High-Resolution Characterization of Enamel Remineralization using Time-of-Flight Secondary Ion Mass Spectrometry and Electron Microscopy.

The aim of this in vitro study was to assess the suitability of high-resolution time-of-flight secondary-ion mass spectrometry (ToF-SIMS) for visualizing cross-sectional changes in human enamel microstructure and chemical composition during treatment and remineralization cycling of artificially generated caries lesions underneath an artificial plaque. Treatments consisted of exposure to twice daily toothpaste/water slurries prepared from 0, 1100, and 5000 µg/g fluoride (F) NaF/Silica toothpastes. In addition, treatments with slurries prepared from 1100 µg/g F SnF2/Silica toothpastes were done using 44Ca in the remineralization solution to allow for differentiation of newly formed mineral and exploration of incorporated metal dopants using ToF-SIMS. Complementary microhardness, scanning electron microscopy, and high-resolution transmission electron microscopy (HR-TEM) investigations were performed on enamel cross-sections. HR-TEM was used for the first time to determine the change in crystallinity during remineralization revealing distinct microstructural zones within one lesion. Chemical mapping using ToF-SIMS demonstrated that the distribution of F, while observed primarily in the new mineral phase, was widespread throughout the lesion with 44Ca substantially limited to the remineralizing mineral. Both penetrated the inter-rod spaces of the sound enamel illustrating how acid damage propagates into the native mineral as the caries lesion deepens. HR-TEM examination revealed different regions within the lesion characterized by distinct micro- and ultra-structures. Importantly, HR-TEM revealed a return of crystallinity following remineralization. Fluoride dose response observations verified the ability of these high-resolution techniques to differentiate remineralization efficacy. The collective results provided new insights such as the visualization of fluoride or calcium penetration pathways, as well as new tools to study the caries process.

In vitro safety evaluation of a hydrogen peroxide whitening emulsion technology on human enamel and dentin.

PURPOSE: To assess effects of a novel hydrogen peroxide leave-on whitening emulsion on surface hardness, fracture susceptibility, surface erosion, and surface morphology of enamel and dentin. METHODS: Human enamel and root dentin sections embedded in resin were leveled and polished for uniformity. A cycling treatment simulating overuse conditions (60 hours over 10 days), coupled with incubation in pooled human saliva at 37°C and two daily toothpaste treatments were used to evaluate the safety of a 3% hydrogen peroxide whitening emulsion treatment (Crest Whitening Emulsions). Controls included a no treatment group, three erosion controls (water, 0.25% citric acid pH 3.6, 1% citric acid pH 3.6), and a bleaching control (8.25% sodium hypochlorite). Color measurements (b*) were taken on select post-treated specimens to confirm bleaching activity. Effects on enamel and dentin physical properties were determined by surface microhardness, fracture toughness, erosion depth, and surface morphology by light and scanning electron microscopy. RESULTS: The hydrogen peroxide emulsion b* value was significantly different versus water control (P< 0.05), confirming bleaching activity. Microhardness and fracture toughness results for hydrogen peroxide emulsions were not significantly different versus baseline (P> 0.2) and no treatment (P= 1.0), respectively. Erosion loss for the hydrogen peroxide emulsion was not observed on enamel (comparable to water) and significantly less than 0.25% citric acid (P< 0.05) on dentin which was verified by microscopic visualization. CLINICAL SIGNIFICANCE: The hydrogen peroxide emulsion had no significant negative effects on enamel and dentin properties after 60 hours of bleaching over 10 days, confirming safety under simulated overuse conditions.

Application of non-inferiority testing in microhardness and its relationship to erosion: Relevance for hard tissue safety evaluations.

