Salivary exRNA biomarkers to detect gingivitis and monitor disease regression.

AIM: This study tests the hypothesis that salivary extracellular RNA (exRNA) biomarkers can be developed for gingivitis detection and monitoring disease regression. MATERIALS AND METHODS: Salivary exRNA biomarker candidates were developed from a total of 100 gingivitis and non-gingivitis individuals using Affymetrix's expression microarrays. The top 10 differentially expressed exRNAs were tested in a clinical cohort to determine whether the discovered salivary exRNA markers for gingivitis were associated with clinical gingivitis and disease regression. For this purpose, unstimulated saliva was collected from 30 randomly selected gingivitis subjects, the gingival and plaque indexes scores were taken at baseline, 3 and 6 weeks and salivary exRNAs were assayed by means of reverse transcription quantitative polymerase chain reaction. RESULTS: Eight salivary exRNA biomarkers developed for gingivitis were statistically significantly changed over time, consistent with disease regression. A panel of four salivary exRNAs [SPRR1A, lnc-TET3-2:1, FAM25A, CRCT1] can detect gingivitis with a clinical performance of 0.91 area under the curve, with 71% sensitivity and 100% specificity. CONCLUSIONS: The clinical values of the developed salivary exRNA biomarkers are associated with gingivitis regression. They offer strong potential to be advanced for definitive validation and clinical laboratory development test.

Clinical investigation of the antiplaque efficacy of a new variant of a commercially available triclosan/copolymer/fluoride dentifrice.

OBJECTIVE: The objective of two single-blind, three-treatment, crossover design, clinical studies was to evaluate the antiplaque efficacy using the Modified Gingival Margin Plaque Index (MGMPI) scores of three dentifrices: 1) a dentifrice containing 0.3% triclosan/2.0% polyvinylmethyl ether/maleic acid (PVM/MA) copolymer/sodium fluoride in a 17% dual silica base (Colgate Total Advanced Toothpaste-Test Dentifrice); 2) a commercially available dentifrice containing 0.3% triclosan/2.0% PVM/MA copolymer/sodium fluoride in a 10% high-cleaning silica base (Colgate Total Toothpaste-Positive Control Dentifrice); and 3) a commercially available dentifrice containing 0.243% sodium fluoride in a regular silica base (Colgate Winterfresh Gel-Negative Control Dentifrice). METHODS: In each study, subjects reported to the clinical facility, and those who met the inclusion/exclusion criteria were given a complete oral prophylaxis, a soft-bristled toothbrush, and a commercially available dentifrice (Colgate Cavity Protection Fluoride Toothpaste). They were instructed to use these products exclusively for seven days (washout period), after which time they reported back to the clinical facility and were randomized into three treatment groups. All subjects then brushed their teeth for one minute with a full ribbon (approximately 1.5 gm) of Colgate Cavity Protection Fluoride Toothpaste, and immediately followed with a one-minute brushing using a full ribbon of one of the three study dentifrices. Subjects then rinsed with a red disclosing solution (Butler Red-Cote) and had their teeth and gums examined to assess their plaque content. They returned to the clinical facility after 24 hours of no oral hygiene to again have their teeth and gums examined to assess their plaque content. As per the crossover clinical design, the same methods and materials were used until all subjects used all three study treatments. RESULTS: Seventeen subjects in the first study and 16 subjects in the second study complied with the protocol and completed all phases of the study. Two-way ANOVA results from both studies showed that there was no difference in mean delta MGMPI scores between the groups using the Test Dentifrice and the Positive Control Dentifrice. Results also showed that there was a statistically significant difference (p < 0.05) in delta MGMPI scores between both the Test Dentifrice treatment and the Positive Control Dentifrice treatment when compared to the Negative Control Dentifrice. CONCLUSION: A new improved dentifrice containing 0.2% triclosan/3.0% PVM/MA copolymer/sodium fluoride in a 17% dual silica base is comparable in controlling dental plaque when compared to a Positive Control Dentifrice containing 0.3% triclosan/2.0% PVM/MA copolymer/sodium fluoride in a 10% high-cleaning silica base, and is statistically significantly better in controlling dental plaque when compared to a Negative Control Dentifrice containing 0.243% sodium fluoride in a silica base.

Clinical evaluation of the anti-plaque effect of a commercial chewing gum.

