Microbial Indicators of Dental Health, Dysbiosis, and Early Childhood Caries.

Dental caries lesions are a clinical manifestation of disease, preceded by microbial dysbiosis, which is poorly characterized and thought to be associated with saccharolytic taxa. Here, we assessed the associations between the oral microbiome of children and various caries risk factors such as demographics and behavioral and clinical data across early childhood and characterized over time the salivary and dental plaque microbiome of children before clinical diagnosis of caries lesions. Children (N = 266) were examined clinically at ~1, 2.5, 4, and 6.5 y of age. The microbiome samples were collected at 1, 2.5, and 4 y. Caries groups consisted of children who remained caries free (International Caries Detection and Assessment System [ICDAS] = 0) at all time points (CFAT) (n = 50); children diagnosed with caries (ICDAS ≥ 1) at 6.5 y (C6.5), 4 y (C4), or 2.5 y of age (C2.5); and children with early caries or advanced caries lesions at specific time points. Microbial community analyses were performed on zero-radius operational taxonomic units (zOTUs) obtained from V4 of 16S ribosomal RNA gene amplicon sequences. The oral microbiome of the children was affected by various factors, including antibiotic use, demographics, and dietary habits of the children and their caregivers. At all time points, various risk factors explained more of the variation in the dental plaque microbiome than in saliva. At 1 y, composition of saliva of the C4 group differed from that of the CFAT group, while at 2.5 y, this difference was observed only in plaque. At 4 y, multiple salivary and plaque zOTUs of genera Prevotella and Leptotrichia were significantly higher in samples of the C6.5 group than those of the CFAT group. In conclusion, up to 3 y prior to clinical caries detection, the oral microbial communities were already in a state of dysbiosis that was dominated by proteolytic taxa. Plaque discriminated dysbiotic oral ecosystems from healthy ones better than saliva.

Home sampling is a feasible method for oral microbiota analysis for infants and mothers.

OBJECTIVES: Large longitudinal cohort studies in infants are needed to understand oral microbiome maturation in relation to general health. The logistics of such studies are complex and costs involved high. Methods like home sampling by caretakers might be a solution to these issues. This study aimed to evaluate feasibility of home sampling by caretakers and to assess which oral niche provides the most reliable sample. METHODS: In this cross-sectional study 30 mothers and their infants aged 2-15 months participated. Swabs of the tongue, buccal mucosa, saliva, and dental plaque of the mother and the infant were collected by the mother after watching an instruction video. Thereafter, the trained researcher repeated the sample collection. Variations on the sampling protocol were listed. Bacterial DNA was quantified and microbial composition was assessed using 16S rDNA amplicon sequencing. RESULTS: None of the sampled niches appeared to be unfeasible based on interviews and observed variations on protocol. No significant differences in bacterial DNA concentration between operators (mother and researcher) were found. In infant's saliva, Shannon diversity of samples collected by the researcher was significantly higher than those collected by mothers (p = 0.0009) and the bacterial composition was influenced by variations on sampling protocol (p = 0.01). CONCLUSIONS: Home sampling by caretakers is a feasible method for oral sample collection in infants and mothers. Oral samples collected by mothers resemble samples collected by a trained researcher, with the tongue sample being the most similar and saliva the least. CLINICAL SIGNIFICANCE: Home sampling can simplify longitudinal oral microbiota collection.

Dental aerosols: microbial composition and spatial distribution.

Background: High-speed dental instruments produce aerosols, which can contribute to the transmission of pathogenic microorganisms. The aim of this study is to describe the microbial load and - composition and spatial distribution of aerosols in dental clinics. Methods: In four dental clinics active and passive sampling methods were used before, during and after treatment and at different locations. Retrieved colony forming units (CFU) were sequenced for taxon identification. Results: The samples contained up to 655 CFU/plate/30 minutes and 418 CFU/m3/30 minutes during dental treatment for active and passive sampling, respectively. The level of contamination after treatment and at 1.5 m distance from the patient's head was similar to the start of the day. The highest contamination was found at the patient's chest area. The aerosols consisted of 52 different taxa from human origin and 36 from water. Conclusion: Contamination in dental clinics due to aerosols is mainly low, although high level of contamination with taxa from both human and water origin was found within 80 cm around the head of the patient. Our results stress the importance of infection control measures on surfaces in close proximity to the head of the patient as well as in dental water lines.

Maturation of the Oral Microbiome in Caries-Free Toddlers: A Longitudinal Study.