PURPOSE: To use non-inferiority statistical testing with simple microhardness measurements (SMH) as a prediction of potential erosive hard tissue damage of topical treatments on enamel. METHODS: Three independent experiments of a simple acid cycling demineralization (ACD) model were used to screen softening effects of various commercial beverages on dental enamel. The cycling model consists of six repeated exposures of enamel slabs with alternating treatments of artificial saliva over the course of 6 hours. After six repeated cycles, effects on surface microhardness were measured. Softening effects of beverages were evaluated using a statistical non-inferiority test of the positive control (water) and negative control (1% citric acid). To confirm whether softening effects as evaluated by a non-inferiority test translated to like differences in enamel erosion susceptibility, selected beverages then underwent more complex erosion cycling model (ECM) evaluation where enamel blocks were cycled with beverages (vs. historically established citric acid) and pooled saliva over a period of 5 days. The ECM also incorporated dentifrice treatments, sodium fluoride (NaF, Crest Cavity Protection, negative control) and a positive control stannous fluoride dentifrice (SnF2, Crest Pro-Health Advanced), to confirm model performance against historically published results of in situ erosion protection benefits of SnF2. RESULTS: There was a spectrum of softening properties of 16 commercial beverages in the ACD test, ranging from a ΔSMH of -22.6 to -316 vs. baseline. Four beverages were evaluated further in ECM testing. Despite a measurable change in SMH, Sprite and beer treatments in the ACD passed the statistical non-inferiority test and both were evaluated in erosion cycling, showing no enamel surface loss. Vinegar (~5% acetic acid) and Gatorade also showed measurable changes in SMH in the ACD, but they failed statistical non-inferiority testing. Both beverages subsequently showed significant enamel tissue loss (erosion) in further erosion cycling testing. This combined set of data suggests that simple surface microhardness evaluation may be used as a proxy for potential erosion surface loss if properly quantified. SnF2 dentifrice significantly reduced erosion from all erosive beverages with greater efficacy than NaF control dentifrice, consistent with prior clinical and in vitro evidence. CLINICAL SIGNIFICANCE: The ACD model with application of non-inferiority statistical testing is proposed as a simple model of hard tissue safety assessment of treatments, including oral hygiene products. Products that pass the non-inferiority test in ACD (surface softening) are proposed as safe for enamel as there is no suggestion from this data that teeth are at risk of tissue loss due to these products. On the other hand, products failing the non-inferiority test require confirmatory safety qualification in erosion cycling. Products equal or worse than citric acid with ACD or with significant erosion in ECM are suggested to warrant reformulation unless favorable safety data for enamel (lack of erosion) or the appropriate justification are provided.

Investigation of oral malodor prevention by dentifrices as measured by VSC reduction.

The oral malodor reduction efficacy of dentifrices containing stannous fluoride (SnF2) or zinc plus arginine (Zn/Arg) was evaluated using a halimeter to measure volatile sulfur compounds (VSCs) in two randomized, controlled, single-blind, cross-over pilot clinical trials. Study 1 (N= 16) had five 1100 ppm F dentifrices and five treatment periods: negative control (NC): 0.243% sodium fluoride (NaF); SnF2A: 0.454% SnF2+ pyrophosphate; SnF2B: 0.454% SnF2+ sodium hexametaphosphate; SnF2C: 0.454% SnF2+ citrate; and SnF2D: experimental 0.454% SnF2with increased bioavailable Sn. Study 2 (N= 16) had four 1450 ppm F dentifrices and four treatment periods: NC: 0.1% NaF + 0.76% sodium monofluorophosphate; Zn/Arg: NaF + zinc + arginine; SnF2E: 0.454% SnF2+ 0.078% NaF + sodium hexametaphosphate; SnF2F: 0.454% SnF2+ 0.078% NaF + citrate. Each period took 96 h; baseline (day 0) morning to day 4 morning. Subjects brushed 2x/day with the treatment dentifrice. VSC levels were measured in the morning, prior to morning brushing, on days 0 and 4. Changes in VSC scores from day 0 to day 4 were assessed using a mixed-model analysis of covariance for cross-over studies. In study 1, SnF2dentifrices B, C and D showed statistically significant reductions (31.3%, 24.8%, 34%, respectively) in VSC scores versus baseline (p< 0.001); the benefits of these treatments versus NC and SnF2dentifrice A were statistically significant (p⩽ 0.001). In study 2, SnF2dentifrices E and F showed statistically significant reductions (31.2%, 22.1%, respectively) in VSC scores versus baseline (p⩽ 0.010); the benefits of these treatments versus NC and the Zn/Arg dentifrice were statistically significant (p⩽ 0.035). The Zn/Arg dentifrice showed a significant increase of 35.2% (p< 0.001) in VSC scores versus baseline. These studies demonstrated that SnF2dentifrices can provide significant oral malodor reductions, but the benefit is formulation dependent. The presence of SnF2is not sufficient to ensure efficacy. The Zn/Arg dentifrice increased VSC levels, indicating greater malodor.Clinical Trial Numbers: 20190429 and 20191028.