OBJECTIVE: The objective of this research was to evaluate the dental plaque control effect of a chewing gum versus brushing with a dentifrice via four clinical studies. METHODOLOGY: Study 1 compared a commercial chewing gum (Colgate Dental Gum, CDG) with a water control after 24 hours post-brushing; Studies 2 and 3 compared CDG to two different brands of commercially available fluoride dentifrices after 24 hours post-brushing; Study 4 examined the anti-plaque effect of CDG plus a regular fluoride dentifrice (Colgate Winterfresh Gel, CWG) versus brushing with CWG alone for five days. The 24-hour clinical tests employed the Modified Gingival Margin Plaque Index (MGMPI), while the Quigley-Hein Plaque Index (QHPI) was used for the five-day study. All studies utilized a randomized, crossover design with a one-week washout period, and were single-blinded to the clinical evaluator. RESULTS: In Study 1, the mean MGMPI score for CDG was significantly lower (p < 0.05) compared to the water control. In Studies 2 and 3, while brushing with regular fluoride dentifrices provided improved plaque control compared to CDG, the chewing gum alone with no tooth brushing delivered a plaque reduction 60% as effective as brushing with a fluoride dentifrice. In Study 4, the group using the combination of chewing with CDG and brushing with CWG provided a significantly lower (p < 0.05) mean QHPI score compared to the group using the dentifrice only, particularly on the hard-to-brush lingual surfaces. CONCLUSIONS: Four clinical studies demonstrated that CDG provides a plaque control benefit. The results suggest that chewing gum may serve as an effective oral hygiene device when brushing may not be possible and, additionally, that chewing gum may serve as an effective adjunct to brushing for enhanced oral health.

Clinical evaluations of sustained calculus control by Colgate Simply White dentifrice using an in-situ appliance.

OBJECTIVE: Two clinical studies were conducted to evaluate the dental calculus control efficacy of Colgate Simply White dentifrice versus a positive and a negative control dentifrice, using a published intra-oral appliance and monitoring the prevention of calcium deposition, an indicator of early dental calculus formation. METHODOLOGY: Healthy human volunteers entered into the two double-blind, cross-over studies. An intra-oral appliance was custom-made for each subject. After brushing with an assigned dentifrice, each subject wore his or her appliance for four daytime hours (study 1) or 12 overnight hours (study 2). When the appliance was removed, it was washed, suspended in 0.1 M HCl to release Ca++ from deposits, and analyzed by Inductively Coupled Plasma (ICP) for deposited calcium. The three dentifrices studied were Colgate Simply White (CSW), Colgate Regular dentifrice (CR), and Colgate Tartar Control Whitening (CTW). There was a one-week wash-out period between each product use. RESULTS: There were no side effects observed or reported in either study. In both the four- and 12-hour studies, CSW and CTW had significantly lower calcium uptake compared to CR (p < 0.05). There was no statistical difference between CSW and CTW in efficacy. CONCLUSION: The results of this research demonstrate that Colgate Simply White dentifrice provides four- and 12-hour calculus control efficacy, superior to a standard dentifrice and comparable to a commercial anti-tartar dentifrice.

New laboratory methods to study tooth surface coverage and interproximal plaque control by dentifrice products.

OBJECTIVE: To develop and test an in vitro tooth model for use in conjunction with laboratory methods to study interproximal effects and efficacy of dentifrices. The application of the model should offer visual evaluation of dentifrice coverage of the tooth surface, and measure dental plaque control at posterior interdental spaces with a dentifrice. METHODOLOGY: The dentifrice products tested with the model were: Colgate Total 2 in 1 Toothpaste and Mouthwash (CTTM), Colgate Total dentifrice (CTD), and Colgate Regular dentifrice (CRD). Extracted human posterior teeth were disinfected, cleaned, aligned, and mounted in denture acrylic. In the area coverage method, tooth surface coverage and penetration of two different forms of dentifrice products (CTTM and CRD) were compared using digital photography. In the interproximal plaque control method, the teeth were coated with human saliva and incubated anaerobically with a mixture of representative oral bacteria for six hours at 37 degrees C. In vitro dental plaque was assessed after brushing the facial surface with one of the three dentifrice products using a clinical plaque scoring index. RESULTS: The area coverage method demonstrated that both dentifrice products tested covered approximately 70% of the facial tooth surface; the CTTM dentifrice coverage on the lingual tooth surface was significantly higher than the coverage for the CRD dentifrice. With the interproximal plaque control method, in the presence of an active ingredient, the CTTM dentifrice had equivalent efficacy to the CTD dentifrice. Both CTTM and CTD were significantly superior to the CRD for interproximal dental plaque control. CONCLUSION: Using the developed tooth model, two assessment methods have been shown to have the potential to demonstrate tooth surface coverage, and to assess the potential efficacy of a dentifrice for the control of interproximal dental plaque. This process can indicate potential clinical evaluation of an oral care product, and support clinical findings with controlled evidence.

