Localized microbially induced inflammation influences distant healthy tissues in the human oral cavity.

Variation in human immune response to the same bacterial or viral pathogen is well established in the literature. Variation in immune response to microbial challenge has also been observed within the human oral cavity. Our recent study focused on characterizing observed variations in microbially induced gingival inflammation-resulting in three distinct clinical Inflammatory Responder Types (IRTs): High-IRT, Low-IRT, and Slow-IRT. Here, we applied a high-resolution temporal multiomic analysis during microbially induced inflammation in order to characterize the effects of localized oral inflammation on distant healthy tissues in young healthy adults. Our results highlight a nonlocalized subclinical effect with alterations in proinflammatory host mediators and an ecological shift toward dysbiosis within the subgingival microbiome in an IRT-dependent manner-despite maintained oral hygiene. Our results provide mechanistic insight into how healthy tissues within humans are influenced by distant localized inflammation and may ultimately become susceptible to disease.

Human variation in gingival inflammation.

Oral commensal bacteria actively participate with gingival tissue to maintain healthy neutrophil surveillance and normal tissue and bone turnover processes. Disruption of this homeostatic host-bacteria relationship occurs during experimental gingivitis studies where it has been clearly established that increases in the bacterial burden increase gingival inflammation. Here, we show that experimental gingivitis resulted in three unique clinical inflammatory phenotypes (high, low, and slow) and reveal that interleukin-1ß, a reported major gingivitis-associated inflammatory mediator, was not associated with clinical gingival inflammation in the slow response group. In addition, significantly higher levels of Streptococcus spp. were also unique to this group. The low clinical response group was characterized by low concentrations of host mediators, despite similar bacterial accumulation and compositional characteristics as the high clinical response group. Neutrophil and bone activation modulators were down-regulated in all response groups, revealing novel tissue and bone protective responses during gingival inflammation. These alterations in chemokine and microbial composition responses during experimental gingivitis reveal a previously uncharacterized variation in the human host response to a disruption in gingival homeostasis. Understanding this human variation in gingival inflammation may facilitate the identification of periodontitis-susceptible individuals. Overall, this study underscores the variability in host responses in the human population arising from variations in host immune profiles (low responders) and microbial community maturation (slow responders) that may impact clinical outcomes in terms of destructive inflammation.

High Glucose Induces Late Differentiation and Death of Human Oral Keratinocytes.

Keratinocytes are essential cells for wound repair. Impaired oral wound healing is common in diabetic patients with periodontal disease. High glucose, or hyperglycemia, impairs the cellular function of different cell types. However, it is unknown whether high glucose has a detrimental effect on the functions of oral keratinocytes. In the current study, a human gingival keratinocyte cell line, telomerase immortalized gingival keratinocytes (TIGK), was treated with high glucose (24 and 48 mM) for up to 120 h. Proliferation, migration, cell viability, and production of markers of differentiation, growth factors and enzymatic antioxidants were assessed after high glucose treatment. The results showed that high glucose significantly inhibited TIGK proliferation and migration. High glucose also induced significant cell death through apoptosis and necrosis as determined by flow cytometry, especially at 120 h after high glucose treatment. Necrosis was the dominant form of cell death induced. Real-time PCR showed that high glucose treatment upregulated mRNA expression of late keratinocyte differentiation makers, such as keratin 1, 10, 13 and loricrin, and downregulated enzymatic antioxidants, including superoxide dismutase 1, catalase, nuclear factor erythroid 2 -related factor 2, heme oxygenase 1. In conclusion, high glucose impairs the proliferation and migration of oral keratinocytes and likely induces cell death through the promotion of late cell differentiation and down-regulation of enzymatic antioxidants.

Recent Development of Active Ingredients in Mouthwashes and Toothpastes for Periodontal Diseases.

Periodontal diseases like gingivitis and periodontitis are primarily caused by dental plaque. Several antiplaque and anti-microbial agents have been successfully incorporated into toothpastes and mouthwashes to control plaque biofilms and to prevent and treat gingivitis and periodontitis. The aim of this article was to review recent developments in the antiplaque, anti-gingivitis, and anti-periodontitis properties of some common compounds in toothpastes and mouthwashes by evaluating basic and clinical studies, especially the ones published in the past five years. The common active ingredients in toothpastes and mouthwashes included in this review are chlorhexidine, cetylpyridinium chloride, sodium fluoride, stannous fluoride, stannous chloride, zinc oxide, zinc chloride, and two herbs-licorice and curcumin. We believe this comprehensive review will provide useful up-to-date information for dental care professionals and the general public regarding the major oral care products on the market that are in daily use.

