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.

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.

Subgingival uptake and retention of stannous fluoride from dentifrice: Gingival crevicular fluid concentrations in sulci post-brushing.

PURPOSE: To examine the delivery of stannous fluoride to subgingival sulci following toothpaste use in a clinical population. METHODS: This was a controlled, single-site study. 23 subjects with at least 20 dental pockets, 2-4 mm with bleeding, who had not used a stannous fluoride dentifrice in the last 3 months were enrolled. After a 2-week washout period, 20 subjects returned for a baseline visit. They were instructed to refrain from brushing the night before the baseline visit. GCF samples were taken from up to 10 sites identified as sampling sites. Subjects were then given a 0.454% stannous fluoride dentifrice and soft manual toothbrush and asked to brush for 1 minute. 30 minutes after brushing, GCF was re-sampled. Subjects continued using the stannous fluoride dentifrice and soft manual toothbrush at home, twice daily for 2 weeks, in place of their usual hygiene products. At Days 1 and 14, subjects returned to the site, and 12 hours post-brushing GCF samples were taken. The samples were analyzed by ICP-MS (inductively coupled plasma mass spectrometry). A Wilcoxon signed-rank test was performed to determine the difference between post-baseline visits and baseline. Statistical tests were 2-sided using a 5% significance level. RESULTS: 20 subjects completed the trial. Significant levels of tin, a marker for stannous fluoride, were detected 30 minutes after brushing at sampling sites of 2-4 mm. The median tin level in gingival crevicular fluid (GCF) was 24.59 ng/µl, which was highly significant versus baseline (P< 0.0001). Tin levels sampled in GCF 12 hours after brushing on Days 1 and 14 were highly significant versus Baseline (P< 0.0001), showing an increasing trend with continued use. CLINICAL SIGNIFICANCE: Stannous fluoride was found to penetrate sampling sites from 2-4 mm and was retained for 12 hours. Subgingival uptake and retention of stannous fluoride following toothbrushing may play a role in detoxification effects on microbial biofilms and may contribute to the therapeutic efficacy of stannous fluoride dentifrices in promoting gingival health.

Clinical effects of stannous fluoride dentifrice in reducing plaque microbial virulence III: Lipopolysaccharide and TLR2 reporter cell gene activation.

PURPOSE: This study expanded the analysis of subgingival dental plaques from previous research to include the evaluation of cohort, site and treatment effects on chemically measured endotoxin and activation of Toll-like receptor (TLR) based gene expression in two additional reporter cell lines: a TLR2 specific cell line and a THP-1 (multi TLR reporter) cell line. METHODS: Participants from high and low bleeding cohorts were sampled at baseline for both supra and subgingival dental plaque at both healthy as well as clinically diseased sites and then provided with intervention hygiene products including a stabilized SnF2 dentifrice and a new soft bristle manual toothbrush. Following 2 and 4 weeks of assigned dentifrice use, participants returned for a re-evaluation of gingival inflammation and bleeding and repeat samplings of dental plaque. Subgingival sampled plaques were chemically analyzed for endotoxin concentration using a Thermo Scientific Pierce LAL chromogenic endotoxin quantitation kit. Samples were also used for inoculation of two reporter cell assays (an HEK293 TLR2 reporter cell line and a THP-1 monocyte cell line). Reporter cell activation was analyzed via luminescence changes of secreted embryonic alkaline phosphatase. RESULTS: The endotoxin content of subgingival plaque could be measured directly with dye assays and plaque isolates activated gene expression in both TLR reporter cell lines. Higher disease cohorts and sites with gingival inflammation generally showed more endotoxins and higher levels of plaque virulence as compared to low disease cohorts or plaque sampled from clinically healthy sites. SnF2 dentifrice treatment was associated with broad scale reductions in endotoxin content and virulence potentiation properties of dental plaque samples collected subgingivally from patients. CLINICAL SIGNIFICANCE: These results collectively support the use of dye or various reporter cell lines in the characterization of plaque virulence in diseased populations and as a potential route for analysis in clinical evaluations of treatment interventions. Subgingival plaque 'detoxification' including effects on microbial pathogenicity as well as metabolic activity may be considered important mechanisms contributing to clinical benefits of SnF2 dentifrice.