Global metabolomic analysis of human saliva and plasma from healthy and diabetic subjects, with and without periodontal disease.

Recent studies suggest that periodontal disease and type 2 diabetes mellitus are bi-directionally associated. Identification of a molecular signature for periodontitis using unbiased metabolic profiling could allow identification of biomarkers to assist in the diagnosis and monitoring of both diabetes and periodontal disease. This cross-sectional study identified plasma and salivary metabolic products associated with periodontitis and/or diabetes in order to discover biomarkers that may differentiate or demonstrate an interaction of these diseases. Saliva and plasma samples were analyzed from 161 diabetic and non-diabetic human subjects with a healthy periodontium, gingivitis and periodontitis. Metabolite profiling was performed using Metabolon's platform technology. A total of 772 metabolites were found in plasma and 475 in saliva. Diabetics had significantly higher levels of glucose and α-hydroxybutyrate, the established markers of diabetes, for all periodontal groups of subjects. Comparison of healthy, gingivitis and periodontitis saliva samples within the non-diabetic group confirmed findings from previous studies that included increased levels of markers of cellular energetic stress, increased purine degradation and glutathione metabolism through increased levels of oxidized glutathione and cysteine-glutathione disulfide, markers of oxidative stress, including increased purine degradation metabolites (e.g. guanosine and inosine), increased amino acid levels suggesting protein degradation, and increased ω-3 (docosapentaenoate) and ω-6 fatty acid (linoleate and arachidonate) signatures. Differences in saliva between diabetic and non-diabetic cohorts showed altered signatures of carbohydrate, lipid and oxidative stress exist in the diabetic samples. Global untargeted metabolic profiling of human saliva in diabetics replicated the metabolite signature of periodontal disease progression in non-diabetic patients and revealed unique metabolic signatures associated with periodontal disease in diabetics. The metabolites identified in this study that discriminated the periodontal groups may be useful for developing diagnostics and therapeutics tailored to the diabetic population.

Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

OBJECTIVE: To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. METHODS: Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. RESULTS: Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. CONCLUSION: The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

Assessment of the effects of dentifrice on periodontal disease biomarkers in gingival crevicular fluid.

BACKGROUND: Periodontal disease has been studied primarily from clinical outcomes in lengthy human studies. Comprehensive biochemical profiling (metabolomics) has become a powerful tool for disease characterization and biomarker discovery. In a previous study, we performed a metabolomic analysis of gingival crevicular fluid collected from healthy, gingivitis, and periodontitis sites. Many metabolites associated with inflammation, oxidative stress, tissue degradation, and bacterial metabolism were found to be significantly induced by the diseases. METHODS: A panel of 10 markers was selected from the previous metabolomic study based on their statistical significance. Thirty-nine chronic periodontitis subjects were randomly assigned to a toothpaste regimen: control dentifrice (n = 21) or triclosan-containing dentifrice ([CT] n = 18). Subjects were instructed to use their assigned dentifrice twice daily for 6 weeks. Gingival crevicular fluid samples from six healthy, six gingivitis, and three periodontitis sites were collected from each subject at baseline, 1 week, and 6 weeks. The relative levels of the markers in the samples were determined by mass spectrometry. One-sided matched-paired t tests were performed to compare data from healthy, gingivitis, and periodontitis sites. RESULTS: Statistical analysis indicates that CT significantly decreased the levels of inosine, lysine, putrescine, and xanthine at the gingivitis sites as early as week 1. In contrast, control dentifrice had little effect. CONCLUSIONS: This result provides biochemical confirmation for the therapeutic effects of CT on gingivitis. Biomarkers were significantly altered by CT before clinical changes were observed, suggesting that the markers have predicative value for disease state assessment.