Understanding the development of the oral microbiota in healthy children is of great importance to oral and general health. However, limited data exist on a healthy maturation of the oral microbial ecosystem in children. Moreover, the data are biased by mislabeling "caries-free" populations. Therefore, we aimed to characterize the healthy salivary and dental plaque microbiome in young children. Caries-free (ICDAS [International Caries Detection and Assessment System] score 0) children (n = 119) and their primary caregivers were followed from 1 until 4 y of child age. Salivary and dental plaque samples were collected from the children at 3 time points (T1, ~1 y old; T2, ~2.5 y old; and T3, ~4 y old). Only saliva samples were collected from the caregivers. Bacterial V4 16S ribosomal DNA amplicons were sequenced using Illumina MiSeq. The reads were denoised and mapped to the zero-radius operational taxonomic units (zOTUs). Taxonomy was assigned using HOMD. The microbial profiles of children showed significant differences (P = 0.0001) over time. Various taxa increased, including Fusobacterium, Actinomyces, and Corynebacterium, while others showed significant decreases (e.g., Alloprevotella and Capnocytophaga) in their relative abundances over time. Microbial diversity and child-caregiver similarity increased most between 1 and 2.5 y of age while still not reaching the complexity of the caregivers at 4 y of age. The microbiome at 1 y of age differed the most from those at later time points. A single zOTU (Streptococcus) was present in all samples (n = 925) of the study. A large variation in the proportion of shared zOTUs was observed within an individual child over time (2% to 42% of zOTUs in saliva; 2.5% to 38% in dental plaque). These findings indicate that the oral ecosystem of caries-free toddlers is highly heterogeneous and dynamic with substantial changes in microbial composition over time and only few taxa persisting across the 3 y of the study. The salivary microbiome of 4-y-old children is still distinct from that of their caregivers.

A reproducible microcosm biofilm model of subgingival microbial communities.

OBJECTIVE: To develop a reproducible subgingival microcosm biofilm model. MATERIAL AND METHODS: Subgingival plaque samples were collected from four deep pockets (probing pocket depth ≥6 mm) in each of seven patients with periodontitis and from shallow pockets (probing pocket depth ≤3 mm) in two periodontally healthy donors. An active attachment model and a peptone medium (Thompson et. al., Appl Environ Microbiol 2015;81:8307-8314) supplemented with 30% serum was used. Biofilms were harvested at 2 and 4 weeks. DNA of dead cells was blocked for amplification by propidium monoazide treatment. Composition was analyzed using 16S rRNA gene amplicon pyrosequencing. Similarities between the biofilm samples were assessed by non-metric multidimensional scaling using the Bray-Curtis similarity index and similarity percentage analysis. Data from duplicate experiments, different biofilm sources and different biofilm age were compared. RESULTS: The non-metric multidimensional scaling revealed a strong clustering by the inoculum source, the donor and their periodontal status. Statistically significant differences were found between the sources of inoculum (P=.0001) and biofilm age (P=.0016). Furthermore, periodontitis biofilms (P) were distinct in composition from health-derived biofilms (H) by genera: Porphyromonas (P=19%; H=0%), Filifactor (P=10%; H=0%), Anaeroglobus (P=3%; H=0%), Phocaeicola (P=1.5%; H=0%), Parvimonas (P=19%; H=14%), Fusobacterium (P=2%; H=26%), Peptostreptococcus (P=20%; H=30%), Veillonella (P=7%; H=8%) and 57 other genera. Similarity distances (Bray-Curtis) (mean 0.73, SD 0.15) and the Shannon diversity index (mean 2, SD 0.2) revealed no differences between duplicate experiments (P=.121). CONCLUSION: This biofilm model allows reproducible production of complex subgingival microbial communities.

Microbial profiles at baseline and not the use of antibiotics determine the clinical outcome of the treatment of chronic periodontitis.

Antibiotics are often used in the treatment of chronic periodontitis, which is a major cause of tooth loss. However, evidence in favour of a microbial indication for the prescription of antibiotics is lacking, which may increase the risk of the possible indiscriminate use of antibiotics, and consequent, microbial resistance. Here, using an open-ended technique, we report the changes in the subgingival microbiome up to one year post-treatment of patients treated with basic periodontal therapy with or without antibiotics. Antibiotics resulted in a greater influence on the microbiome 3 months after therapy, but this difference disappeared at 6 months. Greater microbial diversity, specific taxa and certain microbial co-occurrences at baseline and not the use of antibiotics predicted better clinical treatment outcomes. Our results demonstrate the predictive value of specific subgingival bacterial profiles for the decision to prescribe antibiotics in the treatment of periodontitis, but they also indicate the need for alternative therapies based on ecological approaches.

The effect of propidium monoazide treatment on the measured bacterial composition of clinical samples after the use of a mouthwash.

