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Item The ability of dual whitening anti-caries mouthrinses to remove extrinsic staining and enhance caries lesion remineralization – An in vitro study(Elsevier, 2020) Al-Shahrani, Ahid A.; Levon, John A.; Hara, Anderson T.; Tang, Qing; Lippert, Frank; Cariology, Operative Dentistry and Dental Public Health, School of DentistryObjectives This laboratory study investigated the ability of dual whitening anti-caries mouthrinses to remove extrinsic staining from artificially stained caries lesions and to enhance their remineralization and fluoridation. Materials and Methods Early caries lesions were created in bovine enamel specimens. The lesions were artificially stained and pH cycled for 10 days with the daily cycling regimen consisting of twice daily 60s-treatments with one of 11 mouthrinses, a 4-h demineralization period and artificial saliva treatments in between. Mouthrinses were eight commercially available products, all containing 100 ppm fluoride but utilizing hydrogen peroxide, pyro-, tri- or hexametaphosphate salts and/or sodium bicarbonate. The three control mouthrinses were 100 ppm fluoride, 30 % hydrogen peroxide and deionized water. Enamel color changes (ΔE) were determined spectrophotometrically. Vickers surface microhardness (VHN) was used to determine lesion remineralization. Enamel fluoride content (EFC) was determined using the microbiopsy technique. Data were analyzed using ANOVA. Results ΔE was significantly different among groups (p = 0.0045). Thirty percent hydrogen peroxide was superior to all other mouthrinses, while there were no differences between commercial mouthrinses and deionized water. There were small, directional but non-significant differences between commercial mouthrinses with those containing hydrogen peroxide providing better whitening. There were no significant differences between mouthrinses in their ability to remineralize caries lesions (p = 0.2898). EFC differed among groups (p < 0.0001), with the two mouthrinses containing pyrophosphate salts having lower EFC than all but the deionized water group. Conclusions Artificially stained caries lesions show reduced susceptibility to fluoride remineralization and whitening effects of commercial whitening and anti-caries mouthrinses. Clinical Relevance Artificially stained caries lesions appear to require stronger than over-the-counter interventions to successfully whiten and remineralizing them.Item Artificial biofilm thickness and salivary flow effects on fluoride efficacy – A model development study(2017-10-26) Lippert, Frank; Hara, Anderson T.; Churchley, David; Lynch, Richard J. M.This laboratory model development study investigated the interaction between artificial biofilm thickness and salivary flow rate on fluoride-mediated prevention of enamel caries lesion formation. This 5-day pH cycling study on sound bovine enamel specimens utilized a continuous flow model and followed a 4 (agarose biofilm thickness-‘no biofilm’/1/2/3mm)×2 (remineralizing solution flow rate-0.05/0.5ml/min)×2 (fluoride-0/383ppm as sodium fluoride) factorial design. Vickers surface microhardness change was the outcome measure. Data were analyzed with three-way ANOVA. The three-way interaction gel thickness×flow rate×fluoride concentration was significant (p=0.0006). 383ppm fluoride caused less softening than 0ppm regardless of gel thickness or flow rate. 0.5ml/min flow rate caused less softening than 0.05ml/min for ‘no biofilm’ and 1mm biofilm thickness regardless of fluoride concentration, for 2 and 3mm with 0ppm F but not for 383ppm F. For 0.05ml/min, softening was reduced as gel thickness increased from ‘no biofilm’-1-2mm, but not from 2-3mm. For 0.5ml/min, ‘no biofilm’ caused more softening than 1, 2, and 3mm, but 1, 2, and 3mm were not different from each other for both 0 and 383ppm F. The present findings suggest that the efficacy of fluoride in preventing enamel demineralization is affected by both biofilm thickness and salivary flow rate, with both thicker biofilms and higher flow rate resulting in less demineralization.Item Effect of Green Tea on Streptococcus mutans Metabolic Activity, Planktonic Growth, and Biofilm Activity in the Presence of Nicotine(Office of the Vice Chancellor for Research, 2013-04-05) Gardner, R.; Foltz, J.; Li, Mingyun; Huang, Ruijie; Gregory, Richard L.Streptococcus mutans is the main bacterial cause of dental caries, and it has been proven by previous research that its growth is affected by various concentrations of nicotine and other agents. The amount of S. mutans in the mouth is directly proportional to the number of dental cavities. Studies have shown that smokers have an increased amount of caries, much of which is due to the low concentrations of nicotine the mouth is exposed