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Toothpastes are the most universally accepted form of fluoride delivery for caries prevention. To provide anti-caries benefits, they must be able to release fluoride during the time of tooth brushing or post brushing into the oral cavity. However, there is no standard accepted procedure to measure how much fluoride in a toothpaste may be (bio) available for release. The European Organization for Caries Research proposed and supported a workshop with experts in fluoride analysis in toothpastes and representatives from industry. The objective of the workshop was to discuss issues surrounding fluoride analysis in toothpaste and reach consensus on terminology and best practices, wherever the available evidence allowed it. Participants received a background paper and heard presentations followed by structured discussion to define the problem. The group also reviewed evidence on the validity, reliability and feasibility of each technique (namely chromatography and fluoride electroanalysis) and discussed their strengths and limitations. Participants were able to reach a consensus on terminology and were also able to identify and summarize the advantages and disadvantages of each technique. However, they agreed that most currently available methods were developed for regulatory agencies several decades ago, utilizing the best available data from clinical trials then, but require to be updated. They also agreed that although significant advances to our understanding of the mechanism of action of fluoride in toothpaste have been achieved over the past 4 decades, this clearly is an extraordinarily complex subject and more work remains to be done.

Background: Clinical studies on the caries-preventive properties of chlorhexidine mouthrinses are limited and the results are inconclusive. Aim: The aim of this study was to elucidate the contribution of a 0.2% chlorhexidine mouthrinse to the protection of enamel and dentine against demineralization. Methods: In this randomized two-treatment, two-leg study 14 individuals wearing partial prostheses were enrolled. Sound enamel and dentine specimens were placed in situ for 4 weeks. Twice daily, a mouthrinse was performed with either chlorhexidine or saline (control) depending on the experimental group the participant was allocated to. After the experimental period, plaque samples were collected from the surface of the specimens and from natural tooth surfaces to assess the organic acid production upon a sucrose challenge. The specimens were analyzed for mineral loss by transversal microradiography. Results: This study could not demonstrate a significantly better protection of enamel and dentine against demineralization by the chlorhexidine treatment compared to saline. No differences in acid production of plaque samples from the chlorhexidine-treated and control groups were observed. This result was also found for plaque samples originating from the natural tooth surfaces. Conclusion: Mouth rinsing with 0.2% chlorhexidine did not prevent demineralization of dentine and enamel in situ.

The aim of this clinical study was to evaluate the effect of various rinsing protocols on oral acid production 6 h after tooth brushing with an amine fluoride/stannous fluoride (AmF/SnF2) toothpaste. After a 14-day period of using F-free toothpaste, 30 participants followed three experimental protocols each, followed by F-free washout periods in a randomized crossover trial. They used AmF/SnF2 toothpaste twice daily for 1 week, and after brushing, they either rinsed with tap water, omitted the post-brush rinse, or rinsed with an AmF/SnF2 mouthwash. In the F-free washout periods, the participants brushed their teeth without further instructions. Six hours after the last brushing (± rinsing) of each period, subjects rinsed with 10 ml 10% sucrose solution for 2 min. A tongue film sample and a buccal plaque sample were taken 4 and 8 min after the sucrose challenge, respectively. Metabolic acid ions were determined by capillary electrophoresis. The results show that (1) omitting the post-brush water rinse did not reduce the production of lactic, acetic or minor acids in plaque, nor on the tongue, and that (2) the additional use of AmF/SnF2 mouthwash after brushing reduced the acid production in plaque and tongue samples for at least 6 h. The distributions of acids produced in the plaque or tongue samples were not statistically different between experimental periods. It is concluded that an increase in the antimetabolic effect of AmF/SnF2 toothpaste in between two daily brushing exercises is not achieved by omitting the post-brush water rinse. The additional use of AmF/SnF2 mouthwash after brushing is effective in reducing the acid metabolism in dental plaque and tongue flora.

