M.J. Buijs

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 μm) 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.

Same Exposure but Two Radically Different Responses to Antibiotics: Resilience of the Salivary Microbiome versus Long-Term Microbial Shifts in Feces

ABSTRACT Due to the spread of resistance, antibiotic exposure receives increasing attention. Ecological consequences for the different niches of individual microbiomes are, however, largely ignored. Here, we report the effects of widely used antibiotics (clindamycin, ciprofloxacin, amoxicillin, and minocycline) with different modes of action on the ecology of both the gut and the oral microbiomes in 66 healthy adults from the United Kingdom and Sweden in a two-center randomized placebo-controlled clinical trial. Feces and saliva were collected at baseline, immediately after exposure, and 1, 2, 4, and 12 months after administration of antibiotics or placebo. Sequences of 16S rRNA gene amplicons from all samples and metagenomic shotgun sequences from selected baseline and post-antibiotic-treatment sample pairs were analyzed. Additionally, metagenomic predictions based on 16S rRNA gene amplicon data were performed using PICRUSt. The salivary microbiome was found to be significantly more robust, whereas the antibiotics negatively affected the fecal microbiome: in particular, health-associated butyrate-producing species became strongly underrepresented. Additionally, exposure to different antibiotics enriched genes associated with antibiotic resistance. In conclusion, healthy individuals, exposed to a single antibiotic treatment, undergo considerable microbial shifts and enrichment in antibiotic resistance in their feces, while their salivary microbiome composition remains unexpectedly stable. The health-related consequences for the gut microbiome should increase the awareness of the individual risks involved with antibiotic use, especially in a (diseased) population with an already dysregulated microbiome. On the other hand, understanding the mechanisms behind the resilience of the oral microbiome toward ecological collapse might prove useful in combating microbial dysbiosis elsewhere in the body. IMPORTANCE Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome. Many health care professionals use antibiotic prophylaxis strategies to prevent infection after surgery. This practice is under debate since it enhances the spread of antibiotic resistance. Another important reason to avoid nonessential use of antibiotics, the impact on our microbiome, has hardly received attention. In this study, we assessed the impact of antibiotics on the human microbial ecology at two niches. We followed the oral and gut microbiomes in 66 individuals from before, immediately after, and up to 12 months after exposure to different antibiotic classes. The salivary microbiome recovered quickly and was surprisingly robust toward antibiotic-induced disturbance. The fecal microbiome was severely affected by most antibiotics: for months, health-associated butyrate-producing species became strongly underrepresented. Additionally, there was an enrichment of genes associated with antibiotic resistance. Clearly, even a single antibiotic treatment in healthy individuals contributes to the risk of resistance development and leads to long-lasting detrimental shifts in the gut microbiome.

Inhibition of Dentin Demineralization by Fluoride in vitro

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.

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 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.

Influence of Polymers for Use in Saliva Substitutes on De- and Remineralization of Enamel in vitro

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.

Modification of Amino Acid Residues in Carious Dentin Matrix

The Maillard reaction between sugar and protein has been postulated as the cause for the browning and arrestment of caries lesions. This reaction has been implicated as the cause for decreased degradability of collagen in vivo. The aim of the present study was to verify the occurrence of the reaction in vivo. Carious and sound dentin samples were taken from extracted human teeth and analyzed for the fluorescence characteristic of the Maillard reaction and oxidation and, by HPLC, for Maillard products. In addition, physiological cross-links were analyzed by HPLC. Oxidation- and Maillard reaction-related fluore-scence increased in collagenase digests from carious dentin. Advanced Maillard products (carboxymethyllysine and pentosidine) increased, whereas furosine, a marker for the initial reaction, was not observed consistently. This implies no direct addition of sugars to protein, but rather the addi-tion of smaller metabolites and glycoxidation products. In addition, the physiological crosslinks hydroxylysinonorleucine and dihydroxylysinonorleucine decreased in carious dentin. Also for hydroxylysylpyridinoline, a decrease was observed, but not consistently. In conclusion, the caries process modifies amino acids in dentin collagen, which can lead to increased resistance against proteolysis and ulti-mately to caries arrestment.

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.

Contact Microradiography of Dentine under Wet Conditions to Prevent Lesion Shrinkage

A considerable shrinkage of dentinal lesions is the result of desiccation of dentine sections during contact microradiography. We introduce a simple modification which allows the dentine to be wet during the microradiographic procedure. The volume stability of both sound and demineralized dentine sections microradiographed under wet conditions is compared to sections microradiographed while being exposed to the air. Under wet conditions no shrinkage could be detected of both sound and demineralized sections. Within 15 min exposure to the air, demineralized sections showed their lesion depth to be significantly reduced by 21%, resulting in an underestimation of the mineral loss of 44%. No shrinkage was observed in the air-dried sound dentine. Especially when high-resolution plates are used, which require an extended exposure time, microradiography of dentine sections under wet conditions is recommended. In longitudinal de- and remineralization studies, the use of water instead of impregnation with low-volatility liquids is to be preferred because of the possible effects of these liquids on the mineralization processes.

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.

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 ≤275, 780 and 990 ppm fluoride, respectively. Of the 61 toothpastes with declared fluoride concentration, 75% contained a total F concentration of ≧89% of the declared concentration. In 25% of these toothpastes the free ionisable fluoride was ≤55% of the declared fluoride, and in another 25% of the pastes the free ionisable fluoride concentration was ≧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.