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.

Short- and Long-Term Effects of a Dentifrice Containing Dual Zinc plus Arginine on Intra-Oral Halitosis: Improvements in Breath Quality.

OBJECTIVES: These studies aimed to assess the short-term (12-hour, single use) and long-term (four weeks, continuous use) efficacy of a new Dual Zinc plus Arginine dentifrice against intra-oral halitosis versus a negative control. METHODS: Two clinical studies were conducted to assess the dentifrice: a four-week, continuous use parallel design versus a negative control and a single use crossover design versus a negative control. Both studies used organoleptic and hedonic odor judge scores measured 12 hours overnight after product use as the primary efficacy variable. Additionally, the single use study employed SIFT-MS to quantify the intra-oral concentration of volatile sulfur compounds as a complementary measure of efficacy. RESULTS: In both studies, the Dual Zinc plus Arginine dentifrice provided statistically significant improvements in breath quality across all measures versus a negative control. CONCLUSIONS: Improvements in breath quality were attributed to the effects of zinc cations delivered by the uniquely formulated dentifrice.

Enhanced In Vitro Zinc Bioavailability through Rational Design of a Dual Zinc plus Arginine Dentifrice.

OBJECTIVES: To investigate bioavailability enhancement of zinc on model oral surfaces and in oral biofilms in vitro through strategic formulation with two sources of zinc and L-arginine. METHODS: To modulate the bioavailability of active zinc ions in a zinc citrate dentifrice, an additive research strategy was pursued. A series of zinc citrate dentifrice formulations were prepared with increasing replacement of zinc citrate with zinc oxide (a water insoluble source of zinc ions) to generate a Dual Zinc active system. A screening of isolated zinc and amino acid effects in simple solutions using zeta potential and uptake to model oral surfaces was performed in an effort to determine the effect of particle charge on zinc bioavailability. Zinc delivery and antibacterial efficacy of the Dual Zinc plus Arginine dentifrice formula were tested using in vitro oral epithelial tissue and saliva-derived biofilm models. Furthermore, zinc penetration and retention were determined by subjecting in vitro biofilms to dynamic flow after treatment with the Dual Zinc plus Arginine dentifrice with treated biofilms evaluated for zinc using imaging mass spectrometry (I-MS). Bacterial adhesion to gingival epithelial cells treated with the Dual Zinc plus Arginine dentifrice was imaged upon challenging with Streptococcus gordonii. RESULTS: Addition of zinc oxide into a zinc citrate dentifrice formula enhanced the efficacy of the system against anaerobic biofilms in a concentration- dependent manner. L-arginine further provided a significant positive charge (+36 mV) to the zinc oxide suspension (+16 mV) as measured by zeta potential. Simple solutions of the Dual Zinc active showed increased zinc uptake on model oral surfaces as a direct function of L-arginine concentration. Antibacterial efficacy of a Dual Zinc plus Arginine dentifrice was evaluated through multiple mechanisms. Enhanced antibacterial performance was observed through significant reductions in metabolic activity as measured through bacterial glycolytic function (p = 0.0001) and total oxygen consumption (p = 0.0001). Greater penetration and retention of zinc was observed in bacterial biofilms treated with the Dual Zinc plus Arginine dentifrice in comparison to treatment with a Dual Zinc dentifrice after twelve hours of dynamic flow (10 mL/hour) in an in vitro drip flow biofilm culture. Confocal microscopy showed adherent bacteria on cheek cells treated with the Dual Zinc plus Arginine dentifrice formula. CONCLUSIONS: The combination of zinc citrate, zinc oxide, and the amino acid L-arginine in a dentifrice formula enhances the bioavailability of zinc to model oral tissue surfaces, resulting in unique physicochemical effects. The significant antimicrobial control associated with the Dual Zinc plus Arginine dentifrice provides a unique vehicle toward achieving whole mouth health.

Inflammation and tissue remodeling mediator expression during gingivitis: A comparison between experimental, naturally occurring gingivitis, and periodontal health.