The relationship between the numbers of salivary mutans streptococci and the acid production in dental plaque after a single application of the 40% chlorhexidine varnish EC40® has been studied. Thirteen healthy subjects were treated with EC40 varnish. Saliva samples were taken before and up to 12 weeks after treatment to count mutans streptococci and lactobacilli. At the same time points plaque samples were taken before and after sucrose challenge and analyzed for protein and organic acid. Suppression of salivary mutans streptococci was observed together with a reduced production of lactic acid in sucrose-challenged dental plaque in 9 subjects while inhibition of acid production without significant suppression of mutans streptococci was observed in the other 4 participants. The duration of the effects differed among the individuals but never exceeded 6 weeks. We conclude that a prolonged suppression of mutans streptococci and acid production was not achieved by a single treatment with EC40 varnish in all subjects. Moreover, reduced acidogenicity of dental plaque after chlorhexidine treatment was not necessarily predicted by suppression of mutans streptococci in saliva.

It has been shown that there is a window of infectivity for mutans streptococci between the ages of 19 and 31 months, when many children acquire mutans streptococci transmitted from their mothers. Part of the children that escape this window acquire mutans streptococci at a later age. In this group, maternal transmission is expected to be less prevalent. The present study compared the bacteriocin activity profiles of mutans streptococci isolated from mothers, fathers and children when the children acquired the mutans streptococci between the ages of 5 and 11. Twelve families were randomly selected from a group of 11–year–old children who were known to have acquired mutans streptococci during this age period. From the saliva of the mothers (n = 12), fathers (n = 8) and children (n = 12) approximately 30 mutans streptococci strains were isolated. All isolates were tested twice for bacteriocin activity against 21 indicator strains with a double–layer technique. Bacteriocin activity of strains was considered to be different when the number of strains against which bacteriocin was produced differed >1 or when the width of one or more inhibition zones differed ≥4 mm. In 7/12 mother–child pairs similar profiles were found. In the 8 father–child pairs similar profiles were only found on 2 occasions. In these 2 families, all 3 ( mother, father and child) harboured strains with a similar profile. In 4/8 father–mother pairs similar profiles were found. There was no correlation between the prevalence of mutans streptococci strains, the number of indicator strains against which the strains made bacteriocin, nor the mean size of the inhibition zones and the presence of similarity of bacteriocin activity profiles of mutans streptococci within the family members. The results show that even when a child acquires mutans streptococci after the age of 5, there may be similarity between mutans streptococci in mother, father and child, indicating that transmission between the family members occurs.

The aim of this in situ investigation was to study the effect of an amine fluoride/sodium fluoride mouthrinse (total F = 250 ppm) in addition to an amine fluoride dentifrice (F = 1,250 ppm) on the amount of acquired fluoride in enamel and dentine. In the partial prosthesis of 12 participants a combined specimen of slightly demineralized enamel and dentine was placed. During two consecutive experiments, each lasting 3 weeks, the participants used an amine fluoride dentifrice alone or in combination with a fluoride mouthrinse. After the in situ period the specimens were retrieved and both the enamel and the dentine specimens were analysed for the amount of KOH–soluble fluoride and structurally bound fluoride. The results showed a significant increase in both KOH–soluble and structurally bound fluoride in enamel and dentine when a fluoride mouthrinse was used. Whether the rinsing procedure was performed immediately after toothbrushing or with a delay of 2 h did not influence the results. Furthermore it was shown that dentine acquired substantially more fluoride than enamel during the experimental period. The results indicate that a fluoride mouthrinse used in addition to a fluoride dentifrice may have a beneficial effect on the protection of enamel and dentine against caries.

