Effect of titanium dioxide on Streptococcus mutans biofilm.

BACKGROUND: Streptococcus mutans (S. mutans) participates in the dental caries process. Titanium dioxide (TiO2) nanoparticles produce reactive oxygen species capable of disrupting bacterial DNA synthesis by creating pores in cell walls and membranes. OBJECTIVE: The objective of this study was to determine the effect of TiO2 on the disruption of S. mutans biofilm. METHODS: This study was conducted in four phases involving a TiO2-containing toothbrush and TiO2 nanoparticles. Each phase was completed using 24 h established S. mutans biofilm growth. Phase one data was collected through a bacterial plating study, assessing biofilm viability. Biofilm mass was evaluated in phase two of the study by measuring S. mutans biofilm grown on microtiter plates following crystal violet staining. The third phase of the study involved a generalized oxygen radical assay to determine the relative amount of oxygen radicals released intracellularly. Phase four of the study included the measurement of insoluble glucan/extracellular polysaccharide (EPS) synthesis using a phenol-sulfuric acid assay. RESULTS: Both exposure time and time intervals had a significant effect on bacterial viability counts (p = 0.0323 and p = 0.0014, respectively). Bacterial counts after 6 min of exposure were significantly lower than after 2 min (p = 0.034), compared to the no treatment control (p = 0.0056). As exposure time increased, the amount of remaining biofilm mass was statistically lower than the no treatment control. Exposure time had a significant effect on oxygen radical production. Both the 30 and 100 nm TiO2 nanoparticles had a significant effect on bacterial mass. The silver nanoparticles and the 30 and 100 nm TiO2 nanoparticles significantly inhibited EPS production. CONCLUSION: The TiO2-containing toothbrush kills, disrupts, and produces oxygen radicals that disrupt established S. mutans biofilm. TiO2 and silver nanoparticles inhibit EPS production and reduce biofilm mass. The addition of TiO2 to dental products may be effective in reducing cariogenic dental biofilm.

The influence of biofilm maturation on fluoride's anticaries efficacy.

OBJECTIVES: (1) To explore the influence of biofilm maturation and timing of exposure on fluoride anticaries efficacy and (2) to explore biofilm recovery post-treatment. METHODS: Bovine enamel specimens were utilized in a pH cycling model (28 subgroups [n = 18]). Each subgroup received different treatments [exposure]: sodium fluoride [NaF]; stannous fluoride [SnF2]; amine fluoride [AmF]; and de-ionized water [DIW], at a specific period: early: days 1-4; middle: days 3-6; and late: days 7-10. During non-exposure periods, pH cycling included DIW instead of fluorides. Objective 1: part 1 (cycling for 4, 6, or 10 days). Part 2 (cycling for 10 days). Objective 2: early exposure: three sample collection time points (immediate, 3 days, and 6 days post-treatment); middle exposure: two sample collection time points (immediate, 4 days post-treatment). The enamel and biofilm were analyzed ([surface microhardness; mineral loss; lesion depth]; [lactate dehydrogenase enzyme activity; exopolysaccharide amount; viability]). Data were analyzed using ANOVA (p = 0.05). RESULTS: Objective 1: Early exposure to fluorides produced protective effects against lesion progression in surface microhardness and mineral loss, but not for lesion depth. Objective 2: Early exposure slowed the demineralization process. SnF2 and AmF were superior to NaF in reducing LDH and EPS values, regardless of exposure time. They also prevented biofilm recovery. CONCLUSION: Earlier exposure to SnF2 and AmF may result in less tolerant biofilm. Early fluoride treatment may produce a protective effect against demineralization. SnF2 and AmF may be the choice to treat older biofilm and prevent biofilm recovery. CLINICAL RELEVANCE: The study provides an understanding of biofilm-fluoride interaction with mature biofilm (e.g., hard-to-reach areas, orthodontic patients) and fluoride's sustainable effect hours/days after brushing.

In-Vitro Model of Scardovia wiggsiae Biofilm Formation and Effect of Nicotine.