BACKGROUND: The aim of this study is to evaluate the immune regulation and tissue remodeling responses during experimental gingivitis (EG) and naturally occurring gingivitis (NG) to provide a comprehensive analysis of host responses. Gingival crevicular fluid (GCF) was obtained from 2 human studies conducted in university settings. METHODS: The EG study enrolling 26 volunteers provided controls for the baseline (Day 0) from healthy disease-free participants, while Day 21 (the end of EG induction of the same group) was used to represent EG. Twenty-six NG participants age-matched with those of the EG group were recruited. GCF samples were analyzed for 39 mediators of inflammatory/immune responses and tissue remodeling using commercially available bead-based multiplex immunoassays. The differences in GI and mediator expression among groups were determined at a 95% confidence level (p ≤ 0.05) by a 2-way analysis of variance (ANOVA) with a post-hoc Tukey's test. RESULTS: Our findings showed that EG had a greater gingival index than NG and was healthy (p < 0.01 of all comparisons). Furthermore, EG showed significantly higher levels of MPO (p < 0.001), CCL3 (p < 0.05), and IL-1B (p < 0.001) than NG. In contrast, NG had increased levels of MIF (p < 0.05), Fractalkine (p < 0.001), angiogenin (p < 0.05), C3a (p < 0.001), BMP-2 (p < 0.001), OPN (p < 0.05), RANKL (p < 0.001), and MMP-13 (p < 0.001) than EG. CONCLUSIONS: Consistent with the findings from chronic (NG) versus acute (EG) inflammatory lesions, these data reveal that NG displays greater immune regulation, angiogenesis, and bone remodeling compared to EG.

Effects of a stabilized stannous fluoride dentifrice on clinical, immunomodulatory, and microbial outcomes in a human experimental gingivitis model.

BACKGROUND: Stannous fluoride dentifrice is well established for its beneficial clinical effects. In this study, we evaluated the effects of stannous fluoride on inflammation and oral microbiome. METHODS: In this randomized, parallel-arm, double-blind, controlled clinical trial, we compared clinical resolution of experimental gingivitis by evaluating bleeding on probing, gingival index, and plaque index between stannous fluoride stabilized with zinc phosphate (test) and sodium fluoride (control) dentifrices. Further, these groups were compared for oral neutrophil counts, systemic priming of neutrophils, gingival crevicular fluid (GCF) expression of inflammatory markers, and the oral microbiome. RESULTS: We found significant reduction in bleeding on probing in the test group compared to the control group in experimental gingivitis when participants used the test dentifrice prior to induction of experimental gingivitis. The test group also showed significant reductions in GCF levels of inflammatory markers (matrix metalloproteinase 8 [MMP8], receptor activator of nuclear factor kappa-Β ligand [RANKL]), oral polymorphonuclear neutrophil (PMN) counts, and systemic neutrophil priming (CD11b expression) during experimental gingivitis. Further, significant reductions in the gram-negative genera Porphyromonas, Tannerella, and Treponema were noted in the test group. CONCLUSION: The stannous fluoride stabilized with zinc phosphate dentifrice formulation demonstrated clinical reduction in gingival inflammation and a beneficial effect on microbiome and immune markers. This intervention should be explored as a preventive aid in the progression of plaque-induced gingivitis to periodontitis.

A Dual Zinc plus Arginine formulation protects against tumor necrosis factor-alpha-induced barrier dysfunction and enhances cell proliferation and migration in an in vitro gingival keratinocyte model.

OBJECTIVE: To investigate the effects of Dual Zinc plus Arginine formulations (aqueous solution and dentifrice) on tumor necrosis factor-alpha (TNF-α)-induced barrier dysfunction as well as on cell proliferation and migration in an in vitro gingival keratinocyte model. DESIGN: Gingival keratinocytes were seeded onto the membrane of a double-chamber system in the absence and presence of recombinant TNF-α and the formulations under investigation. The barrier function was assessed by determination of transepithelial electrical resistance (TER) and paracellular transport of fluorescein isothiocyanate (FITC)-dextran. The distribution of zonula occludens-1 (ZO-1) and occludin was visualized by immunofluorescence microscopy. The effects of the formulations on keratinocyte cell proliferation were determined using a fluorescent cell tracker dye, while a migration assay kit was used to investigate their effects on cell migration. RESULTS: Under conditions where TNF-α induces loss of keratinocyte barrier integrity, the Dual Zinc plus Arginine formulations (aqueous solution and dentifrice) protected the keratinocyte tight junction against the damages since they prevented the TNF-α-induced drop in TER and increase in FITC-dextran paracellular flux in the in vitro model. The treatment of keratinocytes with the formulations markedly mitigated the altered distribution of ZO-1 and occludin. Both formulations increased the proliferation of keratinocytes and alleviated the negative impact caused by TNF-α. Lastly, the formulations increased the migration capacity of keratinocytes. CONCLUSIONS: The ability of the Dual Zinc plus Arginine formulations to protect the barrier integrity of gingival keratinocytes from TNF-α-induced damage and to promote their proliferation and migration suggests that they may offer benefits for oral health.