Demineralization of dentin in the presence of fluoride produces lesions with a mineralized surface layer which becomes thicker and more mineralized with higher fluoride concentrations whereas the lesion depth is hardly affected. The aim of this study was to investigate the effects of the time of fluoride treatment and the amount of fluoride taken up on the properties of the mineralized layer. Discs of bovine dentin embedded in methylmethacrylate with one surface exposed were demineralized in 50 mM acetic acid, 2.2 mM CaCl2, 2.2 mM KH2PO4, pH 5.0. At the start and/or later during the demineralization period, the specimens were incubated individually for 1 or 2 days in 10 ml of the same demineralization solution supplemented with 0.5, 2.0 or 5.0 ppm fluoride, which was then assessed for changes in calcium and fluoride concentrations. After 2, 5 and 8 days, specimens were sectioned for microradiographic analysis so as to follow development of the lesions and the mineralized layers. The results were the following: While demineralization with fluoride present at the first day led to the formation of a surface layer, fluoride present only at a later day produced a subsurface layer, not at the lesion front but closer to the surface. This layer resulted from (re)precipitation and not from preservation of the original mineral. The 'integrated mineral content' of the surface layer increased linearly with the uptake of fluoride, which resulted in an apparent fluorapatite content of about 20 vol%. The profiles of the surface layers remained unchanged during continued demineralization in the absence of fluoride. It was concluded that in the presence of fluoride mineral loss is reduced as a result of the reprecipitation of dissolved mineral ions as a layer of fluoride–enriched apatite. This layer does not offer protection of underlying dentin against continued demineralization.

Bovine enamel and dentin specimens were overlaid with acidogenic Streptococcus mutans suspensions in agarose. In this model, the minimal demineralisation-inhibiting concentrations (MDIC) of hexetidine was determined in the presence of fluoride. A commercially available mouthwash containing 0.1% (2.9 mmol/l) hexetidine was diluted serially and added to the bacterial suspensions together with 0, 5.3, or 26.3 mumol/l fluoride (NaF). After 22 h of incubation at 37 degrees C the bacterial suspensions were removed and assessed for calcium and lactate. The results showed significant inhibitory effects of hexetidine on the demineralisation of the enamel specimens with a MDIC between 15 and 31 mumol/l hexetidine. In the presence of fluoride, approximately fourfold higher concentrations of hexetidine were needed for a significant additional protection of the enamel. No synergistic effect between hexetidine and fluoride was observed. For the demineralisation of the dentin specimens, the MDIC of hexetidine had a value between 31 and 61 mumol/l. At both these concentrations the dentin specimens were relatively less protected in the presence than in the absence of fluoride, and some synergistic effect between hexeditine and fluoride was observed.

Background: The treatment of polymicrobial biofilms with antimicrobial compounds results in not only an overall loss of viability, but also compositional shifts. While DNA-based technologies may be more appropriate for the assessment of bacterial composition than culturing, these techniques amplify DNA from both live and dead cells. Propidium monoazide (PMA) has been used to discriminate between live and dead cells by blocking the DNA from membrane-damaged cells from being amplified. Aim: This study evaluated the use of PMA in a saliva-derived polymicrobial biofilm model subjected to a single chlorhexidine (CHX) treatment. Materials and Methods: The effects of PMA on viable cells were tested using both untreated and PMA-treated saliva as an inoculum. Viability was determined by plate counts, metabolic activity was determined by lactic acid production, and biofilm composition was assessed by 16S rRNA gene amplicon sequencing. Results: Exposure to a 0.2% CHX rinse (meridol® perio) reduced the viability and metabolic activity of 48-hour biofilms. The shift in biofilm composition observed after the CHX exposure was enhanced after a post-rinse PMA treatment. PMA treatment had a small effect on the measured composition of water-rinsed biofilms. Treating saliva with PMA reduced bacterial viability and shifted the bacterial composition of saliva and saliva-derived biofilms. Conclusion: The removal of DNA from non-viable cells with PMA treatment was shown to elicit an improvement in the detection of shifts in in vitro polymicrobial biofilms after antimicrobial treatment. However, PMA also influenced the ability of cells to grow, indicating that PMA should be used with caution.