Clinical Effects of Stabilized Stannous Fluoride Dentifrice in Reducing Plaque Microbial Virulence II: Metabonomic Changes.

OBJECTIVES: The clinical effects of stannous fluoride (SnF2) dentifrice in reducing symptoms of gingivitis and reducing the virulence of subgingival plaque through suppression of activation of gene expression in toll receptor based reporter cells were previously reported. This study expanded analysis of the clinical study to include evaluation of dentifrice effects on salivary metabolites using 1H Nuclear Magnetic Resonance (1H NMR) systems biology-based metabonomics. METHODS: The clinical design was reported previously (J Clin Dent2017;28:16-26). Participants included a cohort exhibiting high and low levels of gingival disease as presented at initiation of the study. Participants provided morning lavage saliva samples at baseline. Following this, participants were provided with a hygiene intervention, including a stabilized SnF2 dentifrice and a new soft bristle manual toothbrush. Following two and four weeks of assigned dentifrice use, participants again collected morning lavage saliva samples. Samples were analyzed by 1HNMR spectroscopy on a Bruker 600MHz NMR spectrometer. System-wide analyses were carried out by partial least squared (PLS) comparisons of aggregate spectra, and discrete metabolites with established spectral signatures were likewise directly compared. RESULTS: PLS analysis showed significant differences in saliva composition for saliva collected from high bleeding and low bleeding cohorts. Clear shifts in saliva composition were observed in system-wide PLS analysis following intervention of SnF2 dentifrice for both cohorts. A number of discrete spectral changes were consistently observed with SnF2 dentifrice intervention, most notably including reductions in propionic acid and butyric acid, key short chain fatty acids associated with anaerobic metabolism in dental plaques. CONCLUSIONS: These results collectively demonstrate that SnF2 dentifrice treatment was associated with broad scale modifications in saliva composition following intervention in both high and low diseased cohorts. Changes in overall salivary composition and specific reductions in saliva concentrations of propionic and butyric acid reductions occurred coincident with clinical improvements in gingivitis and gingival bleeding. These results provide support for the hypothesis that the effectiveness of SnF2 dentifrice in improving gingival health is associated with a modification of microbiome metabolism, including suppression of short chain fatty acid metabolites.

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.

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.

Clinical Effects of Stabilized Stannous Fluoride Dentifrice in Reducing Plaque Microbial Virulence I: Microbiological and Receptor Cell Findings.

OBJECTIVES: Lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs), or bacterial endotoxins, bind with Toll-like receptors (TLRs) that are expressed on host cells of the periodontium, thereby contributing to the periodontal pathogenicity of oral bacteria. Stannous fluoride (SnF2), an antibacterial fluoride that treats and controls gingivitis, has been shown to react with lipophilic domains/anionic charges in LPS and LTA. The effects of bacterial species and dental plaque on toll receptors can be studied using genetically engineered cell lines containing linked toll receptors on their surfaces. This randomized, examiner-blinded study examined the clinical effects of stabilized SnF2 dentifrice intervention on gingivitis and dental plaque virulence in populations exhibiting high and low levels of clinical gingivitis. METHODS: Recruited populations were evaluated for gingival inflammation (MGI) and gingival bleeding (GBI) at baseline and assigned into two cohorts of 20 each, those with high (GBI > 20 sites) and low (GBI < 3 sites) levels of observed bleeding/gingivitis. Participants were sampled at baseline for both supra- and subgingival dental plaque at both healthy (no bleeding, PD = 2 mm), as well as clinically diseased sites (bleeding, PD = 3-4 mm), and then provided with an intervention hygiene product including a stabilized SnF2 dentifrice and a new soft bristle manual toothbrush. Following two and four weeks of assigned dentifrice use, participants returned for a re-evaluation of gingival inflammation and bleeding and repeat samplings of dental plaque. Plaque samples were analyzed by anaerobic culturing of gram negative anaerobes (GNA), as well as by incubation of subgingival sampled plaques with TLR4 transfected HEK293 cells, where gene expression was assessed by measurement of a SEAP alkaline phosphatase reporter as a marker of toll receptor activation. RESULTS: Clinical assessments showed statistically significant reductions in MGI (24-26%) and GBI (42-53%) gingivitis in both diseased and healthy cohorts following four weeks of dentifrice intervention. For all clinical examinations, MGI and bleeding sites were statistically significantly different (lower) in the low bleeding versus the higher bleeding cohort. Supragingival and subgingival GNAs were significantly reduced (p < 0.05) in both high and low disease cohorts at bleeding and healthy sites following four weeks of stabilized SnF2 dentifrice use. TLR activation from subgingival sampled plaque was reduced following four weeks of stabilized SnF2 dentifrice use in both high and low disease cohorts and in both healthy, as well as diseased sites. CONCLUSIONS: Collectively, these results support the potential for stabilized SnF2 dentifrice to provide clinical gingivitis benefits via mechanisms beyond control of plaque mass, potentially directly decreasing the pathogenicity of plaque biofilms by blocking reactivity of LPS and LTA ligands with tissue receptors associated with inflammation. Importantly, benefits could be seen in both diseased sites, as well as sites not yet exhibiting symptoms of inflammation, supporting the activity of SnF2 not just in treating diseased sites, but in preventing disease development. These learnings may influence treatment planning for patients susceptible to gingivitis.