Laboratory screening evaluation of the safety of low pH oral care rinse products to dental enamel.

PURPOSE: To assess the hard tissue safety of a variety of low pH oral care rinses to dental enamel in a newly developed screening method. METHODS: Bovine enamel specimens were subjected to a cycling model that consisted of commercial mouthrinse product exposures and artificial saliva soaks based on a previously published screening method. The effect of test products on the surface of treated specimens was measured using surface microhardness (SMH). Results are presented as the change in SMH (between sound enamel baseline and cycling final). An assortment of rinse products were assessed relative to distilled water (positive control) and 1% citric acid (negative control). A priori, a product was considered safe if the change in measured SMH values over the course of six treatment cycles was both significantly greater than the negative control and was not significantly different from the positive control. A non-inferiority statistical test was applied to create a quantitative rule defining product safety. RESULTS: Products tested included two rinses with a pH in excess of 5.5, and eight with a pH less than 5.5. Four of the rinses included fluoride, while six did not. Analyses showed that all of the rinse products tested passed the non-inferiority acceptance criteria. One of the 10 marketed oral care rinses failed to meet the a priori criteria needed to be considered safe as the product was significantly better than the negative control but also significantly lower than the positive control treatment. This product had the lowest pH of all products tested and did not contain fluoride. Application of the non-inferiority statistical test showed the questionable product passing safety criteria. As a proposed method for a screening tool, further testing would be recommended based on these results. CLINICAL SIGNIFICANCE: An in vitro enamel safety screening method was applied as an assessment of the enamel demineralization safety to a number of oral care rinse products. Surface microhardness, coupled with a non-inferiority statistical evaluation, provided a reasonable approach for detecting potential product issues. Products failing this screening laboratory method may require additional testing to verify their safety on hard tissues.

Development of a Screening Method to Establish if pH of Oral Care Products Affects Hard Tissues.

OBJECTIVES: To develop a transferable, simple screening method to evaluate the effect of pH of oral care products on oral hard tissues. METHODS: The method reported here is based on the assessment of oral hard surface changes produced by oral care products measured via Vickers Surface Microhardness (SMH). Two variations of this screening test method were developed, one including the use of salivary pellicle and human teeth and a second using bovine substrates with artificial saliva. The test method using bovine substrates and artificial saliva was replicated in a second laboratory in Beijing, China to verify reproducibility and transferability of the technique. RESULTS: Both approaches confirmed changes on surface hardness with 1% citric acid. All tested marketed products, including those formulated at pH < 5.5, showed no significant %SMH difference from the positive control (water), and demonstrated a significant difference from the negative control (1% citric acid). The two laboratories produced similar results (pH effects, standard deviation, and statistical rank-ordering of treatments). CONCLUSIONS: This simple screening method accurately assesses the influence of positive and negative controls, regardless of the source of hard tissue (human vs. bovine) and saliva (human vs. artificial). It correctly shows that marketed products with pH below 5.5 that demonstrate favorable in vivo safety profiles do not contribute to detrimental hard tissue changes. The method is easily transferable and shows potential as a tool for the safety profile assessment of oral care products.

Fluoride Uptake Profiles of Selected European Toothpastes into Hard Tissues and Plaque.