Although fluoride toothpastes are widely used for caries prevention, little is known about the impact of fluoride dentifrices on plaque composition. Also the issue of adaptation of mutans streptococci to grow in vivo in a fluoride environment has received little attention. Such an adaptation may be of interest as it has been suggested that adapted mutans streptococci may show reduced glycolytic activity thereby being less cariogenic. In the present experiments the impact of the suspension of the use of fluoride toothpastes on plaque composition, fluoride tolerance and acid production of mutans streptococci was studied. Pooled plaque samples from the lingual surfaces of the lower incisors were collected from individuals (n = 13) just before and 7 weeks after they had replaced their fluoride toothpastes (0.1-0.15% F) with a non-fluoride one. The samples were analysed for fluoride and the numbers and proportions of streptococci, Streptococcus mutans, Streptococcus sobrinus, Actinomyces species, and lactobacilli, respectively. The fluoride tolerance of the mutans streptococci was estimated by culture of the plaque samples on TYCSB agar supplemented with of 0, 1, 2, 3, 4, and 5 mmol/l fluoride (NaF) at pH 7.2. From each plaque sample six S. mutans strains were isolated for the measurement of the rate of acid production (Vap) at pH 7 in the presence of 0, 5, and 10 mmol/l F. The overnight final pH was measured in cultures of the S. mutans strains with excess of glucose and 0, 5, and 10 mmol/l F. The results showed that the removal of the fluoride pressure from plaque did not affect the numbers or proportions of the various species and genera of bacteria. The fluoride tolerance of the mutans streptococci, and the Vap or the overnight final pH of the isolated strains had not changed. These results suggest that the use of fluoride toothpaste had not affected plaque composition, nor fluoride tolerance or acidogenicity of mutans streptococci. Probably the amount of fluoride delivered by fluoride dentifrices to dental plaque is too low to induce such adaptations.

Clinical studies on the caries-preventive properties of chlorhexidine mouthrinses are limited and the results are inconclusive.The aim of this study was to elucidate the contribution of a 0.2% chlorhexidine mouthrinse to the protection of enamel and dentine against demineralization.In this randomized two-treatment, two-leg study 14 individuals wearing partial prostheses were enrolled. Sound enamel and dentine specimens were placed in situ for 4 weeks. Twice daily, a mouthrinse was performed with either chlorhexidine or saline (control) depending on the experimental group the participant was allocated to. After the experimental period, plaque samples were collected from the surface of the specimens and from natural tooth surfaces to assess the organic acid production upon a sucrose challenge. The specimens were analyzed for mineral loss by transversal microradiography.This study could not demonstrate a significantly better protection of enamel and dentine against demineralization by the chlorhexidine treatment compared to saline. No differences in acid production of plaque samples from the chlorhexidine-treated and control groups were observed. This result was also found for plaque samples originating from the natural tooth surfaces.Mouth rinsing with 0.2% chlorhexidine did not prevent demineralization of dentine and enamel in situ.

This study addressed the dose response between fluoride toothpastes and in vitro de- and remineralization, to predict the efficacy of toothpastes and understand the mode of action in the range 0–3,000 ppm F. Enamel lesions were pH-cycled with calcium uptake and loss being assessed daily. Both ‘shallow’ (about 50 µm deep) and ‘deep’ (about 200 µm deep) lesions were studied. F treatments were given in 30 (w/v)% toothpaste dilutions for up to 5 min daily. Calcium loss during the demineralization periods showed a dose response, resulting in 72% reduction for 3,000 ppm F compared to 0 ppm F. Calcium uptake during remineralization was increased in the F compared to non-F groups, with F concentration being less important than its mere presence. Significant differences were observed in F response between shallow and deep lesions, suggesting that this parameter should be included when testing caries-preventive products. Microradiographic analysis showed that lesion depth and severity had increased significantly in the non-F groups. In the F groups, the original lesion was partly remineralized, while a new lesion had formed beyond the original lesion front. Mineral loss of this second lesion correlated inversely with the F concentration of the treatments. These data revealed that fluoride can drive demineralization further into enamel by making the surface tissue less soluble, hence by not neutralizing acids penetrating into the tissue. It is also concluded that depth analysis of mineral uptake and loss is important to understand the mode of action of different F products.