Immunomodulatory streptococci that inhibit CXCL8 secretion and NFκB activation are common members of the oral microbiota.

Introduction. Oral tissues are generally homeostatic despite exposure to many potential inflammatory agents including the resident microbiota. This requires the balancing of inflammation by regulatory mechanisms and/or anti-inflammatory commensal bacteria. Thus, the levels of anti-inflammatory commensal bacteria in resident populations may be critical in maintaining this homeostatic balance.Hypothesis/Gap Statement. The incidence of immunosuppressive streptococci in the oral cavity is not well established. Determining the proportion of these organisms and the mechanisms involved may help to understand host-microbe homeostasis and inform development of probiotics or prebiotics in the maintenance of oral health.Aim. To determine the incidence and potential modes of action of immunosuppressive capacity in resident oral streptococci.Methodology. Supragingival plaque was collected from five healthy participants and supragingival and subgingival plaque from five with gingivitis. Twenty streptococci from each sample were co-cultured with epithelial cells±flagellin or LL-37. CXCL8 secretion was detected by ELISA, induction of cytotoxicity in human epithelial cells by lactate dehydrogenase release and NFκB-activation using a reporter cell line. Bacterial identification was achieved through partial 16S rRNA gene sequencing and next-generation sequencing.Results. CXCL8 secretion was inhibited by 94/300 isolates. Immunosuppressive isolates were detected in supragingival plaque from healthy (4/5) and gingivitis (4/5) samples, and in 2/5 subgingival (gingivitis) plaque samples. Most were Streptococcus mitis/oralis. Seventeen representative immunosuppressive isolates all inhibited NFκB activation. The immunosuppressive mechanism was strain specific, often mediated by ultra-violet light-labile factors, whilst bacterial viability was essential in certain species.Conclusion. Many streptococci isolated from plaque suppressed epithelial cell CXCL8 secretion, via inhibition of NFκB. This phenomenon may play an important role in oral host-microbe homeostasis.

A Dual Zinc plus Arginine formulation attenuates the pathogenic properties of Porphyromonas gingivalis and protects gingival keratinocyte barrier function in an in vitro model.

BACKGROUND AND OBJECTIVES: Porphyromonas gingivalis, a late colonizer of the periodontal biofilm, has been strongly associated with the chronic form of periodontitis. The aim of this study was to investigate the effects of a Dual Zinc plus Arginine formulation (aqueous solution and dentifrice) on the pathogenic properties of P. gingivalis and the barrier function of an in vitro gingival epithelium model. RESULTS: The Dual Zinc plus Arginine aqueous solution and dentifrice inhibited the hemolytic and proteolytic activities of P. gingivalis. The Dual Zinc plus Arginine aqueous solution and dentifrice enhanced the barrier function of an in vitro gingival epithelium model as determined by a time-dependent increase in transepithelial electrical resistance and decrease in paracellular permeability. This was associated with an increased immunolabeling of two important tight junction proteins: zonula occludens-1 and occludin. The deleterious effects of P. gingivalis on keratinocyte barrier function as well as the ability of the bacterium to translocate through a gingival epithelium model were attenuated in the presence of either Dual Zinc plus Arginine aqueous solution or dentifrice. CONCLUSIONS: The Dual Zinc plus Arginine formulation may offer benefits for patients affected by periodontal disease through its ability to attenuate the pathogenic properties of P. gingivalis and promote keratinocyte barrier function.

Solving the problem with stannous fluoride: Formulation, stabilization, and antimicrobial action.