OBJECTIVES: To compare the fluoridating potential of selected European toothpastes using a combination of enamel, dentin, and plaque in vitromodels. METHODS: Four in vitromodels were included: 1) Enamel Fluoride (F) Uptake (EFU); 2) Dentin F Uptake (DFU); 3) Enamel Solubility Reduction (ESR); and 4) Plaque F Uptake (PFU). A core set of marketed products was included in all studies, plus a standard toothpaste (1100 ppm F as NaF/silica) and placebo control (the PFU study did not include a placebo control). Test dentifrices: [A] Fluocaril® Bi-Fluoré 250 (1500 ppm F as NaF+1000 ppm F as SMFP); [B] LacerAnticaries (2500 ppm F as SMFP); [C] Elmex® Caries Professional™ (1450 ppm F as SMFP+1.5% arginine); [D] Colgate® Triple Action (1450 ppm F as SMFP); [E] Placebo (0 ppm F); and [F] standard toothpaste (1100 ppm as NaF/silica). In all studies (EFU, DFU, ESR, and PFU), assessments were compared for each pair using the Tukey-Kramer HSD test (p < 0.05). RESULTS: In all studies of fluoride uptake, the Fluocaril dentifrice [A] provided the greatest numerical benefit, regardless of the substrate. Statistical groupings were EFU: A > F = B = C = D > E; DFU: A > F = B = C = D = E; PFU: A = B > F = C = D). In demineralization prevention, the Fluocaril dentifrice [A] also provided the greatest benefit (ESR: A = F = C = B = D > E). In all studies that included a placebo control, all of the F-containing dentifrices performed better than the placebo control. CONCLUSIONS: While these results demonstrate that all of the marketed products tested provide effective anticaries benefits, the Fluocaril Bi-Fluoré 250 dentifrice consistently delivered unsurpassed performance. It delivered the highest level of F to plaque, provided greater measures of efficacy in both remineralization and inhibition of demineralization, and delivered substantial improvement in fluoridation of dentin, suggesting the potential for delivering both coronal and root caries benefits.

A Profilometry-Based Dentifrice Abrasion Method for V8 Brushing Machines Part III: Multi-Laboratory Validation Testing of RDA-PE.

OBJECTIVES: We have previously reported on progress toward the refinement of profilometry-based abrasivity testing of dentifrices using a V8 brushing machine and tactile or optical measurement of dentin wear. The general application of this technique may be advanced by demonstration of successful inter-laboratory confirmation of the method. The objective of this study was to explore the capability of different laboratories in the assessment of dentifrice abrasivity using a profilometry-based evaluation technique developed in our Mason laboratories. In addition, we wanted to assess the interchangeability of human and bovine specimens. METHODS: Participating laboratories were instructed in methods associated with Radioactive Dentin Abrasivity-Profilometry Equivalent (RDA-PE) evaluation, including site visits to discuss critical elements of specimen preparation, masking, profilometry scanning, and procedures. Laboratories were likewise instructed on the requirement for demonstration of proportional linearity as a key condition for validation of the technique. Laboratories were provided with four test dentifrices, blinded for testing, with a broad range of abrasivity. In each laboratory, a calibration curve was developed for varying V8 brushing strokes (0, 4,000, and 10,000 strokes) with the ISO abrasive standard. Proportional linearity was determined as the ratio of standard abrasion mean depths created with 4,000 and 10,000 strokes (2.5 fold differences). Criteria for successful calibration within the method (established in our Mason laboratory) was set at proportional linearity = 2.5 ± 0.3. RDA-PE was compared to Radiotracer RDA for the four test dentifrices, with the latter obtained by averages from three independent Radiotracer RDA sites. Individual laboratories and their results were compared by 1) proportional linearity and 2) acquired RDA-PE values for test pastes. RESULTS: Five sites participated in the study. One site did not pass proportional linearity objectives. Data for this site are not reported at the request of the researchers. Three of the remaining four sites reported herein tested human dentin and all three met proportional linearity objectives for human dentin. Three of four sites participated in testing bovine dentin and all three met the proportional linearity objectives for bovine dentin. RDA-PE values for test dentifrices were similar between sites. All four sites that met proportional linearity requirement successfully identified the dentifrice formulated above the industry standard 250 RDA (as RDA-PE). The profilometry method showed at least as good reproducibility and differentiation as Radiotracer assessments. It was demonstrated that human and bovine specimens could be used interchangeably. CONCLUSIONS: The standardized RDA-PE method was reproduced in multiple laboratories in this inter-laboratory study. Evidence supports that this method is a suitable technique for ISO method 11609 Annex B.

Comparative Antiplaque Effect of Two Antimicrobial Dentifrices: Laboratory and Clinical Evaluations.

