Surface porosity of enamel white spot lesions and penetration of fluorapatite nanocrystals into their subsurface: proof-of-concept study.

OBJECTIVES: In this study, we used atomic force microscopy (AFM) to quantify the size of surface pore apertures of enamel white spot lesions and then demonstrated the penetration of fluorapatite nanocrystals (nFA) into the subsurface of these lesions. METHODS: For the porosity study, enamel lesions were created on three sound human teeth using a demineralizing gel for 8 days. The interface between sound enamel and the artificial lesion was analyzed by AFM. To visualize the penetration of nFA tagged with a calcium-binding fluorophore (Fluo-4) into the subsurface of white spot lesions, we used two-photon microscopy. Sixteen extracted human teeth with either active, natural, or in vitro-created carious lesions in enamel were randomly divided into three groups. The teeth were treated for 2 min with either a suspension of tagged nFA crystals, Fluo-4 alone, or deionized water, and left for 30 min before being washed with distilled water and examined microscopically. RESULTS: A greater concentration of surface pores with larger areas was observed on the in vitro demineralized enamel (29 % of pores greater than 1.0 µm2) when compared with the adjacent sound enamel (8 % of pores greater than 1.0 µm2) (p=0.012, Fisher exact test). In vitro and natural lesions treated with tagged nFA showed fluorescence at depths ranging from 50 to 170 µm, demonstrating penetration of the nFA into the lesion subsurface. The lesions treated with Fluo-4 alone with no crystals showed mostly surface fluorescence (restricted to the outer 25 µm), while those treated with deionized water showed minimal (restricted to the outer 20 µm) to no fluorescence. CONCLUSION: We have demonstrated the use of AFM to quantify the surface pore apertures and two-photon microscopy to visualize nFA crystals in the subsurface of non-cavitated enamel lesions. CLINICAL SIGNIFICANCE: The restoration of the subsurface of non-cavitated caries lesions is a clinical challenge. This study demonstrated that a 2 min application of nFA could penetrate through the surface apertures of non-cavitated enamel lesions into their subsurface.

Measurement and analysis of microbial fluoride resistance in dental biofilm models.

The interactions between communities of microorganisms inhabiting the dental biofilm is a major determinant of oral health. These biofilms are periodically exposed to high concentrations of fluoride, which is present in almost all oral healthcare products. The microbes resist fluoride through the action of membrane export proteins. This chapter describes the culture, growth and harvest conditions of model three-species dental biofilm comprised of cariogenic pathogens Streptococcus mutans and Candida albicans and the commensal bacterium Streptococcus gordonii. In order to examine the role of fluoride export by S. mutans in model biofilms, procedures for generating a strain of S. mutans with a genetic knockout of the fluoride exporter are described. We present a case study examining the effects of this mutant strain on the biofilm mass, acid production and mineral dissolution under exposure to low levels of fluoride. These general approaches can be applied to study the effects of any gene of interest in physiologically realistic multispecies oral biofilms.

Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models.

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLCF F-/H+ antiporter and FEX fluoride channel, respectively, whereas oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride but to a lesser degree. Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time. Biochemical purification of the S. mutans CLCF transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity. IMPORTANCE: Dental caries is a globally prevalent condition that occurs when pathogenic species, including Streptococcus mutans and Candida albicans, outcompete beneficial species, such as Streptococcus gordonii, in the dental biofilm. Fluoride is routinely used in oral hygiene to prevent dental caries. Fluoride also has antimicrobial properties, although most microbes possess fluoride exporters to resist its toxicity. This work shows that sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms. These results suggest that the development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses. This is a novel strategy to treat dental caries.

Time-lapse confocal microscopy to study in vitro Streptococcus mutans surface colonization.