to. It is known that S. mutans thrives in low-moderate concentrations of nicotine, and that nicotine is a promoting agent for S. mutans. S. mutans has also been proven as a contributor to atherosclerosis, resulting from dental plaque entering the bloodstream. Green Tea is a commonly consumed beverage, which has been known to reduce the number of dental cavities. Previous research has concluded that green tea contains polyphenols, which have antimicrobial effects, including an inhibitory effect on S. mutans. The objective of this research is to observe how green tea affects S. mutans metabolic activity, as well as biofilm and planktonic growth, in the presence of nicotine. The experiments compared S. mutans treated with nicotine concentrations (0-8 mg/ml), and S. mutans treated with a 2.5 g/200 mL concentration of Sencha Jade Reserve Japanese green tea in conjunction with the various nicotine concentrations. The assays were performed in a microtiter plate; the XTT and biofilm assays measured absorbance, and the planktonic assay measured kinetic growth. The experiments conclude that green tea has an inhibitory effect on nicotine-treated S. mutans metabolic activity and planktonic growth, with higher concentrations of green tea inhibiting more effectively. It was also concluded that green tea increases biofilm formation. These conclusions provide evidence of the inhibitory effect green tea has on nicotine-treated S. mutans, and may indicate a way to reduce the incidence of caries and atherosclerosis.Item Effect of nicotine on cariogenic virulence of Streptococcus mutans(Springer, 2016-11) Li, Mingyun; Huang, Ruijie; Zhou, Xuedong; Qiu, Wei; Xu, Xin; Gregory, Richard L.; Department of Biomedical and Applied Sciences, IU School of DentistryNicotine has well-documented effects on the growth and colonization of Streptococcus mutans. This study attempts to investigate the effects of nicotine on pathogenic factors of S. mutans, such as the effect on biofilm formation and viability, expression of pathogenic genes, and metabolites of S. mutans. The results demonstrated that addition of nicotine did not significantly influence the viability of S. mutans cells. The biofilms became increasingly compact as the concentrations of nicotine increased. The expression of virulence genes, such as ldh and phosphotransferase system (PTS)-associated genes, was upregulated, and nlmC was upregulated significantly, while ftf was downregulated. The lactate concentration of S. mutans grown in 1 mg/mL of nicotine was increased up to twofold over either biofilm or planktonic cells grown without nicotine. Changes in the metabolites involved in central carbon metabolism from sucrose indicated that most selected metabolites were detectable and influenced by increased concentrations of nicotine. This study demonstrated that nicotine can influence the pathogenicity of S. mutans and may lead to increased dental caries through the production of more lactate and the upregulation of virulence genes.Item EFFECT OF NICOTINE ON METABOLISM OF STARVED AND UNSTARVED STREPTOCOCCUS MUTANS(Office of the Vice Chancellor for Research, 2011-04-08) RODENBECK, JESSICA; GREGORY, RICHARD L.Streptococcus mutans is the major etiological agent of dental caries. Nicotine is the addictive ingredient present in most tobacco products that has been shown to have an effect on the growth and metabolism of oral bacteria, specifically S. mutans. This same bacterium has been recently linked to heart disease. Smokers regularly introduce this chemical into their system which causes an increased growth and metabolic rate of the bacteria, thus increasing their chances for dental caries. This research worked to further qualify the increase of metabolic rates by subjecting the bacteria to nicotine in a starved environment, on the basis that humans do not constantly have nutrients available to oral bacteria. Metabolic rates of an established biofilm of S. mutans were measured through an XTT and menadione assay with a spectrophotometer. The unstarved bacteria were grown in a full concentration of TSBS while the starved were grown in a 1:10 dilution of TSBS with sterile saline in various nicotine concentrations (0.0, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, and 128.0 mg/ml). As the concentration of nicotine increase, the metabolic rates of S. mutans also increased. The high concentrations in which the bacteria were no longer metabolically active are very high and a normal smoker would not be able to reach these concentrations. However, as more nicotine is present in smokers these more metabolically active bacteria would be more likely to cause caries.Item The effect of theobromine on the in vitro de- and remineralization of enamel carious lesions(Elsevier, 2020) Thorn, Anna K.; Lin, Wei-Shao; Levon, John