Fluoride Mode of Action: Once There Was an Observant Dentist . . .

The discovery and implementation of fluoride in the prevention of dental caries is often praised as one of the most important achievements in health care. In the early 20th century, it took 30 y to identify fluoride as the cause of enamel mottling but also of reduced caries prevalence in a population drinking water containing fluoride. Similarly, from 1960 to 1990, it took major efforts to unravel the working mode of fluoride in such detail that a rational scheme of caries prevention could be formulated. This article describes the scientific struggle leading to a consensus on the topic. For a historic purpose, the field, the actors, and their main research achievements are described. Ultimately it was generally agreed that the effect of fluoride is primarily topical by fluorides in the oral fluids rather than systemic by incorporation of fluoride in the enamel mineral crystals. Fluoride concentrations, even <1 mg/L, enhance the deposition of calcium phosphates during remineralization of enamel (and dentin). Similarly, such low levels of fluoride are effective in reducing the dissolution of the calcified tissues. This understanding has led to the development of fluoride-containing caries-preventive products that had an undisputed beneficial effect on the levels of dental caries.

Second Era of OMICS in Caries Research: Moving Past the Phase of Disillusionment.

Novel approaches using OMICS techniques enable a collective assessment of multiple related biological units, including genes, gene expression, proteins, and metabolites. In the past decade, next-generation sequencing ( NGS) technologies were improved by longer sequence reads and the development of genome databases and user-friendly pipelines for data analysis, all accessible at lower cost. This has generated an outburst of high-throughput data. The application of OMICS has provided more depth to existing hypotheses as well as new insights in the etiology of dental caries. For example, the determination of complete bacterial microbiomes of oral samples rather than selected species, together with oral metatranscriptome and metabolome analyses, supports the viewpoint of dysbiosis of the supragingival biofilms. In addition, metabolome studies have been instrumental in disclosing the contributions of major pathways for central carbon and amino acid metabolisms to biofilm pH homeostasis. New, often noncultured, oral streptococci have been identified, and their phenotypic characterization has revealed candidates for probiotic therapy. Although findings from OMICS research have been greatly informative, problems related to study design, data quality, integration, and reproducibility still need to be addressed. Also, the emergence and continuous updates of these computationally demanding technologies require expertise in advanced bioinformatics for reliable interpretation of data. Despite the obstacles cited above, OMICS research is expected to encourage the discovery of novel caries biomarkers and the development of next-generation diagnostics and therapies for caries control. These observations apply equally to the study of other oral diseases.

MicroCT study on the enamel mineral density of primary molars.

AIM: The aim of this study is to report on the mineral density of the enamel of primary molars related to the age of the child and to compare the mineral density of sound and carious enamel in those molars. MATERIALS AND METHODS: This study included 23 children and 41 extracted primary molars. The primary molars of 21 children met all of the inclusion criteria, and these were studied and scanned using microCT. The teeth were embedded in Impregum (3M ESPE) and stored in a solution of tap water with thymol crystals. Sixteen primary molars from 7 children were used to compare the mineral density in sound and carious areas, and 13 primary molars from 11 children were used for the comparison between mineral density and time in situ. RESULTS: A statistically significant difference (31%) was found between the mineral density in carious enamel and sound enamel (p = 0.0006). In addition, a significant relationship was observed between the mineral density of sound enamel and the time the teeth had been in situ (r = 0.698). We also found two teeth with radiolucencies in the dentin with the enamel clinically showing only a non-cavitated carious lesion in the enamel. No significant differences were found between the mean mineral density in sound enamel surfaces and unaffected areas in surfaces of molars with enamel caries (p = 0.4373). CONCLUSION: Local and general differences in enamel mineralisation are presented. Post-eruptive maturation seems to be present not only in permanent teeth but also in primary molars. Carious enamel has significantly less mineral density than clinically sound enamel.

Biofilm layers affect the treatment outcomes of NaF and Nano-hydroxyapatite.