BACKGROUND: Stannous fluoride (SnF2) is a compound present in many commercially available dentifrices; however, oxidative decomposition negatively impacts its efficacy. Stannous oxidation is often mitigated through the addition of complexing agents or sources of sacrificial stannous compounds. The authors have found that the addition of zinc phosphate significantly improved stannous stability more effectively than other stabilization methods. The authors evaluated the chemical speciation of stannous compounds within a variety of formulations using x-ray absorption near edge spectroscopy (XANES), a technique never used before in this manner. These data were compared and correlated with several antimicrobial experiments. METHODS: XANES data of various commercially available compounds and Colgate TotalSF were performed and analyzed against a library of reference compounds to determine the tin chemical speciation. The antibacterial assays used were salivary adenosine triphosphate, short-interval kill test, plaque glycolysis, and anaerobic biofilm models. RESULTS: XANES spectra showed a diverse distribution of tin species and varying degrees of SnF2 oxidation. In vitro antimicrobial assessment indicated significant differences in performance, which may be correlated to the differences in tin speciation and oxidation state. CONCLUSIONS: Driven by the excipient ingredients, SnF2 dentifrices contain a distribution of tin species in either the SnF2 or Sn(IV) oxidation state. The addition of zinc phosphate provided significant robustness against oxidation, which directly translated to greater efficacy against bacteria. PRACTICAL IMPLICATIONS: The choice of inactive ingredients in a dentifrice with active SnF2 can dramatically impact product stability.

Analysis of the antibacterial activity and plaque control benefit of colgate total dentifrice via clinical evaluation and real-time polymerase chain reaction.

OBJECTIVE: This study analyzed, from a combined clinical and molecular biologic perspective, the antibacterial and antiplaque efficacy of Colgate Total dentifrice (CTD). METHODOLOGY: A single-blind crossover study design utilized 11 healthy human subjects. After a one-week washout period, subjects donated dental plaque, received a dental prophylaxis, and subsequently brushed with a test product. Twenty-four hours postbrushing, dental plaque was collected and a clinical plaque score determined. Dental plaque was submitted for Real-time Polymerase Chain Reaction (Real-time PCR) analysis. The same procedure was repeated in accordance with a crossover design for the use of the second test product. Following a one-week washout, a plaque donation, prophylaxis, and brushing with the test product ensued for each subject. Twenty-four hours post-brushing, the subjects returned for a plaque score and plaque donation. RESULTS: Twenty-four hours after brushing, dental plaque coverage increased 17.88% +/- 8.27% with CTD, compared to 30.42% +/- 9.97% with Colgate Cavity Protection (CCP; p = 0.005). Real-time PCR found plaque collected 24 hours after brushing with CTD exhibited, on average, fewer representative periodontal pathogens (Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, and Porphyromonas gingivalis) and fewer early colonizers (Actinomyces naeslundii) than plaque collected before brushing, whereas CCP showed a moderate effect on oral bacteria. CONCLUSION: The study provides clinical and molecular biological evidence to substantiate the antibacterial and plaque control benefits of Colgate Total, and suggests the value of combining a molecular biological method with clinical research to corroborate clinical benefits.

Effectiveness of a triclosan/copolymer dentifrice on microbiological and inflammatory parameters.

According to the US Surgeon General's report, "Oral Health in America," published in 2000, most adults in the United States show some degree of periodontal pathology, with severe periodontal diseases affecting about 14% of middle-aged adults. Periodontal diseases are polymicrobial-induced inflammatory diseases, and they vary from mild gingival inflammation to severe deterioration of the periodontium, ie, loss of periodontal supportive tissues and, ultimately, tooth loss. New evidence shows that periodontal diseases may impact systemic health. For this reason, the maintenance of a healthy mouth is becoming increasingly important for the overall health of the body. This article summarizes laboratory research conducted during the development of a novel, multibenefit, oral-care technology based on triclosan--a broad-spectrum antibacterial agent--and a polyvinylmethylether/maleic acid copolymer. This unique combination of agents is found in Colgate Total, a clinically proven efficacious dentifrice for control of dental plaque and gingivitis. Data are presented that demonstrate the unique antibacterial properties of this dentifrice: (1) a broad-spectrum antimicrobial profile; (2) the long-lasting retention of triclosan on hydroxyapatite and epithelial cells; and (3) molecular evidence of antibacterial activity against specific pathogens in clinical dental plaque. In addition, data are presented that demonstrate the anti-inflammatory effects of triclosan on specific cytokines, the interruption of inflammatory pathways, and the inhibition of bone resorption. Overall, these data support the multibenefit clinical effects of Colgate Total and suggest a plurality of mechanisms of action.

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.