Quantitative analysis of the effects of essential oil mouthrinses on clinical plaque microbiome: a parallel-group, randomized trial.

BACKGROUND: The rich diversity of microorganisms in the oral cavity plays an important role in the maintenance of oral health and development of detrimental oral health conditions. Beyond commonly used qualitative microbiome metrics, such as relative proportions or diversity, both the species-level identification and quantification of bacteria are key to understanding clinical disease associations. This study reports the first-time application of an absolute quantitative microbiome analysis using spiked DNA standards and shotgun metagenome sequencing to assess the efficacy and safety of product intervention on dental plaque microbiome. METHODS: In this parallel-group, randomized clinical trial, essential oil mouthrinses, including LISTERINE® Cool Mint Antiseptic (LCM), an alcohol-containing prototype mouthrinse (ACPM), and an alcohol-free prototype mouthrinse (AFPM), were compared against a hydroalcohol control rinse on clinical parameters and the oral microbiome of subjects with moderate gingivitis. To enable a sensitive and clinically meaningful measure of bacterial abundances, species were categorized according to their associations with oral conditions based on published literature and quantified using known amounts of spiked DNA standards. RESULTS: Multivariate analysis showed that both LCM and ACPM shifted the dysbiotic microbiome composition of subjects with gingivitis to a healthier state after 4 weeks of twice-daily use, resembling the composition of subjects with clinically healthy oral conditions recruited for observational reference comparison at baseline. The essential oil-containing mouthrinses evaluated in this study showed statistically significant reductions in clinical gingivitis and plaque measurements when compared to the hydroalcohol control rinse after 6 weeks of use. CONCLUSIONS: By establishing a novel quantitative method for microbiome analysis, this study sheds light on the mechanisms of LCM mouthrinse efficacy on oral microbial ecology, demonstrating that repeated usage non-selectively resets a gingivitis-like oral microbiome toward that of a healthy oral cavity. TRIAL REGISTRATION: The trial was registered on ClinicalTrials.gov on 10/06/2021. The registration number is NCT04921371.

Targeting macrophages and their recruitment in the oral cavity using swellable (+) alpha tocopheryl phosphate nanostructures.

The phosphorylation of (+) alpha tocopherol produces adhesive nanostructures that interact with oral biofilms to restrict their growth. The aim of this work was to understand if these adhesive (+) alpha tocopheryl phosphate (α-TP) nanostructures could also control macrophage responses to the presence of oral bacteria. The (+) α-TP planar bilayer fragments (175 nm ±â€¯21 nm) formed in a Trizma®/ethanol vehicle swelled when exposed to the cell lines (maximum stabilized size = 29 µm). The swelled (+) α-TP aggregates showed selective toxicity towards THP-1 macrophages (LD50 = 304 µM) compared to human gingival fibroblasts (HGF-1 cells; LD50 > 5 mM), and they inhibited heat killed bacteria stimulated MCP-1 production in both macrophages (control 57.3 ±â€¯18.1 pg/mL vs (+) α-TP 6.5 ±â€¯3.2 pg/mL) and HGF-1 cells (control 673.5 ±â€¯133 pg/mL vs (+) α-TP - 463.9 ±â€¯68.9 pg/mL).

Soft, adhesive (+) alpha tocopherol phosphate planar bilayers that control oral biofilm growth through a substantive antimicrobial effect.

'Soft' nanomaterials have the potential to produce substantive antibiofilm effects. The aim of this study was to understand the oral antimicrobial activity of soft nanomaterials generated from alpha-tocopherol (α-T) and alpha-tocopherol phosphate (α-TP). (+) α-TP formed planar bilayer islands (175 ± 21 nm, -14.9 ± 3.5 mV) in a Trizma® buffer, whereas (+) α-T formed spherical liposomes (563 ± 1 nm, -10.5 ± 0.2 mV). The (+) α-TP bilayers displayed superior Streptococcus oralis biofilm growth retardation, a more substantive action, generated a superior adsorption to hydroxyapatite and showed an enhanced inhibition of multi-species bacterial saliva biofilm growth (38 ± 7µm vs 58 ± 18 µm, P ˂ 0.05) compared to (+) α-T. Atomic force microscopy data indicated that the ability of the 'soft' α-TP nanomaterials to transition into planar bilayer structures upon contact with interfaces facilitated their adhesive properties and substantive antimicrobial effects.

A randomized trial of ethyl lauroyl arginate-containing mouthrinse in the control of gingivitis.