Characterization of cornified oral mucosa for iontophoretically enhanced delivery of chlorhexidine.

Topical administration of chlorhexidine for periodontal disease can provide advantages over systemic delivery, but is limited by the permeability of the cornified oral mucosal tissue. In the present study, passive and iontophoretic transport of tetraethylammonium, salicylate, mannitol, dexamethasone, fluoride, and chlorhexidine across bovine palate was investigated to (a) determine the intrinsic barrier properties of bovine palate for its eventual use as a model of human cornified oral mucosa, (b) examine the feasibility of iontophoretically enhanced transport of chlorhexidine into and across bovine palate, and (c) identify the transport mechanisms involved in iontophoretic transport across the palate. The histology study suggests that bovine and human palates have similar cornified epithelium structures; bovine palate could be a model tissue of human hard palate for drug delivery studies. Transport study of tetraethylammonium, salicylate, and mannitol suggests that bovine palate was net negatively charged and the cornified epithelial layer was the rate-determining barrier. The direct-field effect (electrophoresis) was shown to be the dominant flux-enhancing mechanism in iontophoretic transport of ionic compounds. Electroosmosis also contributed to the iontophoretic transport of both neutral and ionic permeants. Anodal iontophoresis enhanced the delivery of chlorhexidine into and across the palate, reduced the transport lag time, and provided tissue concentration above the drug minimum inhibitory concentration, and therefore could be a promising method to assist in the treatment of periodontal disease.

Lipopolysaccharide and Lipoteichoic Acid Virulence Deactivation by Stannous Fluoride.

OBJECTIVES: Oral bacterial pathogens promote gingivitis and periodontal disease. Bacterial endotoxins, also known as lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs), are known to enhance bacterial pathogenicity through binding with Toll-like receptors (TLRs), a group of pattern recognition receptors critical to the activation of innate immunity, that are expressed on host cells. Both LPS and LTA contain lipophilic domains and anionic charges that may be susceptible to reactivity with stannous fluoride, a commonly used ingredient clinically proven for the treatment and prevention of gingivitis. This study examined the effects of stannous fluoride on Toll-like receptor activation in response to bacterially derived LPS and LTA in select cell lines and secretion of inflammatory cytokines from human primary peripheral monocytes likewise exposed to LPS. METHODS: TLR4 and TLR2 transfected HEK293 cells and THP1-Dual™ cells were exposed to bacterial LPS and LTA in the presence of increasing concentrations of stannous fluoride. Gene expression was assessed by measurement of secreted embryonic alkaline phosphatase (SEAP) reporter gene for HEK293 cells and SEAP and luciferase for THP-1 cells. Cell viability was confirmed using PrestoBlue. Human primary monocytes were treated with LPS with various concentrations of supplemented stannous fluoride, and cytokine expression was directly measured. RESULTS: Stannous fluoride inhibited gene expression response of TLR4 and TLR2 in HEK293 cells and THP1-Dual™ cells in a dose response fashion, producing complete inhibition at micromolar concentrations. The addition of stannous fluoride suppressed production of TNF-a, IFN-g, IL12p70, IL10, IL-1b, IL2, and IL-6, and also increased secretion of Il-8 in dose response fashion. Viability assays confirmed no effects of LPS or stannous fluoride on viability of HEK293, THP-1, and primary human monocytes. CONCLUSIONS: These results support the potential for stannous fluoride to provide clinical gingivitis benefits by directly decreasing the pathogenicity of plaque biofilms by blocking reactivity of LPS and LTA ligands with tissue receptors associated with inflammation. These learnings may influence recommendations for patients at risk for plaque-related diseases.