Recently, Scardovia wiggsiae has been reported to be strongly associated with caries formation. This study aimed to establish an in vitro model of S. wiggsiae biofilm and to investigate the effect of nicotine on S. wiggsiae colony-forming units (CFUs) growth. S. wiggsiae biofilm was grown overnight using brain-heart infusion (BHI) broth supplemented with 5 g of yeast extract/L (BHI-YE). The overnight culture was used as an inoculum to grow S. wiggsiae biofilm on standardized enamel and dentin samples. Samples were incubated with different nicotine concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL) for 3 days. The dissociated biofilms were diluted, spiral plated on blood agar plates, and incubated for 24 h. CFUs/mL were quantified using an automated colony counter. A two-way ANOVA was used to compare the effect of different nicotine concentrations on S. wiggsiae CFUs. This study demonstrated that S. wiggsiae biofilm could be initiated and formed in vitro. Increased CFUs was observed through 0.5-4 mg/mL and 0.5-8 mg/mL of nicotine using enamel and dentin substrates, respectively. 16 and 32 mg/mL of nicotine were determined as the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively. S. wiggsiae formed greater biofilm on enamel than dentin specimens in response to the nicotine stimulus. This study demonstrated the negative effect of smoking on increasing S. wiggsiae biofilm. Establishing S. wiggsiae biofilm in vitro may allow researchers in the future to have a better understanding of caries pathogenesis and bacterial interaction.

In-vitro model of scardovia wiggsiae biofilm formation and effect of nicotine

Abstract Recently, Scardovia wiggsiae has been reported to be strongly associated with caries formation. This study aimed to establish an in vitro model of S. wiggsiae biofilm and to investigate the effect of nicotine on S. wiggsiae colony-forming units (CFUs) growth. S. wiggsiae biofilm was grown overnight using brain-heart infusion (BHI) broth supplemented with 5 g of yeast extract/L (BHI-YE). The overnight culture was used as an inoculum to grow S. wiggsiae biofilm on standardized enamel and dentin samples. Samples were incubated with different nicotine concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL) for 3 days. The dissociated biofilms were diluted, spiral plated on blood agar plates, and incubated for 24 h. CFUs/mL were quantified using an automated colony counter. A two-way ANOVA was used to compare the effect of different nicotine concentrations on S. wiggsiae CFUs. This study demonstrated that S. wiggsiae biofilm could be initiated and formed in vitro. Increased CFUs was observed through 0.5-4 mg/mL and 0.5-8 mg/mL of nicotine using enamel and dentin substrates, respectively. 16 and 32 mg/mL of nicotine were determined as the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively. S. wiggsiae formed greater biofilm on enamel than dentin specimens in response to the nicotine stimulus. This study demonstrated the negative effect of smoking on increasing S. wiggsiae biofilm. Establishing S. wiggsiae biofilm in vitro may allow researchers in the future to have a better understanding of caries pathogenesis and bacterial interaction.

Resumo Recentemente, foi relatado que Scardovia wiggsiae está fortemente associado à formação de cáries. Este estudo teve como objetivo estabelecer um modelo in vitro de biofilme de S. wiggsiae e investigar o efeito da nicotina no crescimento de unidades formadoras de colônias (UFC) de S. wiggsiae. O biofilme de S. wiggsiae foi cultivado durante a noite usando caldo de infusão de cérebro-coração (BHI) suplementado com 5 g de extrato de levedura / L (BHI-YE). A cultura noturna foi usada como um inóculo para cultivar biofilme de S. wiggsiae em amostras padronizadas de esmalte e dentina. As amostras foram incubadas com diferentes concentrações de nicotina (0, 0,5, 1, 2, 4, 8, 16 e 32 mg/mL) por 3 dias. Os biofilmes dissociados foram diluídos, semeados em espiral em placas de ágar sangue e incubados por 24 h. UFC/mL foram quantificados usando um contador de colônias automatizado. Uma ANOVA de duas vias foi usada para comparar o efeito de diferentes concentrações de nicotina em UFCs de S. wiggsiae. Este estudo demonstrou que o biofilme de S. wiggsiae pode ser iniciado e formado in vitro. UFCs aumentadas foram observadas com 0,5-4 mg/mL e 0,5-8 mg/mL de nicotina usando substratos de esmalte e dentina, respectivamente. A concentração inibitória mínima (CIM) e a concentração bactericida mínima (CBM) de nicotina foram determinadas, respectivamente, como 16 e 32 mg/mL. S. wiggsiae formou maior biofilme no esmalte do que espécimes de dentina em resposta ao estímulo de nicotina. Este estudo demonstrou o efeito negativo do tabagismo no aumento do biofilme de S. wiggsiae. O estabelecimento do biofilme de S. wiggsiae in vitro pode permitir que os pesquisadores no futuro tenham uma melhor compreensão da patogênese da cárie e da interação bacteriana.

The anti-caries efficacy of three fluoride compounds at increasing maturation of a microcosm biofilm.