During caries formation, dental biofilms function not only as acid producers but also as reservoirs and diffusion barriers for active caries-preventive components. The aim of this study was to investigate the influence of biofilms as a stagnant layer on the efficacy of NaF and nano-hydroxyapatite (nHA). Biofilms of Streptococcus mutans C180-2 were formed on the surfaces of artificially demineralized enamel in an active attachment biofilm model. After 2 days of biofilm formation, the model was subjected to a pH-cycling schedule, together with a control group without biofilms. Specimens were treated for 5 min twice daily with water, a 10% nHA slurry, or 18.4 mM NaF. At the end of the pH-cycling period, the biofilms were removed for the determination of the viable counts, the lactic acid production, and the calcium content. The mineral changes in the demineralized enamel blocks were analyzed by transversal microradiography. No differences in the biofilm viable counts and lactic acid production were found in the different treatment groups. The mean calcium content of the biofilms in the nHA group was 60.7 ± 15.3 mmol/g wet weight, which was approximately 8-fold higher than in the other 2 groups. The application of NaF resulted in net remineralization, but in the presence of a biofilm, net demineralization was observed. In contrast, nHA treatment reduced further demineralization compared with the water treatment, but the presence of a biofilm enhanced this effect. In conclusion, the presence of biofilms clearly influenced the treatment outcomes of anticaries products. Biofilms could either enhance or impede their efficacy. This result implies that biofilms should be included in the in vitro tests for the preclinical screening of caries-protective agents.

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.

Candida and other fungal species: forgotten players of healthy oral microbiota.

In the last half-decade or so, interest in the bacterial part of the human microbiome and its role in maintaining health have received considerable attention. Since 2009, over 300 publications have appeared describing the oral bacterial microbiome. Strikingly, fungi in the oral cavity have been studied exclusively in relation to pathologies. However, little to nothing is known about a role of fungi in establishing and maintaining a healthy oral ecology. In a healthy ecology, balance is maintained by the combined positive and negative influences between and among its members. Interactions between fungi and bacteria occur primarily at a physical and chemical level. Physical interactions are represented by (co-)adhesion and repulsion (exclusion), while chemical interactions include metabolic dependencies, quorum-sensing, and the production of antimicrobial agents. Information obtained from oral model systems and also from studies on the role of fungi in gastro-intestinal ecology indicates that fungi influence bacterial behavior through these different interactions. This review describes our current knowledge of the interactions between fungi and bacteria and aims to illustrate that further research is required to establish the role of fungi in maintaining a healthy oral cavity.

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.

Determination of arginine catabolism by salivary pellet.

To determine the formation of ammonium from arginine by oral bacteria residing in saliva and dental plaque, an arginolytic activity assay based on the work described by Nascimento et al. [2] was developed. Following the original methodology, insufficient ammonium production could be determined. To improve the method for our research goal, the following modifications were made to the original protocols:•The following changes were made to the arginine catabolism assay resulting in a 1000-fold increase in sensitivity: (i) the salivary pellet was washed and concentrated five times resulting in the removal of low density compounds interfering with the assay, (ii) the pH of the Tris-maleate buffer was increased from 6.0 to 7.5 resulting in a better conversion of arginine to ammonium and (iii) the incubation time was increased to 3 h to ensure that non-responders and salivary pellets low in cell numbers could yield detectable levels of ammonium.•Removal of a centrifuge step from the protein determination resulted in a higher protein yield improving the accuracy of the assay.•Changing from the use of the toxic, environmentally hazardous, mercury containing Nessler's reagent to a colorimetric enzyme assay achieved a safer and greener determination of ammonium concentration.

Mineral content in teeth with deciduous molar hypomineralisation (DMH).

OBJECTIVES: We report the mineral (hydroxyapatite) density of sound and opaque areas in DMH molars with sound parts of (carious) deciduous teeth serving as controls. METHODS: Twenty-nine extracted second primary molars obtained from 15 children were studied. Thirteen of these molars were DMH molars with yellow opacities, seven were DMH molars with white opacities, three DMH molars with brown opacities and eleven were molars without DMH. Prior to microCT scanning, the teeth were mounted in impression material (Impregum(®)) and stored in water with a thymol crystal. Spot analysis and line scans were performed in areas with opacities and in sound areas. An ANOVA test and t-tests were used to test if there were significant differences between the groups. RESULTS: The average densities of the hydroxyapatite in yellow and brown opacities (1368mg HA/cm(2) and 1407mg HA/cm(2), respectively) were significantly lower than in clinically unaffected enamel (1747mg HA/cm(2)) of DMH molars or of sound molars (1758mg HA/cm(2)). The mineral density in white opacities (1737mg HA/cm(2)) was not different from that in the enamel of sound molars. The mineral density values in yellow and brown enamel opacities were in between those of dentine (1018mg HA/cm(2)) and enamel. CONCLUSIONS: DMH molars with yellow or brown opacities had a 20-22% lower mineral density in the hypomineralised enamel compared with sound molars. White opacities do not show a lower mineral content. The reduction in enamel mineral content in DMH molars stressed the need for a preventive approach in DMH.

Contemporary perspective on the use of fluoride products in caries prevention.