OBJECTIVES: The use of an anti-microbial mouthwash results not only in a reduction of the number of viable cells in dental plaque but potentially also in a shift in the oral microbiome. DNA-based techniques may be appropriate to monitor these shifts, but these techniques amplify DNA from both dead and living cells. Propidium monoazide (PMA) has been used to overcome this problem, by preventing the amplification of DNA from membrane-damaged cells. The aim of this study was to evaluate the use of PMA when measuring compositional shifts in clinical samples after mouthwash use. MATERIALS AND METHODS: On two consecutive days, baseline samples from buccal surfaces, tongue, and saliva were obtained from six volunteers, after which they used a mouthwash (Meridol, GABA, Switzerland) twice daily for 14 days. Subsequently similar samples were obtained on two consecutive days. The microbial composition of the samples, with or without ex vivo PMA treatment, was assessed with 16S rRNA gene amplicon sequencing. RESULTS: Data showed a clear effect of mouthwash usage on the tongue and saliva samples. PMA treatment enhanced the observed differences only for the saliva samples. Mouthwash treatments did not affect the composition of the plaque samples irrespective of the use of PMA. CONCLUSION: The necessity to use a PMA treatment to block the DNA from dead cells in clinical studies aimed at measuring compositional shifts after the use of a mouthwash is limited to salivary samples. CLINICAL RELEVANCE: Measuring shifts in the oral microbiome could be hampered by the presence of DNA from dead cells.

The effects of propidium monoazide treatment on the measured composition of polymicrobial biofilms after treatment with chlorhexidine.

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.

Chlorhexidine efficacy in preventing lesion formation in enamel and dentine: an in situ study.

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.

Elevated fluoride products enhance remineralization of advanced enamel lesions.

Caries prevention might benefit from the use of toothpastes containing over 1500 ppm F. With few clinical studies available, the aim of this pH-cycling study was to investigate the dose response between 0 and 5000 ppm F of de- and remineralization of advanced (> 150 microm) enamel lesions. Treatments included sodium and amine fluoride, and a fluoride-free control. Mineral uptake and loss were assessed from solution calcium changes and microradiographs. Treatments with 5000 ppm F both significantly enhanced remineralization and inhibited demineralization when compared with treatments with 1500 ppm F. Slight differences in favor of amine fluoride over sodium fluoride were observed. The ratio of de- over remineralization rates decreased from 13.8 to 2.1 in the range 0 to 5000 ppm F. As much as 71 (6)% of the remineralized mineral was calculated to be resistant to dissolution during subsequent demineralization periods. With 5000-ppm-F treatments, more demineralizing episodes per day (10 vs. 2 for placebo) would still be repaired by remineralization.

Effects of ozone and sodium hypochlorite on caries-like lesions in dentin.

The hypothesis that ozone promotes remineralization of dentinal lesions was tested in vitro. Artificial caries-like lesions in dentin were treated with ozone gas, with another potent oxidizer (sodium hypochlorite, NaOCl, 10%) or with water. The specimens were then remineralized and subsequently demineralized again. Mineral content was assessed by transverse microradiography. NaOCl-treated samples showed damaged surface and, after being remineralized, demineralized significantly more than water- or ozone-treated groups. No difference was found between ozone and water groups. The exposure to ozone had thus no effect on remineralization and subsequent demineralization of remineralized dentinal lesions.

Effect of an intensified treatment with 40% chlorhexidine varnish on plaque acidogenicity.

Previous work showed that a single application of 40% chlorhexidine varnish, EC40, reduced plaque acidogenicity upon sucrose challenge during less than 3 weeks. It was questioned whether lactic acid production could be reduced significantly longer when the treatment was intensified. Therefore, the effects of three consecutive EC40 applications on plaque acidogenicity were evaluated. Nine subjects who participated in the previous study received three full mouth EC40 applications within 1 week. Before the first application and up to 9 weeks after the third application, plaque samples were taken after a 10% sucrose rinse and analyzed for organic acids with capillary electrophoresis. At baseline, the mean provoked lactic acid concentration was 1.64 (+/-0.69) micromol/mg protein. At the first and seventh day after the third application, there was too little plaque to measure acid concentrations. At 2 weeks after the third application, lactic acid concentrations were significantly reduced (p < 0.05). The acid concentrations 3 weeks after the third application (1.61 (+/-0.99) micromol/mg protein) did not differ from the values at baseline (paired T test, p > 0.05). We conclude that a triple 40% chlorhexidine varnish treatment did not affect plaque acidogenicity for more than 3 weeks. From comparison with a previous study, we conclude that the triple treatment with EC40 within 1 week was not more effective in reducing plaque acidogenicity than the single one.

Increased salivary fluoride concentrations after post-brush fluoride rinsing not reflected in dental plaque.