OBJECTIVE: To explore the anti-caries efficacy of three fluoride compounds at increasing maturation of a microcosm biofilm. DESIGN: Microcosm biofilm, obtained from saliva collected from three donors (IRB #1406440799), was grown on enamel samples (n = 18/group) for 24-h (Brain Heart Infusion; 0.2 % sucrose). Then, pH cycling model started. Three maturations were explored (4d, 8d, and 12d). The pH cycling consisted of daily 2 × 5 min treatments (NaF, SnF2, AmF: 287.5 ppm F, and de-ionized water [DIW]), 4 × 10 min remineralization (BHI, no sucrose, pH 7.0), and 3 × 2:15 h demineralization (BHI, 1% sucrose, pH 4.5). We analyzed the enamel (surface microhardness [VHNchange], integrated mineral loss [ΔZ], lesion depth [L]), and the biofilm (viability [log10 CFU/mL], lactic acid production [LDH], and exopolysaccharide [EPS] amount). Data were analyzed using two-way ANOVA (p = 0.05). RESULTS: The interaction between tested variables was significant for VHNchange, viability, LDH, EPS (p = 0.0354, p = 0.0001, p < 0.0001, p < 0.0001), but not for L (p = 0.2412) or ΔZ (p = 0.6811). LDH and EPS analyses exhibited more tolerance of mature biofilm against NaF (LDH and EPS p < 0.0001); NaF-treated groups demonstrated significantly lower results than the control in the 12d group. The effect of SnF2 and AmF continued over time. VHNchange, L, and ΔZ: The effect of SnF2 and AmF was higher than NaF and DIW. L and ΔZ did not result in significant differences over time (all treatments). Within each maturation, fluoride compounds demonstrated statistically significantly lower L and ΔZ values than DIW. CONCLUSIONS: Biofilm's maturation may influence the selection of fluoride compounds to achieve an optimum cariostatic effect.

Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization.

INTRODUCTION: The acquired pellicle formation is the first step in dental biofilm formation. It distinguishes dental biofilms from other biofilm types. OBJECTIVE: To explore the influence of salivary pellicle formation before biofilm formation on enamel demineralization. METHODOLOGY: Saliva collection was approved by Indiana University IRB. Three donors provided wax-stimulated saliva as the microcosm bacterial inoculum source. Acquired pellicle was formed on bovine enamel samples. Two groups (0.5% and 1% sucrose-supplemented growth media) with three subgroups (surface conditioning using filtered/pasteurized saliva; filtered saliva; and deionized water (DIW)) were included (n=9/subgroup). Biofilm was then allowed to grow for 48 h using Brain Heart Infusion media supplemented with 5 g/l yeast extract, 1 mM CaCl2.2H2O, 5% vitamin K and hemin (v/v), and sucrose. Enamel samples were analyzed for Vickers surface microhardness change (VHNchange), and transverse microradiography measuring lesion depth (L) and mineral loss (∆Z). Data were analyzed using two-way ANOVA. RESULTS: The two-way interaction of sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange (p=0.0079), while sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001). Surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values for both sucrose concentrations. The differences between filtered/pasteurized subgroups and the two other surface conditionings were significant (filtered saliva p=0.006; DIW p=0.0075). Growing the biofilm in 1% sucrose resulted in lesions with higher ∆Z and L values when compared with 0.5% sucrose. The differences in ∆Z and L between sucrose concentration subgroups was significant, regardless of surface conditioning (both p<0.0001). CONCLUSION: Within the study limitations, surface conditioning using human saliva does not influence biofilm-mediated enamel caries lesion formation as measured by transverse microradiography, while differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm-mediated demineralization of the enamel surface.

Comparison of human and bovine enamel in a microbial caries model at different biofilm maturations.

OBJECTIVES: To compare human versus bovine enamel when used in microbial caries models; and to evaluate the use of nylon mesh to support biofilm growth over enamel. METHODS: Twenty-four sub-subgroups were included (time factor: 4, 8, and 12 days; substrate factor: human/bovine; mesh factor: yes/no; treatment factor: 18.4 mM NaF (350 ppm F), de-ionized water [DIW]; n = 9/sub-subgroup). Microcosm biofilm from human saliva (IRB approval #1,406,440,799) was grown on enamel specimens for 24-h (Brain Heart Infusion media; 0.2 % sucrose), using active attachment model. Then, pH-cycling took place. At the end of each pH-cycling period, enamel specimens were analyzed: surface microhardness (VHNchange); transverse microradiography (integrated mineral loss [ΔZ], lesion depth [L]). Biofilm was analyzed: lactic acid production (LDH activity); exopolysaccharide (EPS) amount; and viability (12-day sub-groups). Data were analyzed using ANOVA at a 5 % level of significance. RESULTS: The three-way interaction between pH-cycling duration, substrate type, and treatment type was significant for (VHNchange [p < 0.0005], ΔZ [p = 0.0027], and L [p < 0.0001]). VHNchange exhibited increased lesion severity as pH-cycling time increases, in both treatments. VHNchange data indicated a treatment effect in all timepoints. ΔZ and L exhibited higher values with more mature biofilms. ANOVA analyses for LDH and EPS indicated a significance between variables (LDH p = 0.0100; EPS p < 0.0001). Mesh-covered specimens resulted in lower LDH and EPS values in all maturations. ANOVA analyses of viability (12 days) between variables was significant. CONCLUSION: within the study's limitations, human or bovine enamel can be used in microbial in vitro caries models to study biofilm's maturation and anticaries agents. CLINICAL SIGNIFICANCE: This study demonstrated how a known cariostatic effect of a fluoride concentration in toothpastes can be modulated by the maturation stage of oral biofilm. This can represent hard to reach areas in the oral cavity (e.g. in orthodontic patients or patients with intermaxillary fixation following oral and maxillofacial surgeries).

Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization

Abstract The acquired pellicle formation is the first step in dental biofilm formation. It distinguishes dental biofilms from other biofilm types. Objective To explore the influence of salivary pellicle formation before biofilm formation on enamel demineralization. Methodology Saliva collection was approved by Indiana University IRB. Three donors provided wax-stimulated saliva as the microcosm bacterial inoculum source. Acquired pellicle was formed on bovine enamel samples. Two groups (0.5% and 1% sucrose-supplemented growth media) with three subgroups (surface conditioning using filtered/pasteurized saliva; filtered saliva; and deionized water (DIW)) were included (n=9/subgroup). Biofilm was then allowed to grow for 48 h using Brain Heart Infusion media supplemented with 5 g/l yeast extract, 1 mM CaCl2.2H2O, 5% vitamin K and hemin (v/v), and sucrose. Enamel samples were analyzed for Vickers surface microhardness change (VHNchange), and transverse microradiography measuring lesion depth (L) and mineral loss (∆Z). Data were analyzed using two-way ANOVA. Results The two-way interaction of sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange (p=0.0079), while sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001). Surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values for both sucrose concentrations. The differences between filtered/pasteurized subgroups and the two other surface conditionings were significant (filtered saliva p=0.006; DIW p=0.0075). Growing the biofilm in 1% sucrose resulted in lesions with higher ∆Z and L values when compared with 0.5% sucrose. The differences in ∆Z and L between sucrose concentration subgroups was significant, regardless of surface conditioning (both p<0.0001). Conclusion Within the study limitations, surface conditioning using human saliva does not influence biofilm-mediated enamel caries lesion formation as measured by transverse microradiography, while differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm-mediated demineralization of the enamel surface.

The Use of Fluorescence Technology versus Visual and Tactile Examination in the Detection of Oral Lesions: A Pilot Study.

PURPOSE: This study compared the effectiveness of the VELscope® Vx versus visual and tactile intraoral examination in detecting oral lesions in an adult, high risk population. METHODS: The pilot study compared the intra oral findings between 2 examination types. The sample was comprised of 30 participants who were addicted to either cigarettes or a dual addiction (cigarettes plus hookah). High risk population was defined as males who were current cigarette smokers or had a dual addiction. Two trained and experienced licensed dental hygienists conducted all examinations. Throughout the study, all visual and tactile intraoral examinations were conducted first by one dental hygienist first, followed by the VELscope® Vx fluorescence examinations by the second dental hygienist. All subjects received an inspection of the lips, labial and buccal mucosa, floor of the mouth, dorsal, ventral and lateral sides of the tongue, hard and soft palate, and visual inspection of the oropharynx and uvula. Both evaluations took place in 1 visit in the Dental Hygiene Research Center at Old Dominion University and external sites. All participants received oral cancer screening information, recommendations, referrals for tobacco cessation programs and brochures on the 2 types of examinations conducted. RESULTS: Participants were considered high risk based on demographics (current smokers and mostly males). Neither visual and tactile intraoral examination nor the VELscope® Vx examination showed positive lesions. No lesions were detected; therefore, no referrals were made. Data indicated the duration of tobacco use was significantly higher in cigarette smokers (14.1 years) than dual addiction smokers (5 years) (p>0.005). The average numbers of cigarettes smoked per day were 13.5 compared to 14.2 cigarettes for dual addiction smokers. CONCLUSION: Results from this study suggest the visual and tactile intraoral examination produced comparative results to the VELscope® Vx examination. Findings from this study support that the VELscope® Vx is still considered an adjunct technology and cannot be used exclusively for oral cancer screening.