Dental caries has declined in the 40 years since fluoridated toothpastes were introduced. Much has been learned about why fluoride is so effective and how this knowledge can be used to optimise programmes for caries prevention. Fluoride works through enhancing the remineralisation of early stages of caries and by inhibiting demineralisation, which would lead to dental caries. Remineralisation involves the deposition of calcium phosphates from saliva to rebuild partly dissolved enamel crystallites. When fluoride is incorporated the dissolution of these reinforced crystallites will be reduced during a subsequent sugar-induced and bacteria-mediated acid attack. Fluoride works primarily when it is present in the oral cavity. Based on our understanding of the fluoride mode of action the following advice can be given from clinicians to their patients: The fluoride concentration in oral products is related to efficacy but the concentration does not necessarily need to be high to be efficacious. Fluoride availability throughout the day is important; this can be achieved when fluoride products are used as part of the daily hygiene routine (F-brushing or rinsing). Alternatively, when fluoride is provided in the drinking water or through professionally applied F-varnishes or gels, the patient will benefit without requiring daily compliance to its use. The latter methods are particularly effective as additional treatments in high caries individuals.

Fluoride toothpaste containing 1.5% arginine and insoluble calcium as a new standard of care in caries prevention.

In spite of obvious achievements in prevention, caries remains a prevalent disease. Fluorides are effective by inhibiting enamel and dentin demineralization and enhancing remineralization, but have little or no influence on bacterial processes in dental plaque. Dental caries is a continuum of stages from reversible, early lesions to irreversible, pre-cavitated lesions and, ultimately, to cavities. Prevention should focus on strengthening protective and reducing pathological factors, and careful monitoring of the disease state. While fluoride and the mineral aspects of caries have been in focus for decades, new insights into the etiology of caries have generated novel concepts and approaches to its prevention and treatment. The observation that some plaque bacteria can produce alkali metabolites and, thus, raise pH or neutralize acid formed in plaque has long been known. Such pH rise factors are related to caries susceptibility. Nourishing the plaque with substrates that encourage alkali-producing reactions is a protective factor in the caries continuum. This article reviews the results of clinical studies with a novel toothpaste containing 1.5% arginine, an insoluble calcium compound, and fluoride which have demonstrated superior remineralization of white spot enamel lesions and rehardening of root surface lesions, favorable effects on the de-/remineralization balance, as well as superior cavity prevention efficacy compared to toothpaste with fluoride alone. Studies have also confirmed formation of ammonia and elevated pH levels in subjects using the arginine-containing toothpaste. This novel toothpaste effectively combines the established effects of fluoride on de- and remineralization with reduction of caries-inducing pathological factors resulting from plaque metabolism.

Factors associated with alkali production from arginine in dental biofilms.

Alkali production by oral bacteria in the oral cavity has been linked to protection against dental caries. The current study assessed various parameters associated with ammonium produced during arginine catabolism in dental biofilms. Polymicrobial biofilms were formed with saliva as the inoculum. The NH(3) level and the pH of the spent medium were used to monitor and quantitate the bacterial reactions. The presence of sucrose, a low buffer capacity, and a low pH (≤ pH 4.5) were found to hamper alkali production from arginine. The rate of alkali production exhibited an optimum around pH 5.5. Biofilms were found to produce NH(3) also from polypeptides and proteins in the medium. The biofilm age affected these processes. The experimental model proved valuable for the assessment of the collective bacterial reactions determining the overall pH outcome. This experimental approach could bridge the gap in our knowledge between pH-rise phenomena and caries susceptibility from clinical observations and studies performed on alkali-producing bacteria in well- controlled, though simplified, in vitro models. Analysis of our data supports the hypothesis that the initiation and progression of dental caries may be influenced by the relative rates of acid and base formation, which critically depend on the aforementioned parameters.

The numerous microbial species in oral biofilms: how could antibacterial therapy be effective?

Hundreds of bacterial species inhabit the oral cavity. Many of these have never been cultivated and can be assessed only with DNA-based techniques. This new understanding has changed the paradigm of the etiology of oral disease from that associated with 'traditional pathogens' as being primarily responsible for all diseases. Increasingly, associations between oral bacteria and systemic diseases are being reported. The emergence of antibiotic resistance is alarming and calls for in-depth studies of biofilms, bacterial physiology, and a body-wide approach to infectious diseases. We propose that the borderline between commensal bacteria and pathogens is no longer discrete. In a field of science where so many of the established paradigms are being undermined, a thorough analysis of threats and opportunities is required. This article addresses some of the questions that can be raised and serves to identify research opportunities and needs to leverage the prevention of oral diseases through novel antimicrobial strategies.

Novel anticaries and remineralizing agents: prospects for the future.