The aim of the present study was to assess fluoride concentrations in unstimulated saliva and buccal dental plaque 6 h after an oral hygiene procedure that consisted of brushing with an AmF/SnF2 dentifrice and different post-brush rinsing protocols: expectorating the excess of dentifrice foam and rinsing with tap water, expectorating only, or rinsing with 10 ml AmF/SnF2 mouthwash. The fluoride concentrations in plaque and saliva were increased after all three experimental protocols compared to F-free periods. The increase of the fluoride concentration in saliva was more pronounced after AmF/SnF2 mouthrinse as compared to rinsing with water and expectorating the excess of dentifrice foam. Such an effect was not seen in dental plaque. It is concluded that the potentially beneficial effect of not rinsing or fluoride rinsing after tooth brushing is not reflected in an increased fluoride concentration in newly formed dental plaque 6 h after brushing.

Effects of various rinsing protocols after the use of amine fluoride/stannous fluoride toothpaste on the acid production of dental plaque and tongue flora.

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 relative efficacy of fluoride toothpastes assessed with pH cycling.

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 microm deep) and 'deep' (about 200 microm 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.

Total and free fluoride in toothpastes from some non-established market economy countries.

In 101 fluoride toothpastes, bought in local shops in Burkina Faso (n = 3), China (n = 5), Myanmar (n = 22), Nepal (n = 19), Philippines (n = 13), Syria (n = 22), Togo (n = 7) and Vietnam (n = 10), the total and free ionisable fluoride concentrations were established. The total fluoride concentration of the toothpastes was determined by gas liquid chromatography. The amount of soluble fluoride was measured after dilution in artificial saliva and treatment of the supernatants with acidic phosphatase. The free fluoride concentration in this mixture was measured with a fluoride electrode. Twenty-five percent of all toothpastes contained less than 738 ppm total fluoride, and another 25% contained between 738 and 977 ppm fluoride. Regarding free ionisable fluoride the 25th, 50th, and 75th percentile contained < or =275, 780 and 990 ppm fluoride, respectively. Of the 61 toothpastes with declared fluoride concentration, 75% contained a total F concentration of > or =89% of the declared concentration. In 25% of these toothpastes the free ionisable fluoride was < or =55% of the declared fluoride, and in another 25% of the pastes the free ionisable fluoride concentration was > or =89% of the declared fluoride. In conclusion, deficiencies were found regarding the total as well as the free ionisable fluoride concentration of toothpastes bought in non-established market economy countries. Unclear labelling concerning the type and concentration of fluoride was often encountered. A need for quality control of fluoride toothpastes is recommended.

The effect of a single application of 40% chlorhexidine varnish on the numbers of salivary mutans streptococci and acidogenicity of dental plaque.

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.

The effects of the solubility of artificial fissures on plaque pH.

Dissolution of the fissure walls may buffer acids formed in plaque and thus prevent the penetration of acids into the fissure. To test this, five volunteers wore dentin, enamel, and polyacrylate specimens with narrow grooves for 7 days to accumulate plaque. Temporal (pre- and post-glucose) and spatial (0-0.7 mm) pH profiles were recorded in the grooves in a flow-through reactor with pH microsensors. Mineral loss was assessed by transverse microradiography. We observed that resting pH did not differ among substrata. The median pH 1 hr post-glucose at the bottoms of dentin, enamel, and polyacrylate grooves was 6.7, 6.2, and 5.7, respectively (p < 0.01). On subject level, lesions formed in dentin correlated with pH changes in polyacrylate, where no buffering of acids due to mineral dissolution occurred. We conclude that fluoride-deficient tissue at the bottom of a fissure is at increased risk for caries, if acids are not buffered near the entrance to the fissure.

Acidogenicity of buccal plaque after a single rinse with amine fluoride - stannous fluoride mouthrinse solution.

Caries and gingivitis prevention may benefit from chemotherapeutic plaque control, therefore we compared in a cross-over study with 5 subjects the anti-acidogenic effects of a single use of AmF-SnF2 mouthrinse solutions (Meridol with and without 5% alcohol) with baseline and with the effects of a placebo and a chlorhexidine mouthrinse (CHX). Buccal plaque was collected 0.5, 3 and 8 h after the subjects used one of the mouthrinses, each time before and after a rinse with 10% sucrose to induce lactic acid production. Samples were analysed for acid anions by capillary electrophoresis and for protein. At 0.5 h after the use of AmF-SnF2 or CHX, the concentration of acetate in resting plaque was 70% lower than at baseline or after using the placebo. Average post-sucrose acetate and lactate concentrations in the placebo group were 30-80% higher than at baseline; up to 3 h this difference was significant. 8 h after using AmF-SnF2 or CHX, the post-sucrose acetate and lactate concentrations were still 30-50% lower than after the placebo, and up to 40% lower than at baseline. To conclude, AmF-SnF2 in both Meridol formulations and CHX were shown to have a similar potency to inhibit acid production after a single rinse.

In situ fluoride retention in enamel and dentine after the use of an amine fluoride dentifrice and amine fluoride/sodium fluoride mouthrinse.

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.