Quantitation of endotoxin and lipoteichoic acid virulence using toll receptor reporter gene.

PURPOSE: To apply quantitative Toll-like receptors (TLR) cell assays to compare lipopolysaccharides (LPSs) and lipoteichoic acids (LTAs) from different oral bacterial strains for potential pathogenicity in vitro. METHODS: The potency of LPS and LTA from different bacteria on activation of TLR reporter genes in HEK-tlr cell lines was examined. P. gingivalis LPS mix, P. gingivalis 1690 LPS, P. gingivalis 1435/50 LPS, E. coli LPS (E. coli K12), B. subtilis LTA, S. aureus LTA, E. hirae LTA and S. pyogenes LTA were examined in both TLR2 and TLR4 HEK cell line reporter assays. Solutions of LPS and LTA from selected bacteria were applied in a dose response fashion to the TLR reporter cells under standard culture conditions for mammalian cells. Reporter gene secreted-embryonic-alkaline-phosphatase (SEAP) was measured, and half maximal effective concentration (EC50) was determined for each sample. Concentration dependent TLR activation was compared to similar responses to LPS and LTA for commercial BODIPY-TR-Cadaverine and LAL biochemical (non cell based) assays. RESULTS: All LPS from P. gingivalis activated both TLR2 and TLR4 responses. E. coli LPS is a strong activator for TLR4 but not for TLR2 responses. In contrast, both B. subtilis and S. aureus LTA provoked responses only in TLR2, but not in the TLR4 assay. Interestingly, E. hirae LTA and S. pyogenes LTA did not stimulate strong TLR2 responses. Instead, both E. hirae LTA and S. pyogenes LTA mounted a reasonable response in TLR4 reporter gene assay. Both LPS and LTA showed deactivation of fluorescence in BODIPY-TR-Cadaverine while only LPS was active in LAL. As with biochemical assays, an EC50 could be determined for LPS and LTA from various bacterial strains. The EC50 is defined as a concentration of LPS or LTA that provokes a response halfway between the baseline and maximum responses. Lower EC50 means higher potency in promoting TLR responses, and in principle indicates greater toxicity to the host. CLINICAL SIGNIFICANCE: InvivoGen TLR2 and TLR4 assays distinguish specific types of microbial products, such as LPS and LTA from different bacteria. Application of EC50 determinations creates a means for quantitative and comparisons of LPS and LTA virulence in a cellular-based assay and combinations of TLR reporter cell assays along with biochemical evaluation of LPS#47;LTA in BODIPY-TR-Cadaverine and LPS in LAL assays provides a means to quantitate virulence of plaque samples with respect to both LPS and LTA. These learnings have long-term implications for patient care in that understanding the virulence of patients' plaque provides important information to assess risk of oral diseases.

Lipopolysaccharide and lipoteichoic acid binding by antimicrobials used in oral care formulations.