Although the use of fluorides has been successful in reducing dental caries, the need remains to develop and evaluate new approaches and promising products for caries prevention. Comprehensive caries-prevention protocols should encompass fluoride and other agents affecting the de-/remineralization balance but also antimicrobial strategies. Different from the traditional restorative approach, the current opinion is that caries should be detected and monitored in its earliest stages, when a nonsurgical reversal can still be achieved. This paradigm shift has implications for methods of caries diagnosis, the choice of preventative materials and the design of randomized clinical trials. This article summarizes the highlights of a special conference dedicated to the topic of novel anticaries and remineralizing agents (ICNARA 2), and identifies the current consensus and remaining questions on pivotal issues in this field.

Effect of pH on Galla chinensis extract's stability and anti-caries properties in vitro.

OBJECTIVES: Considering that Galla chinensis extract (GCE) solution has a low pH, which might dissolve dental enamel, we investigated the effects of elevation of pH on GCE stability, and on its anti-caries properties. DESIGNS: Stability of GCE solutions, either in H(2)O (pH less than 4.0) or when buffered at pH 5.5, 7.0 and 10.0, was assessed from UV-VIS spectra. Inhibition of enamel demineralization was determined in a pH-cycling set up, comprising treatments with either GCE solutions or negative control buffers and acid and neutral buffer immersions. Demineralization was assessed by calcium in the acetate buffers. To determine antimicrobial properties, polymicrobial biofilms were formed after saliva inoculation on glass surfaces which were treated after 48 h. Treatment output parameters were lactic acid formation and viability, the latter by colony forming unit (CFU) counts. RESULTS: At pH 7.0 and higher GCE solutions changed colour and absorption spectra in UV-VIS, indicative of chemical changes. Regarding enamel demineralization, significant inhibitions (P<0.05) were found for all GCE treatments when compared with corresponding controls. In polymicrobial biofilms, GCE reduced the acid production, compared with the negative controls (P<0.05). However, this difference was only significant at the lower pH values. CONCLUSIONS: GCE solutions were unstable under neutral and alkaline conditions. pH did not significantly influence the inhibiting effect of GCE on enamel demineralization. However, GCE was not effective on polymicrobial biofilms at alkaline pH (8.5). To avoid enamel damage due to acidic treatment, GCE solutions should be used at about pH 5.5.

Deciduous molar hypomineralization and molar incisor hypomineralization.

This study was embedded in the Generation R Study, a population-based prospective cohort study from fetal life until young adulthood. This study focused on the relationship between Deciduous Molar Hypomineralization (DMH) and Molar Incisor Hypomineralization (MIH). First permanent molars develop during a period similar to that of second primary molars, with possible comparable risk factors for hypomineralization. Children with DMH have a greater risk of developing MIH. Clinical photographs of clean, moist teeth were taken with an intra-oral camera in 6,161 children (49.8% girls; mean age 74.3 mos, SD ± 5.8). First permanent molars and second primary molars were scored with respect to DMH or MIH. The prevalence of DMH and MIH was 9.0% and 8.7% at child level, and 4.0% and 5.4% at tooth level. The Odds Ratio for MIH based on DMH was 4.4 (95% CI, 3.1-6.4). The relationship between the occurrence of DMH and MIH suggests a shared cause and indicates that, clinically, DMH can be used as a predictor for MIH.

Child dental anxiety, parental rearing style and referral status of children.

OBJECTIVE: Treating children can be difficult for both dentist and child. In some cases treatment fails and those children are referred to a specialist paediatric dentist. Different factors can be put forward for referral of children, such as factors relating to the child, dentist and parent. Possible child-related factors can be dental anxiety and the child's temperament. A possible parental factor is the parental rearing style. The objective of this study was to assess the possible associations between dental anxiety, parental rearing style and referral status of children. METHODS: Parents of 120 non-referred and 335 referred paediatric dental patients were asked to fill out the Child Rearing Practices Report (CRPR) and the Child Fear Survey Schedule Dental Subscale (CFSS-DS) on behalf of their children. RESULTS: The questionnaires were filled out by 115 (96%) parents of primary schoolchildren and by 331 (99%) parents of referred children. Referred children were younger than non-referred children, t(442) = 6.9, p < 0.01, and had significantly more dental anxiety, t(430) = -8.7, p < 0.01. No differences existed between parents of referred children and parents of non-referred children on parental rearing-style. No differences existed between fearful and non-fearful children on parental rearing-style and also no correlation existed between children's dental anxiety and their parent's rearing style. However, non-referred children with parents using an authoritarian parenting style were more anxious than the other non-referred children. CONCLUSIONS: In the present study, referral status and dental anxiety of 4-12 year old children were not associated with parental rearing style.