OBJECTIVES: To compare the effect of a stannous fluoride dentifrice versus a triclosan-containing dentifrice on the reduction of plaque using in vitro and clinical models. METHODS: Both investigations evaluated a novel 0.454% stabilized stannous fluoride dentifrice (Crest® Pro-Health™ smooth formula) versus a sodium fluoride/triclosan positive control dentifrice (Colgate® Total®). The in vitro evaluation utilized the Plaque Glycolysis and Regrowth Model (PGRM), wherein the metabolic effects (acid production/glycolysis inhibition) of the dentifrices were assessed on plaque biofilms grown on glass rods after three days growth and a single dentifrice treatment. Treatments were evaluated via analysis of variance, Student's t-test. The clinical trial was a four-week, single-center, randomized and controlled, double-blind, parallel group study, where 120 adults were randomized to one of the two dentifrices for use at home according to manufacturer's instructions. Plaque was evaluated at baseline and Week 4 with the Rustogi Modified Navy Plaque Index (RMNPI). Statistical analyses were via analysis of covariance. RESULTS: In vitro PGRM: The stannous fluoride dentifrice provided 43.3% glycolysis inhibition compared to 27.5% for the triclosan control, and the pH decrease associated with acid production was significantly less for stannous fluoride (0.87) versus triclosan (1.11); p < 0.05. Clinical trial: One hundred eighteen (118) subjects completed the study with fully evaluable data. Both dentifrice groups demonstrated statistically significant (p < 0.0001) reductions in plaque at Week 4 compared with baseline, with the stannous fluoride dentifrice producing a significantly lower adjusted mean Week 4 plaque score (p < 0.0001) versus the triclosan positive control for whole mouth plaque (23.1% lower) and interproximal plaque (43.5% lower). Both dentifrices were well-tolerated. CONCLUSIONS: The stabilized stannous fluoride dentifrice provided statistically significant reductions in plaque glycolysis in vitro and plaque growth in vivo compared to the triclosan dentifrice. Results for both studies were consistent.

In Vitro and In Vivo Evaluations of the Anticalculus Effect of a Novel Stabilized Stannous Fluoride Dentifrice.

OBJECTIVES: To evaluate the effect of a novel stannous fluoride dentifrice with zinc citrate on calculus inhibition using both in vitro and clinical models. METHODS: Each investigation tested a novel stabilized 0.454% stannous fluoride dentifrice with zinc citrate as an anticalculus agent (Crest® Pro-Health™ smooth formula) compared to a negative control fluoride dentifrice. The in vitro study used the modified Plaque Growth and Mineralization Model (mPGM). Plaque biofilms were prepared and mineralized by alternate immersion of glass rods in human saliva and artificial mineralization solution. Treatments of 25% w/w dentifrice/water slurries were carried out for 60 seconds daily for 6 days, between saliva and mineralization solution immersions. Plaque calcium levels were determined by digestion and inductively coupled plasma optical emission spectroscopy. Student's t-test (p < 0.05) was used for statistical analysis. The clinical study was a parallel group, double-blind, randomized, and controlled trial. Following a dental prophylaxis, subjects entered a two-month run-in phase. At the end, they received a Volpe-Manhold Index (V-MI) calculus examination. Eighty (80) qualified subjects who had formed at least 9 mm of calculus on the linguals of the mandibular anterior teeth were re-prophied and randomly assigned to either the stannous fluoride dentifrice or the negative control. Subjects brushed twice daily, unsupervised, during the three-month test period, returning at Weeks 6 and 12 for safety and V-MI examinations. Statistical analyses were via ANCOVA. RESULTS: In vitro mPGM: The stabilized stannous fluoride dentifrice showed 20% less in vitro tartar formation, measured as calcium accumulation normalized by biofilm mass, versus the negative control (106.95 versus 133.04 µg Ca/mg biofilm, respectively, p < 0.05). Clinical Trial: Seventy-eight (78) subjects completed with fully evaluable data. The stannous fluoride dentifrice group had 15.1% less adjusted mean calculus at Week 6 compared to the negative control group (p = 0.05) and 21.7% less calculus at Week 12 (p < 0.01). Both dentifrices were well-tolerated. CONCLUSIONS: The stannous fluoride dentifrice produced significant anticalculus benefits in vitro and in a clinical trial compared to a negative control.

A profilometry-based dentifrice abrasion Method for V8 brushing machines. Part I: Introduction to RDA-PE.