AIM: This 4-week, single-centre, randomized, examiner-blind, controlled study investigated the efficacy and safety of 0.15% ethyl lauroyl arginate (LAE)-containing mouthrinse in adults with mild-to-moderate gingivitis. MATERIAL AND METHODS: Subjects were randomized to use 0.15% LAE-containing mouthrinse or 5% hydroalcohol-negative control twice daily after brushing with standard fluoride toothpaste. Plaque, gingivitis and bleeding were assessed at baseline and Weeks 2 and 4. The oral microflora was analysed at baseline and Week 4. RESULTS: Eighty-seven subjects were randomized to treatment. The 0.15% LAE-containing mouthrinse was associated with statistically significantly (p < 0.001) greater reductions in mean plaque and gingivitis scores versus the negative control at Week 2 (difference [95% confidence interval]: plaque 0.83 [0.64, 1.02], 29.1%; gingivitis 0.11 [0.07, 0.14], 4.8%) and Week 4 (co-primary endpoints: plaque 1.23 [1.07, 1.39], 42.6%; gingivitis 0.23 [0.19, 0.28], 10.7%). Bleeding-index scores were significantly (p < 0.001) reduced versus the control at Weeks 2 (by 0.04 [0.03, 0.06], 36.3%) and 4 (by 0.06 [0.04, 0.08], 50.9%). No shifts were detected in the oral microflora. There were no treatment-related adverse events. CONCLUSIONS: The 0.15% LAE-containing mouthrinse was well tolerated and significantly reduced plaque, gingivitis and bleeding when used as an adjunct to tooth brushing for 4 weeks.

Diminishing biofilm resistance to antimicrobial nanomaterials through electrolyte screening of electrostatic interactions.

The extracellular polymer substances (EPS) generated by biofilms confers resistance to antimicrobial agents through electrostatic and steric interactions that hinder molecular diffusion. This resistance mechanism is particularly evident for antibacterial nanomaterials, which inherently diffuse more slowly compared to small organic antibacterial agents. The aim of this study was to determine if a biofilm's resistance to antibacterial nanomaterial diffusion could be diminished using electrolytes to screen the EPS's electrostatic interactions. Anionic (+) alpha-tocopherol phosphate (α-TP) liposomes were used as the antimicrobial nanomaterials in the study. They self-assembled into 700 nm sized structures with a zeta potential of -20 mV that were capable of killing oral bacteria (S. oralis growth inhibition time of 3.34 ± 0.52 h). In a phosphate (-ve) buffer the -ve α-TP liposomes did not penetrate multispecies oral biofilms, but in a Tris (hydroxymethyl)aminomethane (+ve) buffer they did (depth - 12.4 ± 3.6 µm). The Tris did not modify the surface charge of the α-TP nanomaterials, rather it facilitated the α-TP-biofilm interactions through electrolyte screening (Langmuir modelled surface pressure increase of 2.7 ± 1.8 mN/ m). This data indicated that EPS resistance was mediated through charge repulsion and that this effect could be diminished through the co-administration of cationic electrolytes.

In vitro efficacy of essential oil mouthrinse versus dentifrices.

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 48h on hydroxyapatite discs using pooled, homogenized saliva from 8 healthy volunteers as the inoculum. The mean thickness of these biofilms was 84µm (range, 23-241µm). CLSM with viability mapping was used to visualize the antimicrobial kill penetration of each treatment regime within a biofilm. RESULTS: At 30s 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 30s 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 120s) resulted in better penetration and an increased reduction of viable cells, comparable to EO mouthrinse treatment at 30s duration. Further, CLSM imaging showed that the combined treatment of a non-antimicrobial dentifrice (45s) with EO mouthrinse (30s) 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.

Anticaries efficacy of a new dentifrice for hypersensitivity.

PURPOSE: To evaluate the expected anticaries efficacy of a new dentifrice containing stannous fluoride as the anticaries agent and potassium nitrate as the antihypersensitivity agent using a series of laboratory and animal studies. METHODS: Four surrogate studies were performed in this assessment including fluoride uptake in sound enamel, enamel solubility reduction, fluoride bioavailability and animal caries. RESULTS: Each of these studies indicated the new dentifrice for hypersensitivity (Colgate Sensitive Maximum Strength) was effective in inhibiting the caries process. The data demonstrate that this new dentifrice is predicted to be highly effective against caries and equivalent to a positive control dentifrice.

Relationship between dentifrice fluoride concentration and clinical caries reduction.

PURPOSE: To review the published literature on clinical caries trials of fluoride dentifrices and to combine their results in order to evaluate the correlation between fluoride concentration in dentifrice formulations and their anticaries efficacy, i.e., the dose-response relationship. METHODS: All available dentifrice caries clinical trials reported in the literature of products having at least two fluoride concentrations greater than 0 ppm were reviewed, graphed and statistically analyzed. RESULTS: The evaluation supported a linear correlation between the fluoride concentration of dentifrices between 0 and 5000 ppm F and clinical caries efficacy.