A.; Morton, Dean; Eckert, George J.; Lippert, Frank; Cariology, Operative Dentistry and Dental Public Health, School of DentistryObjectives This in vitro study investigated the effect of theobromine on the de- and remineralization of enamel carious lesions under plaque fluid-like conditions. Methods Early carious lesions were created in 272 bovine enamel specimens and assigned to sixteen groups (n = 17) based on Knoop surface microhardness (SMH). Lesions were demineralized again under plaque fluid-like conditions in the presence of fluoride (0.2 or 1 ppm) and theobromine (0; 10; 100 or 200 ppm) at different pH values (5.5 or 7.0) in a factorial design. SMH was determined again and percent SMH recovery (%SMHr) calculated. Three-way ANOVA was used for the fixed effects of fluoride, theobromine and pH levels to compare the differences between each level. Results The three-way interaction was not significant (p = 0.712). The two-way interaction between fluoride and pH was significant (p = 0.030), whereas those between fluoride and theobromine as well as that for pH and theobromine were not (p = 0.478 and p = 0.998, respectively). Theobromine did not affect %SMHr at any of the tested concentrations. There were trends for the higher fluoride concentration and the higher pH resulting in more rehardening with the lesions exposed to 0.2 ppm fluoride at pH 5.5 displaying significantly less rehardening than those exposed to 0.2 ppm fluoride at pH of 7.0 and lesions exposed to 1 ppm fluoride at pH of 5.5. Conclusion Theobromine, when continuously present in a plaque fluid-like medium at various concentrations and at different pH values, does not affect de- or remineralization of enamel carious lesions under the presently studied conditions.Item Effect of Violet-Blue Light on Streptococcus mutans-Induced Enamel Demineralization(MDPI, 2018-03-21) Felix Gomez, Grace Gomez; Lippert, Frank; Ando, Masatoshi; Zandona, Andrea Ferreira; Eckert, George J.; Gregory, Richard L.; Biomedical and Applied Sciences, School of DentistryBackground: This in vitro study determined the effectiveness of violet-blue light (405 nm) on inhibiting Streptococcus mutans-induced enamel demineralization. Materials and Methods: S. mutans UA159 biofilm was grown on human enamel specimens for 13 h in 5% CO2 at 37 °C with/without 1% sucrose. Wet biofilm was treated twice daily with violet-blue light for five minutes over five days. A six-hour reincubation was included daily between treatments excluding the final day. Biofilms were harvested and colony forming units (CFU) were quantitated. Lesion depth (L) and mineral loss (∆Z) were quantified using transverse microradiography (TMR). Quantitative light-induced fluorescence Biluminator (QLF-D) was used to determine mean fluorescence loss. Data were analyzed using one-way analysis of variance (ANOVA) to compare differences in means. Results: The results demonstrated a significant reduction in CFUs between treated and non-treated groups grown with/without 1% sucrose. ∆Z was significantly reduced for specimens exposed to biofilms grown without sucrose with violet-blue light. There was only a trend on reduction of ∆Z with sucrose and with L on both groups. There were no differences in fluorescence-derived parameters between the groups. Conclusions: Within the limitations of the study, the results indicate that violet-blue light can serve as an adjunct prophylactic treatment for reducing S. mutans biofilm formation and enamel mineral loss.Item Effects of PVP-Iodine pH and Calcium Concentration on Fluoride Varnish Anti-Caries Efficacy In Vitro(Quintessence, 2019) Aljamah, Ali F.; Hara, Anderson T.; Levon, John A.; Eckert, George J.; Lippert, Frank; Cariology, Operative Dentistry and Dental Public Health, School of DentistryPurpose: This laboratory study investigated the effects of PVP-iodine solutions with varying pH and calcium concentrations on enamel remineralization and fluoridation by subsequent treatment with fluoride varnish. Materials and Methods: Caries-like lesions were created in bovine enamel specimens (n = 15 per group) and characterized using Vickers surface microhardness (VHN). Specimens were treated with 10% PVP-iodine solutions varying in calcium concentration (0/10/100 mM) and pH (3.0/4.0/5.0), followed by 5% sodium fluoride varnish. A fluoride varnish-only control group was included. Specimens were then placed into artificial saliva for 16 h. The varnish was removed, hardness measured and enamel fluoride uptake (EFU) determined using the microbiopsy technique. Data were analyzed with one-way ANOVA. Results: Groups receiving PVP-iodine pre-treatments exhibited directionally greater rehardening (range: p = 0.0001 - 0.7008) and EFU (p = 0.0001-0.2670) than the control group. The presence of calcium in the