Virginia Monsul Barnes

Acceleration of Purine Degradation by Periodontal Diseases

Periodontal diseases, such as gingivitis and periodontitis, are characterized by bacterial plaque accumulation around the gingival crevice and the subsequent inflammation and destruction of host tissues. To test the hypothesis that cellular metabolism is altered as a result of host-bacteria interaction, we performed an unbiased metabolomic profiling of gingival crevicular fluid (GCF) collected from healthy, gingivitis, and periodontitis sites in humans, by liquid and gas chromatography mass spectrometry. The purine degradation pathway, a major biochemical source for reactive oxygen species (ROS) production, was significantly accelerated at the disease sites. This suggests that periodontal-disease-induced oxidative stress and inflammation are mediated through this pathway. The complex host-bacterial interaction was further highlighted by depletion of anti-oxidants, degradation of host cellular components, and accumulation of bacterial products in GCF. These findings provide new mechanistic insights and a panel of comprehensive biomarkers for periodontal disease progression.

Metabolomics Reveals Elevated Macromolecular Degradation in Periodontal Disease

Periodontitis is a chronic inflammatory disease characterized by tissue destruction. In the diseased oral environment, saliva has primarily been considered to act as a protectant by lubricating the tissue, mineralizing the bones, neutralizing the pH, and combating microbes. To understand the metabolic role that saliva plays in the diseased state, we performed untargeted metabolomic profiling of saliva from healthy and periodontitic individuals. Several classes of biochemicals, including dipeptide, amino acid, carbohydrate, lipids, and nucleotide metabolites, were altered, consistent with increased macromolecular degradation of proteins, triacylglycerol, glycerolphospholipids, polysaccharides, and polynucleotides in the individuals with periodontal disease. These changes partially reflected the enhanced host-bacterial interactions in the diseased state as supported by increased levels of bacterially modified amino acids and creatine metabolite. More importantly, the increased lipase, protease, and glycosidase activities associated with periodontitis generated a more favorable energy environment for oral bacteria, potentially exacerbating the disease state.

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 Metabolons 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.

Assessing fluoride treatment and resistance of dental enamel to soft drink erosion in vitro: Applications of focus variation 3D scanning microscopy and stylus profilometry

OBJECTIVES This study assesses the application of the focus variation 3D microscopy for the evaluation of dental erosion and fluoride treatment for prevention of enamel erosion in vitro. METHODS Human dental enamel disks were treated with Prevident 5000 (PV, n=15) for 1 week and compared with a reference group (PN, n=15) after orange juice erosion in vitro. A focus variation 3D scanning microscope (IFM) and a stylus type profilometer (SSP) were used to evaluate the erosion depths on enamel. 3D topographic images were taken with vertical resolutions of 0.1 and 0.02 microm. Scratch marks depths from SSP were measured on IFM images. Measurements were compared between the SSP and IFM and between the two study groups. RESULTS The SSP and IFM measurements of eroded enamel surfaces showed similar trends between the two methods and between the two study groups. The SSP and the IFM measurements were statistically significantly different but correlated with each other. PV group showed consistently lower erosion depth than PN in all profile measures using both SSP and IFM. The stylus tip created scratch marks that were significantly different in depths between the eroded and the reference surfaces in both groups. CONCLUSIONS The focus variation 3D microscopy is a powerful tool in evaluating surface topography associated with enamel erosion and in assessing the treatment effects of anti-erosive therapies. Topical treatment with Prevident 5000 significantly increased enamel resistance to erosion by orange juice and should be considered as a treatment choice in patients susceptible to acidic dental erosion.

Preventive effects of dentifrice containing 5000 ppm fluoride against dental erosion in situ