Compared with the knowledge accumulated on enamel-fluoride interactions, relatively little data is available regarding fluoride effects on dentin. This applies to both laboratory and clinical studies into the efficacy of fluoride schemes for the prevention of root surface caries. This study aimed to determine the effects of fluoride and pH on the demineralization of dentin, such as to provide information necessary to develop preventive programmes. Bovine dentin blocks were subjected to undersaturated calcium- and phosphate-containing solutions in the pH range 4.0–6.0 with fluoride added at concentrations between 0.5 and 10 ppm. Non-fluoride solutions served as controls. Mineral loss was assessed chemically and by transversal microradiography. Comparisons were made with similar studies on enamel demineralization. The results showed that demineralization of dentin depends on both pH and fluoride concentration in the demineralizing solution. Inhibition of demineralization that could be relevant from a clinical point of view was found at fluoride values 5–10 times the corresponding values for enamel. Also rapid depletion of fluoride from the solutions was observed, indicating the high uptake capacity of dentin for fluoride. Lesion depth depended on pH of the solution while the fluoride levels were associated with the surface layer, both in mineral content and depth. For dentin we propose a demineralization mechanism where acid penetrates rapidly into the tissue, presumably through the tubules, after which the released calcium and phosphate is partly trapped by the inward diffusing fluoride. This leads to the formation of a surface layer, which may even be hypermineralized compared to sound dentin.

A number of polymers which have previously been tested for their applicability as thickening agents in saliva substitutes were studied in vitro for their caries-protective properties. These were: polyacrylic acid, carboxymethylcellulose, xanthan gum, guar gum, hydroxyethylcellulose and porcine gastric mucin. The polymers were tested for their effects on: (1) growth of hydroxyapatite crystals in a supersaturated calcium phosphate solution, (2) dissolution of hydroxyapatite crystals in 50 mM acetic acid, pH 5.2 and (3) demineralization and remineralization of bovine enamel in a pH-cycling model. Growth of hydroxyapatite crystals was strongly inhibited by polyacrylic acid and carboxymethylcellulose at very low concentrations (0.005% w/v). Other polymers displayed lower inhibition of hydroxyapatite crystal growth. Hydroxyapatite dissolution was inhibited by all polymers except by hydroxymethylcellulose and xanthan gum. This occurred both in the presence of the polymers as well as after a 30-min preincubation. In the pH-cycling experiment, bovine enamel specimens with preformed lesions were alternately exposed to a demineralization buffer and a remineralization buffer containing the polymers hydroxyethylcellulose, carboxymethylcellulose, xanthan gum, polyacrylic acid, or porcine gastric mucin. A remineralization buffer containing 1 ppm NaF was used as a positive control. Under the experimental conditions, the control experiment without additives resulted in a net mineral loss (30.6 mumol Ca/cm2 after 14 days of pH cycling). In the presence of 1 ppm NaF, a small mineral gain was observed (8.6 mumol/cm2). All polymers largely inhibited further demineralization (1.2-12.3 mumol/cm2) except polyacrylic acid which, inhibited of its high calcium-binding capacity, caused demineralization, especially in the remineralization buffer (17.1 mumol/cm2). In conclusion, polymers tested in this study, except the polyacrylic acid, reduced the demineralization of enamel in vitro. The precise mechanism of the protective effect is not clear but it is speculated that formation of an absorbed polymer layer on the hydroxyapatite or enamel surface may provide protection against acidic attacks.