PURPOSE: To study the reactivity of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) with the cationically charged agents cetylpyridinium chloride, stannous fluoride, and the non-cationic agent triclosan. We also assessed the effect of these agents to inhibit LPS and LTA binding to cellular Toll-like Receptors (TLRs) in vitro. METHODS: The ability of these antimicrobials to bind with LPS and/or LTA was assessed in both the Limulus amebocyte lysate and BODIPY-TR-cadaverine dye assays. Mass spectroscopy was then used to confirm that stannous fluoride directly binds with LPS and to determine stoichiometry. Lastly, we looked for possible inhibitory effects of these antimicrobial agents on the ability of fluorescently conjugated LPS to bind to TLR4 expressed on HEK 293 cells. RESULTS: Cetylpyridinium chloride (CPC) and stannous salts including stannous fluoride interfered with LPS and LTA reactivity in both dye assays, while triclosan had no effect. Mass spectroscopy revealed direct binding of stannous fluoride with E. Coli LPS at 1:1 stoichiometric ratios. In the cellular assay, cetylpyridinium chloride and stannous fluoride, but not triclosan, inhibited LPS binding to TLR4. CLINICAL SIGNIFICANCE: These results support a potential mechanism of action for stannous fluoride and CPC formulated in oral products in which these ingredients bind bacterial toxins and potentially render them less toxic to the host. These results may influence home care recommendations for patients at risk for plaque-related diseases.

Response of chronic gingivitis to hygiene therapy and experimental gingivitis. Clinical, microbiological and metabonomic changes.

PURPOSE: To compare the clinical, microbiological and metabonomic profiles of subjects with high and low levels of chronic gingival bleeding during a controlled oral hygiene regimen intervention including sequential phases of rigorous therapeutic oral hygiene followed by experimental gingivitis (EG). METHODS: Two cohorts of qualified study subjects with differences in gingival bleeding on probing levels at their baseline clinical examination were entered into the study. These two cohorts were followed through three separate study phases including a 1-week baseline phase, a 2-week phase of rigorous oral hygiene including dental prophylaxis, and a 3-week EG phase of no oral hygiene to encourage relapse of gingivitis. The 58 subjects were assessed during each phase of the study for clinical presentation of gingivitis and concurrently had plaque sampled for real-time polymerase chain reaction (RTPCR) microbiological characterization and salivary lavage samples for 'systems biology' metabonomics assessment by 1H-NMR. RESULTS: Subjects presenting with different levels of gingival bleeding on probing when they entered the study responded differently to rigorous oral hygiene and EG. Specifically, the high bleeding cohort responded sluggishly to rigorous oral hygiene and exhibited markedly greater relapse to gingivitis during EG. RTPCR analysis showed changes in bacterial populations that were associated with study phases, particularly the increases in putative periodontal pathogens during EG. However, the microbiological profiles of high- and low-susceptibility gingival bleeding patients were largely similar. Metabonomic analysis likewise revealed significant changes in metabolite composition during study phases associated with differences in plaque toxicity, especially the short chain carboxylic acids propionate and n-butyrate, which tracked clinical changes in gingivitis severity. Systems analysis of metabonomic changes suggested differences between cohorts, although analysis to date has not elucidated whether these differences are causative (population predictive) or simply diagnostic of clinical status within populations.

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.

Comparative bioavailability and antimicrobial activity of cetylpyridinium chloride mouthrinses in vitro and in vivo.

PURPOSE: Commercialized cetylpyridinium chloride (CPC) mouthrinses were compared for antimicrobial substantivity/bioavailability in an in vitro disk retention assay (DRA) and clinical antimicrobial activity in vivo in the plaque glycolysis and regrowth method (PGRM). METHODS: Formulations compared in this testing included commercially available CPC mouthrinses: Crest Pro Health (CPH), (containing 700 ppm formulated CPC); Colgate Total Puerto Rico (CT450), (containing 450 ppm formulated CPC); Colgate Total US (CT750), (containing 750 ppm formulated CPC); and Scope Mouthwash (SCP), (containing 450 ppm formulated CPC). A water control (CTR) was included in one of the PGRM clinical trials. Two separate clinical PGRM studies employed a controlled, double-blind, randomized, crossover design where qualified adult PGRM panelists were supplied with acclimation NaF dentifrice for use throughout the trials. On treatment days, subjects sampled baseline plaque and then rinsed with assigned mouthrinse following morning toothbrushing. Treated plaque samples were collected 15 and 45 minutes after rinsing. Sampled plaques were vortexed, normalized for biomass and incubated under standard conditions to assess glycolysis. pH response of treated plaques in incubation buffers were compared to baseline untreated plaque values and an Area Under Curve (AUC) composite/aggregate analysis of glycolysis inhibition was used for treatment comparisons. A laboratory disk retention substantivity/bioavailability assay measured adsorption affinity of CPC in mouthrinse for anionic cellulose disks in vitro. RESULTS: Clinical PGRM studies showed significant differences in antibacterial clinical efficacy of commercialized mouthrinses. Combining clinical study results reveals rank ordered efficacy CPH > CT750 > SCP > CT450 > CTR. Comparison of DRA to PGRM glycolysis showed a linear relation highlighting correlation of CPC bioavailability to clinical antimicrobial performance of CPC mouthrinses.