The cariogenicity of Streptococcus mutans relates to its ability to form biofilms on dental surfaces. The aim of this work was to develop a flowcell system compatible with time-lapse confocal microscopy to compare the adhesion and accumulation of S. mutans cells on surfaces in unsupplemented media against media containing sucrose or sucralose (a non-metabolized sweetener) over a short period of time. Fluorescent S. mutans 3209/pVMCherry was suspended in unsupplemented media or media supplemented with 1% sucrose or 1% sucralose and passed through a 3D-printed flowcell system. Flowcells were imaged over 60 minutes using a confocal microscope. Image analysis was performed, including a newly developed object-movement-based method to measure biomass adhesion. Streptococcus mutans 3209/pVMCherry grown in 1% sucrose-supplemented media formed small, dense, relatively immobile clumps in the flowcell system measured by biovolume, surface area, and median object centroid movement. Sucralose-supplemented and un-supplemented media yielded large, loose, mobile aggregates. Architectural metrics and per-object movement were significantly different (P < 0.05) when comparing sucrose-supplemented media to either unsupplemented or sucralose-supplemented media. These results demonstrate the utility of a flowcell system compatible with time-lapse confocal microscopy and image analysis when studying initial biofilm formation and adhesion under different nutritional conditions.

Fluoride export is required for competitive fitness of pathogenic microorganisms in dental biofilm models.

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLCF F-/H+ antiporter and FEX fluoride channel, respectively, whereas commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride, but to a lesser degree. Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time, and biochemical purification the S. mutans CLCF transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms, and that S. mutans is especially susceptible to fluoride toxicity.

Período de wash-out en diseños experimentales cruzados con dentífrico de alta concentración de fluoruro / Wash-out period for crossover design experiments using high fluoride concentration dentifrice

OBJETIVO La mayoría de los estudios cruzados con dentífrico fluoretado (DF) de concentración estándar (1.000-1.500 ppm F) han empíricamente utilizado un periodo de wash-out de 7 días para eliminar el efecto residual del tratamiento. Para DF de alta concentración (5.000 ppm F) este periodo es desconocido y sería necesario un tiempo mayor para la remoción de fluoruro (F) de la saliva. Este estudio verificó si menos de 7 días sería suficiente para eliminar el F residual de la saliva después de uso DF de 5.000 ppm F. METODOLOGÍA Estudio in vivo, análisis ciego, donde voluntarios (n = 6) cepillaron sus dientes 3 veces por día en la secuencia: a) periodo inicial o lead-in de 3 días con uso de dentífrico placebo de fluoruro (DP) (0 ppm F); b) uso de DF de alta concentración (5.000 ppm F) por 4 días; y c) wash-out con uso de DP por 3 días. Durante los 3 periodos, saliva estimulada y no estimulada fue colectada en ayuno (después del periodo overnight del último cepillado). La concentración de F en la saliva fue evaluada utilizando electrodo específico. RESULTADOS El F en la saliva después de suspendido el uso de DF (periodo de wash-out) fue similar a los valores basales. Concentraciones de F no presentaron diferencias entre saliva estimulada y no estimulada. CONCLUSIÓN Dos días de wash-out con dentífrico no fluorado fueron suficientes para eliminar F residual en la saliva después de haber utilizado dentífrico de alta concentración. Estos resultados son válidos también para dentífrico de concentración estándar.

OBJECTIVE Most crossover studies using fluoride dentifrice (FD) of standard concentration (1000-1500 ppm F) have empirically used a wash-out period of 7 days to remove the residual effect of the treatment. For higher concentrations of FD (5000 ppm F) the period is unknown, and a longer time may be required to remove fluoride (F) from saliva. Therefore, the aim of the study was to determine if less than 7 days of wash-out would be sufficient to remove residual F in saliva after using 5000 ppm F FD. METHODOLOGY An in vivo study, blind analysis, was conducted on volunteers (n = 6) who brushed their teeth 3 times per day in the following sequence: a) initial or lead-in period of 3 days using placebo fluoride dentifrice (PD) (0 ppm F); b) using a high concentration FD (5000 ppm F) for 4 days; and c) wash-out using PD for 3 days. During the 3 periods, samples of non-stimulated and stimulated saliva were collected after fasting (one overnight period from the last brushing). Fluoride concentration was assessed in saliva using a fluoride specific electrode. RESULTS F concentrations in saliva after discontinued use of FD (wash-out period of 2 and 3 days) were similar to baseline values. F concentrations did not differ between unstimulated and stimulated saliva. CONCLUSION A two day wash-out period using non-fluoridated dentifrice was sufficient to eliminate residual F in saliva after use of a high concentration F dentifrice. These results are also valid for standard concentrations of dentifrice.