OBJECTIVE To evaluate the effectiveness of a dentifrice with 5000ppm fluoride in preventing dental erosion by orange juice in situ in comparison to a control dentifrice with 1450ppm fluoride. METHODS This was a double-blind and randomized clinical study with a cross-over design. Sixteen subjects wore an intra-oral appliance containing two enamel disks with an exposed surface of approximately 2mm×5mm. Enamel disks in the study group were treated with a dentifrice with 5000ppm fluoride and in the control group with 1450ppm fluoride. The subjects rinsed with slurries of study dentifrices for one minute before immersing the enamel disks in 250ml orange Juice four times in an 8-h period daily. The treatment procedure was repeated for three 5-day phases for each dentifrice. Enamel erosion was measured after each 5-day treatment phase using a focus-variation 3D scanning microscopy. Medians and inter-quartile ranges (IQR) of mean erosion depth were compared between the groups. RESULTS The mean erosion depths of enamel varied greatly amongst the subjects. Enamel treated with 5000ppm fluoride had less erosion (median 5.7μm, IQR 4.5μm) as compared to the control (median 12.6μm, IQR 12.3μm) after 15 days of fluoride treatment and erosive challenge cycles (p<0.05). CONCLUSIONS Enamel treated with 5000ppm fluoride had significantly improved resistance to erosion by orange juice. Periodic application of 5000ppm fluoride may be beneficial in individuals at risk of acidic erosion associated with soft drink consumptions.

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.

Effects of dentin tubule occlusion by dentifrice containing a PVM/MA bioadhesive copolymer in a silica base

OBJECTIVE To study the effectiveness of a dentifrice containing polymethyl vinyl ether-maleic acid (PVM/MA) copolymer in a silica base in occluding dentin tubules for treatment of dentin sensitivity. METHODS Thirty-two human dentin discs were divided into two groups and brushed in the morning for 30s each to study the dentifrices with and without PVM/MA copolymer. Dentin tubule occlusion and dentin permeability were evaluated with a focus variation three dimensional vertical scanning microscope (IFM) and electrochemical impedance spectroscopy (EIS). After second brushing for 30s in the afternoon the dentin discs were immersed in saliva for 16 h and then subjected to erosion using orange juice for 10 min. The effects of saliva and orange juice on tubule occlusion used in the study of dentifrices were further evaluated with IFM. RESULTS On average 97.7% of the dentin tubules were occluded after brushing in the PVM/MA group, as compared to 13.3% in the control group (p<0.0001). EIS showed that the impedance of the dentin disc increased after treatment with PVM/MA but not in the control group (p<0.05). After 16 h of storage in saliva and 10 min of erosion by orange juice, 86% of the dentin tubules remained occluded in the PVM/MA treated dentifrice. The sizes of the tubule openings were increased after orange juice erosion in the control group but not in the PVM/MA group. CONCLUSION Dentifrice containing PVM/MA copolymer in a silica base effectively occluded dentin tubules. The intra-tubular plugs were resistant to saliva and orange juice challenges.

Triclosan Blocks MMP-13 Expression in Hormone-Stimulated Osteoblasts

BACKGROUND Matrix metalloproteinase-13 (MMP-13) is an important enzyme for the modulation of bone turnover and gingival recession. Elevated levels of MMP-13 are associated with alveolar bone resorption, periodontal ligament breakdown, and gingival attachment loss, which are the clinical symptoms of periodontal disease. Evidence continues to suggest that periodontal disease contributes to oral tissue breakdown and is linked to numerous systemic conditions. Triclosan (TCN) is a long-standing, proven antibacterial and anti-inflammatory agent found in the only Food and Drug Administration-approved dentifrice for the treatment of plaque and gingivitis. METHODS This study examines the inhibitory effects of TCN on lipopolysaccharide-, parathyroid hormone (PTH)-, and prostaglandin E2 (PGE2)-induced expression of MMP-13 in UMR 106-01 cells, an osteoblastic osteosarcoma cell line. The cells were stimulated with PTH or PGE2 to induce MMP-13 mRNA expression, and real-time reverse transcription-polymerase chain reaction was performed to determine gene expression levels. Western blot analysis assessed the presence or absence of protein degradation or inhibition of protein synthesis. MMP-13 promoter reporter assay was used to explore possible direct effects of TCN on the MMP-13 promoter. RESULTS TCN significantly reduced PTH or PGE2 elevated expression of MMP-13 in osteoblastic cells without affecting basal levels of the mRNA. Surprisingly, TCN enhanced the expression of c-fos and amphiregulin mRNA. A promoter assay indicated that TCN directly inhibits the activation of the PTH-responsive minimal promoter of MMP-13. CONCLUSION The present study appears to have identified a nuclear mechanism of action of TCN that accounts for the ability of TCN to inhibit PTH- or PGE2-induced MMP-13 expression in osteoblastic cells.