Depth profile analysis of non-specific fluorescence and color of tooth tissues after peroxide bleaching.

PURPOSE: To examine laboratory changes of endogenous non-specific fluorescence and color throughout subsurface of tooth structures prior to and following peroxide bleaching. METHODS: Extracted human teeth were cross sectioned and mounted on glass slides. Cross sections were examined for internal color (digital camera) and nonspecific fluorescence (microRaman spectroscopy) throughout the tooth structure at specified locations. Surfaces of sections were then saturation bleached for 70 hours with a gel containing 6% hydrogen peroxide. Cross sections were reexamined for color and non-specific fluorescence changes. RESULTS: Unbleached enamel, dentin-enamel junction and dentin exhibit different CIELab color and non-specific fluorescence properties. Bleaching of teeth produced significant changes in color of internal cross sections and substantial reductions of non-specific fluorescence levels within enamel dentin and DEJ. Enamel and dentin non-specific fluorescence were reduced to common values with bleaching with enamel and the DEJ showing larger reductions than dentin.

Plaque levels of patients with fixed orthodontic appliances measured by digital plaque image analysis.

INTRODUCTION: A digital plaque image analysis system was developed to objectively assess dental plaque formation and coverage in patients treated with fixed orthodontic appliances. METHODS: The technique was used to assess plaque levels of 52 patients undergoing treatment with fixed appliances in the Department of Orthodontics at Johannes Gutenberg University in Mainz, Germany. RESULTS: Plaque levels ranged from 5.1% to 85.3% of the analyzed tooth areas. About 37% of the patients had plaque levels over 50% of the dentition, but only 10% exhibited plaque levels below 15% of tooth coverage. The mean plaque coverage was 41.9% ± 18.8%. Plaque was mostly present along the gum line and around the orthodontic brackets and wires. CONCLUSIONS: The digital plaque image analysis system might provide a convenient quantitative technique to assess oral hygiene in orthodontic patients with multi-bracket appliances. Plaque coverage in orthodontic patients is extremely high and is 2 to 3 times higher than levels observed in high plaque-forming adults without appliances participating in clinical studies of the digital plaque image analysis system. Improved hygiene, chemotherapeutic regimens, and compliance are necessary in these patients.

Construction and purification of His-tagged staphylococcal ArsB protein, an integral membrane protein that is involved in arsenical salt resistance.

Bacterial resistance to arsenical salts encoded on plasmid pI258 occurs by active extrusion of toxic oxyanions from cells of Staphylococcus aureus. The operon encodes for three gene products: ArsR, ArsB and ArsC. The gene product of arsB is an integral membrane protein and it is sufficient to provide resistance to arsenite and antimonite. A poly His-ArsB fusion protein was generated to purify the staphylococcal ArsB protein. Cells containing the His-tagged arsB gene were resistant to arsenite and antimonite. The levels of resistance to these toxic oxyanions by the His-tagged construct were greater than the levels obtained with the wild type gene. These data would indicate that the His-tagged protein is functionally active. A new 36 kDa protein band was visualized on 10% SDS-polyacrylamide gel electrophoresis (PAGE), which was confirmed as the His-ArsB protein by immunodetection with polyclonal Hisantibodies. The His-ArsB fusion protein was purified by the use of metal-chelate affinity chromatography with a Ni(+2)-nitrilotriacetic acid column and size-exclusion chromatography suggests that the protein was a homodimer.