pre-treatment enhanced rehardening. the groups '10mM Ca/pH 3.0' (∆VHN = 10.5 ± 6.3), '100mM Ca/pH 3.0' (∆VHN = 9.7 ± 4.1) and '10mM Ca/pH 5.0' (∆VHN = 8.7 ± 7.0) displayed the highest numerical gain vs the control (∆VHN=3.6±2.2). Different pH values had a minor effect on rehardening and EFU. The calcium effect was more pronounced for EFU than for rehardening with all three '100 mM Ca' groups exhibiting higher EFU (7.0 - 7.2 µg F/cm2) than all other groups (6.1 - 6.9 µg F/cm2). Conclusion: PVP-iodine pre-treatments can be modified to enhance the rehardening and fluoridating effect of fluoride varnishes, thereby potentially improving their ability to prevent caries in vivo. Although numerical differences between groups were small, the addition of high concentrations of calcium paired with a low pH appears most favorable under the present conditions.Item Fluoride Dentifrice Overcomes the Lower Resistance of Fluorotic Enamel to Demineralization(Karger, 2019) Almeida, L. F.; Marín, L. M.; Martínez-Mier, E. A.; Cury, J. A.; Cariology, Operative Dentistry and Dental Public Health, School of DentistryWe evaluated if the low resistance of fluorotic enamel to demineralization could be overcome by fluoride dentifrice (FD) treatment. Paired enamel slabs of sound and fluorotic enamel (n = 20/group) from human teeth presenting Thylstrup and Fejerskov index (TF) scores from 0 to 4 were obtained. Half of the anatomic surface of the enamel slabs was isolated and used as a control (baseline) regarding enamel mineralization and fluoride concentration. The slabs were submitted to a pH-cycling model simulating a high cariogenic challenge, and 2×/day they were treated with placebo dentifrice (PD) or FD (1,100 µg F/g, as NaF). After 10 days, the slabs were cut into two halves. Enamel demineralization was evaluated by cross-sectional microhardness in one half, and the fluoride formed (FF) concentration was determined in the other half. For statistical analysis, the data on net demineralization area (ΔΔS) and FF (µg F/g) were grouped into TF0, TF1–2, and TF3–4, and analyzed by two-way ANOVA followed by Tukey’s test (α = 5%). The factors studied were TF (0, 1–2, and 3–4) and dentifrice treatment (PD or FD). The effect of the factors was statistically significant for ΔΔS and FF (p < 0.05). In the PD group, the following pattern for ΔΔS was observed: TF3–4 > TF1–2 > TF0 (p < 0.05); however, the groups did not differ (p > 0.05) when FD was used. Regarding FF, the groups treated with PD did not differ (p > 0.05), but the greatest (p < 0.05) FF concentration was found in group TF3–4 treated with FD. These findings suggest that the higher susceptibility of fluorotic enamel to demineralization lesions is decreased by the use of FD.Item Fluoride Dose-Response of Human and Bovine Enamel Artificial Caries Lesions under pH-Cycling Conditions(Springer, 2015-11) Lippert, Frank; Juthani, Kalp; Department of Cariology, Operative Dentistry and Dental Public Health, School of DentistryObjectives This laboratory study aimed to (a) compare the fluoride dose-response of different caries lesions created in human and bovine enamel (HE/BE) under pH-cycling conditions and (b) investigate the suitability of Knoop and Vickers surface microhardness (K-SMH/V-SMH) in comparison to transverse microradiography (TMR) to investigate lesion de- and remineralization. Materials and methods Caries lesions were formed using three different protocols (Carbopol, hydroxyethylcellulose-HEC, methylcellulose-MeC) and assigned to 24 groups using V-SMH, based on a 2 (enamel types) × 3 (lesion types) × 4 (fluoride concentrations used during pH-cycling-simulating 0/250/1100/2800 ppm F as sodium fluoride dentifrices) factorial design. Changes in mineral content and structural integrity of lesions were determined before and after pH-cycling. Data were analyzed using three-way ANOVA. Results BE was more prone to demineralization than HE. Both enamel types showed similar responses to fluoride with BE showing more remineralization (as change in integrated mineral loss and lesion depth reduction), although differences between tissues were already present at lesion baseline. Carbopol and MeC lesions responded well to fluoride, whereas HEC lesions were almost inert. K- and V-SMH correlated well with each other and with the integrated mineral loss data, although better correlations were found for HE than for BE and for MeC than for Carbopol lesions. Hardness data for HEC lesions correlated only with surface zone mineral density data. Conclusion BE is a suitable surrogate for HE under pH-cycling conditions. Clinical relevance The in vitro modeling of dental caries is complex and requires knowledge of lesion behavior, analytical techniques, and employed hard tissues.
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