s each; and the triclosan copolymer dentifrice slurries for 30, 45, 60, and 120 s. Biofilms were treated with LISTERINE Antiseptic for 30 s as per label instructions. Experiments were done in triplicate and neutralizers were used to halt all antimicrobial activity after treatment. Details are provided in the Supplementary Methods section.
2.6
Viability staining
A mixture of Sytox Green (Invitrogen Life Sciences, Massachusetts, USA) and CellTrace™ Calcein Red-Orange, AM- Special packaging (Invitrogen Life Sciences, Massachusetts, USA) was used for viability staining. The staining process was performed for each treatment group immediately before imaging. Details are provided in the Supplementary Methods section.
2.7
CLSM imaging and image processing
A Leica TCS SP5 upright confocal laser scanning microscope (Leica microsystems, Wetzlar, Germany) and a HCX APO L/0.90W U-V-1 water immersion lens was used for image acquisition. Stains were excited with 488 nm and 633 nm laser lines and emissions were each detected with separate photomultipliers set to 505–535 nm and 620–670 nm respectively. Two z-stacks were acquired on each disc and fields of view were chosen at random (disc perimeter was avoided).
Optical sections of 1.01 μm were acquired and z-stacks were imported into Volocity Image Analysis Software (PerkinElmer, Massachusetts, USA). One representative z-stack was chosen for each treatment group in each experiment; these were chosen visually based on lowest deviation from treatment group median. All representative z-stacks were processed identically for enhanced visibility: brightness was increased 2 x and black level was set to a 1% threshold for both channels. From each z-stack, 3 types of images were produced and exported as TIFFs: an XZ oriented extended focus/maximum projection, a 3D rendered image from above the biofilm looking down onto the top of the biofilm, and a 3D rendered image of the biofilm from the bottom looking up at the bottom of the biofilm.
2.8
Statistical analysis
Percent non-viable biovolume was quantified for the bottom 10 slices of each biofilm in Volocity. A measurement protocol was prepared to threshold each channel individually to 15% black level for Calcein Red-orange and 5% black level for Sytox Green, crop the thresholded image to 10 slices (10.1 μm) above the biofilm bottom, and measure the total biovolume of each channel. Percentage non-viable biovolume was calculated by dividing the biovolume of Sytox Green by the total combined-channel biovolume. All data were reported as mean ± standard deviation. Statistical comparisons were made using either one-way ANOVA with Tukey’s post-hoc analysis or Pearson correlations. Statistical significance was defined as P < 0.05.
2
Materials and methods
2.1
Subjects
Healthy adults (aged >18 years) with a minimum of 20 natural teeth; no partial dentures, removable orthodontic appliances, or tongue piercings; and no antibiotic or anti-inflammatory therapy 30 days before saliva donation, dental prophylaxis during the 2 weeks preceding saliva donation, or any dental procedure (surgical/restorative), were included in the study.
Written informed consent was obtained from all subjects prior to saliva collection.
2.2
Saliva collection and processing
Subjects refrained from smoking, eating, drinking, and any oral hygiene procedure for 12 h before saliva collection. Donors chewed a 4″ × 4″ piece of Parafilm™; subsequently, 5–25 mL of saliva was collected in a sterile 50 mL capped centrifuge tube and stored at 4 °C. The saliva from each donor was then pooled into a sterile container and homogenized using a non-aerating mixer (Glass-Col, Indiana, USA). Pooled saliva was only used on the day it was collected.
2.3
Biofilm growth
Autoclaved hydroxyapatite discs (Himed Hitemco Medical Applications Inc., New York, USA) were incubated with homogenized, pooled human saliva statically at 35 °C for 30 min to form a pellicle and provide an initial organism source. Thereafter, further incubation steps were carried out with additional saliva and basic media/phosphate buffer saline mixture for 24 h; media was exchanged 2 times after the initial 24 h with a saliva-free basic media/phosphate buffer saline mixture for an overall incubation period of 48 h. Further details are provided in the Supplementary Methods section.
2.4
Preparation of dentifrice slurries
Three commercially available dentifrices, including Crest ® Pro-Health ® (0.45% stannous fluoride), Crest ® Cavity Protection (0.24% sodium fluoride) (The Procter & Gamble Co., Ohio, USA), and Colgate ® Total ® (0.30% triclosan) (Colgate-Palmolive Company, New York, USA) were used for this study. An equal weight of sterile saliva (Complete Saliva, Northeast laboratory services, Maine, USA) and dentifrice (10 g each) was combined in a 1:1 by weight ratio . Conical tubes were vortexed at maximum speed for 5 min each and then centrifuged at 7000 RPM for 10 min. The liquid supernatant of each dentifrice slurry was subsequently used for biofilm treatment.
2.5
Biofilm treatment
Biofilms were treated with: filtered sterilized water (as a negative control); LISTERINE ® Antiseptic mouthrinse (essential oils: 0.092% eucalyptol, 0.042% menthol, 0.060% methyl salicylate, and 0.064% thymol), the stannous fluoride dentifrice slurry, and the sodium fluoride dentifrice slurry for 30 s each; and the triclosan copolymer dentifrice slurries for 30, 45, 60, and 120 s. Biofilms were treated with LISTERINE Antiseptic for 30 s as per label instructions. Experiments were done in triplicate and neutralizers were used to halt all antimicrobial activity after treatment. Details are provided in the Supplementary Methods section.
2.6
Viability staining
A mixture of Sytox Green (Invitrogen Life Sciences, Massachusetts, USA) and CellTrace™ Calcein Red-Orange, AM- Special packaging (Invitrogen Life Sciences, Massachusetts, USA) was used for viability staining. The staining process was performed for each treatment group immediately before imaging. Details are provided in the Supplementary Methods section.
2.7
CLSM imaging and image processing
A Leica TCS SP5 upright confocal laser scanning microscope (Leica microsystems, Wetzlar, Germany) and a HCX APO L/0.90W U-V-1 water immersion lens was used for image acquisition. Stains were excited with 488 nm and 633 nm laser lines and emissions were each detected with separate photomultipliers set to 505–535 nm and 620–670 nm respectively. Two z-stacks were acquired on each disc and fields of view were chosen at random (disc perimeter was avoided).
Optical sections of 1.01 μm were acquired and z-stacks were imported into Volocity Image Analysis Software (PerkinElmer, Massachusetts, USA). One representative z-stack was chosen for each treatment group in each experiment; these were chosen visually based on lowest deviation from treatment group median. All representative z-stacks were processed identically for enhanced visibility: brightness was increased 2 x and black level was set to a 1% threshold for both channels. From each z-stack, 3 types of images were produced and exported as TIFFs: an XZ oriented extended focus/maximum projection, a 3D rendered image from above the biofilm looking down onto the top of the biofilm, and a 3D rendered image of the biofilm from the bottom looking up at the bottom of the biofilm.
2.8
Statistical analysis
Percent non-viable biovolume was quantified for the bottom 10 slices of each biofilm in Volocity. A measurement protocol was prepared to threshold each channel individually to 15% black level for Calcein Red-orange and 5% black level for Sytox Green, crop the thresholded image to 10 slices (10.1 μm) above the biofilm bottom, and measure the total biovolume of each channel. Percentage non-viable biovolume was calculated by dividing the biovolume of Sytox Green by the total combined-channel biovolume. All data were reported as mean ± standard deviation. Statistical comparisons were made using either one-way ANOVA with Tukey’s post-hoc analysis or Pearson correlations. Statistical significance was defined as P < 0.05.
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