OBJECTIVE: This paper describes the development and standardization of a profilometry-based method for assessment of dentifrice abrasivity called Radioactive Dentin Abrasivity - Profilometry Equivalent (RDA-PE). METHODS: Human dentine substrates are mounted in acrylic blocks of precise standardized dimensions, permitting mounting and brushing in V8 brushing machines. Dentin blocks are masked to create an area of "contact brushing." Brushing is carried out in V8 brushing machines and dentifrices are tested as slurries. An abrasive standard is prepared by diluting the ISO 11609 abrasivity reference calcium pyrophosphate abrasive into carboxymethyl cellulose/glycerin, just as in the RDA method. Following brushing, masked areas are removed and profilometric analysis is carried out on treated specimens. Assessments of average abrasion depth (contact or optical profilometry) are made. RESULTS: Inclusion of standard calcium pyrophosphate abrasive permits a direct RDA equivalent assessment of abrasion, which is characterized with profilometry as Depth test/Depth control x 100. Within the test, the maximum abrasivity standard of 250 can be created in situ simply by including a treatment group of standard abrasive with 2.5x number of brushing strokes. RDA-PE is enabled in large part by the availability of easy-to-use and well-standardized modern profilometers, but its use in V8 brushing machines is enabled by the unique specific conditions described herein. CONCLUSION: RDA-PE permits the evaluation of dentifrice abrasivity to dentin without the requirement of irradiated teeth and infrastructure for handling them. In direct comparisons, the RDA-PE method provides dentifrice abrasivity assessments comparable to the gold industry standard RDA technique.

A Profilometry-Based Dentifrice Abrasion Method for V8 Brushing Machines Part II: Comparison of RDA-PE and Radiotracer RDA Measures.

OBJECTIVE: The purpose of this study was to compare the abrasivity of commercial dentifrices by two techniques: the conventional gold standard radiotracer-based Radioactive Dentin Abrasivity (RDA) method; and a newly validated technique based on V8 brushing that included a profilometry-based evaluation of dentin wear. This profilometry-based method is referred to as RDA-Profilometry Equivalent, or RDA-PE. METHODS: A total of 36 dentifrices were sourced from four global dentifrice markets (Asia Pacific [including China], Europe, Latin America, and North America) and tested blindly using both the standard radiotracer (RDA) method and the new profilometry method (RDA-PE), taking care to follow specific details related to specimen preparation and treatment. RESULTS: Commercial dentifrices tested exhibited a wide range of abrasivity, with virtually all falling well under the industry accepted upper limit of 250; that is, 2.5 times the level of abrasion measured using an ISO 11609 abrasivity reference calcium pyrophosphate as the reference control. RDA and RDA-PE comparisons were linear across the entire range of abrasivity (r2 = 0.7102) and both measures exhibited similar reproducibility with replicate assessments. RDA-PE assessments were not just linearly correlated, but were also proportional to conventional RDA measures. CONCLUSION: The linearity and proportionality of the results of the current study support that both methods (RDA or RDA-PE) provide similar results and justify a rationale for making the upper abrasivity limit of 250 apply to both RDA and RDA-PE.

Optimization of preparative electrophoretic chiral separation of ritalin enantiomers.

Continuous free flow electrophoresis (CFFE) was applied to the preparative chiral separation of ritalin enantiomers. Sulfated beta-cyclodextrin (sbeta-CD) was used as the chiral additive. Liquid chromatography-mass spectrometry (LC-MS) experiments were applied to study the time averaged concentration of sbeta-CD in the separation chamber. The distribution of sbeta-CD in the separation chamber greatly influenced resolution and the angle of deflection. To optimize the separation, several parameters (methanol, concentration of sbeta-CD in the cathodic wash and in the separation buffer, and the introduction of a low conductivity zone) were investigated. The dependence of the resolution and deflection angles of ritalin enantiomers on the concentration of sbeta-CD in both the separation buffer and in the cathode wash solution appeared to be non-linear. Under close to optimal conditions, resolution of ritalin enantiomers was about 0.8 with an average processing rate of 0.5 mg/h. Overall, the enantiomeric purity of the individual isomers was approximately 83%; however, of the 20 vials containing ritalin, the presence of both enantiomers was only detected in three vials.