The effects of outward forced convective flow on inward diffusion of potassium across human dentin.

PURPOSE: To measure the inward flux of potassium across human dentin disks, in vitro, in the absence and then in the presence of a simulated pulpal pressure (PP), before and after brushing with two desensitizing dentifrices. MATERIALS AND METHODS: Dentin discs were made from extracted unerupted third molars and etched with 6% citric acid to remove the smear layers. The hydraulic conductance (Lp or outward fluid movement) of the discs was measured before and after brushing for 2 minutes with two desensitizing dentifrices, Colgate Sensitive Maximum Strength or Sensodyne Fresh Mint, both dentifrices containing 5% potassium nitrate. The potassium flux was measured at a pressure of 0 cm H2O, and a simulated pulpal pressure of 20 cm H2O. RESULTS: The results showed the Colgate-brushed specimens had lower Lp (P < 0.05) than specimens brushed with Sensodyne or with saline. The Colgate-brushed specimens had a corresponding lower K+ flux, at a PP = 0 cm H2O, than specimens brushed with saline, but were not significantly different than specimens brushed with Sensodyne dentifrice. However, when the pressure was increased from 0 to 20 cm H2O, to simulate outward dentin fluid pressure, there were significant decreases in K+ flux across all three treatment groups, but the K+ flux of the Colgate-brushed specimens were affected the least, resulting in the Colgate-brushed specimens having significantly (P < 0.05) greater K+ fluxes than the saline or Sensodyne dentifrice-brushed groups. The magnitude of reduction in K+ flux in going from 0 cm H2O to 20 cm H2O was demonstrated to be linearly related to the magnitude of the Lp of the brushed specimens. Hence, a small Lp corresponded to a small reduction in K+ flux, and a correspondingly large K+ flux at 20 cm H2O. An explanation of this phenomenon is that a decrease in the Lp of dentin, produced by brushing, corresponds to a decreased outward dentin fluid flow. This lowers the outward K+ convective flow and also lowers the inward K+ diffusion. However, since the decrease of the outward K+ convective flow is greater than the decrease of the inward K+ diffusion, and the net flux is the algebraic sum of these two terms, the result is greater net inward K+ flux through the dentin. Therefore, the Colgate-brushed specimens, which exhibited the lowest Lp, provided the greatest inward K+ flux at simulated dentin fluid pressure.

Impact of an anticaries mouthrinse on in vitro remineralization and microbial control.

Objective. The objective of this research was to evaluate the caries control potential of a new fluoride mouthrinse that also contained antimicrobial agents and a biofilm disrupting agent using different in vitro models. Methods. Four in vitro studies were conducted to assess the performance of this three pronged approach to caries control: (1) traditional enamel fluoride uptake, (2) surface microhardness study using pH cycling model and subsequent fluoride uptake, (3) a salivary biofilm flow-through study to determine the anti-microbial activity, and (4) a single species biofilm model measuring effect on biofilm matrix disruption. Results. The data showed that a LISTERINE rinse with fluoride, essential oils and xylitol was superior in promoting enamel fluoride uptake and in enhancing antimicrobial activity over traditional commercially available fluoridated products. An increase of the surface microhardness was observed when the LISTERINE rinse was used in combination with fluoridated toothpaste versus the fluoridated toothpaste alone. Finally, it was demonstrated that xylitol solutions disrupted and reduced the biovolume of biofilm matrix of mature Streptococcus mutans. Conclusion. These in vitro studies demonstrated that a fluoride mouthrinse with antimicrobial agent and biofilm matrix disrupting agent provided multifaceted and enhanced anti-caries efficacy by promoting remineralization, reducing acidogenic bacteria and disrupting biofilm matrix.

Biomimetic ultrathin whitening by capillary-force-induced random clustering of hydrogel micropillar arrays.

Capillary-force-induced collapse of high-aspect-ratio microstructures has often been considered a failure mechanism in device fabrication. Here, we study capillary-force-induced clustering behavior of highly ordered hydrogel micropillar arrays from 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) and explore their utility as ultrathin whitening layers (less than 9 mum thick). When exposed to water, followed by drying in an air stream, the micropillars were softened, bent, and randomly clustered together because of competition between the capillary force and elastic restoring force of the pillars. By varying the relative composition of the water-swellable PHEMA and glassy PMMA, we modulated the elastic modulus of the pillars in the wet state spanning over 3 orders of magnitude. By minimizing the sum of the capillary meniscus interaction energy and the elastic bending energy of the pillars for a cluster, we estimated the average cluster size as a function of the elastic modulus of the pillars, which agreed well with the experimental observation. The randomly clustered micropillar arrays appeared white in color because of random light scattering from the clusters, similar to the observation in the white beetles, whose scales consist of a few micrometer-thick random networks of microfilaments.