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Objective: To describe the caries lesion transition pattern in permanent tooth surfaces over 2 years among a convenience sample of children in a fluoridated (0.8 ppm F) low-socioeconomic community of Brazil. Material and methods: One hundred forty-nine schoolchildren (7–12 years) were examined for caries using Nyvad criteria at baseline and after 2 years. Descriptive analysis was used to evaluate caries lesion transition patterns. Results: Less than 1% of sound surfaces and non-cavitated caries lesions at baseline progressed to cavitation stage within 2 years. 12.7% of the active non-cavitated (ANC) lesions became inactive, 34.7% regressed to sound, 48.0% remained active, and 4.6% progressed to cavitated/filled stages at follow-up. Similarly, 55.2% of the inactive non-cavitated (INC) lesions at baseline remained inactive, 33.3% regressed to sound, 8.0% progressed to cavitated/filled lesions, while only 3.5% progressed to ANC lesions. Conclusion: The caries lesion transition pattern in this child population exposed to water fluoride and fluoride toothpaste showed that a low proportion of sound surfaces and non-cavitated lesions progressed to cavitation within the 2-year follow-up. Caries arrest was mainly ascribed to a high proportion of active non-cavitated lesions regressing to sound or inactive lesions. Clinical relevance: Caries activity can be controlled by regular exposure to fluoridated water and fluoridated toothpaste.

Objectives: To describe a new molar-incisor hypomineralization (MIH) severity scoring system (MIH-SSS) that focuses on the defects’ severity and to assess the system’s validity and reliability over 3 years. Materials and methods: One hundred eighty-one children diagnosed with MIH were examined by MIH-SSS. For reliability assessment, 15–20 children were examined twice, and analyses were performed at the tooth level at four different cutoff points. Follow-up examinations were performed over 36 months. Only teeth presenting MIH opacities at baseline were assessed. Odds ratios were calculated to evaluate the chance of post-eruptive breakdown (PEB) occurrence related to the colors of MIH defects. Survival curves were created for different types of teeth (molars and incisors) based on white and yellow opacities. The Kaplan-Meier method was used with PEB as the outcome. Results: According to the MIH-SSS, kappa values ranged from 0.82 to 0.88. Regarding the longitudinal evaluation, for molars and incisors, yellow/brown opacities had a significantly higher chance to evolve to dentin breakdown compared with white/creamy opacities (OR = 2.54, OR = 10.58, respectively). Survival analysis showed that the occurrence of PEB was more frequent in the first evaluation period (12 months). Conclusion: MIH-SSS, which provides detailed information about MIH severity, is a valid instrument presenting high reliability. Yellow/brown opacities progressed more than did white/creamy opacities. Clinical relevance: It is of paramount importance to detect any surface breakdown as early as possible to be able to avoid pain and caries progression. Moreover, this is the first paper that shows survival curves for MIH enamel breakdown over time.

This review considers an integrated hypothesis of dental caries and periodontal diseases that builds on theoretical ecological principles. The backbone of the hypothesis is based on the dynamic stability stage of the oral microbiota, at which intrinsic (mainly saliva and gingival crevicular fluid) and bacterial (mainly metabolic) resilience factors maintain ecological dynamic stability, compatible with clinical health. However, loss of intrinsic resilience factors and/or prolonged changes in the availability of microbial metabolic substrates may shift the ecological balance of the microbiota into either saccharolytic (acidogenic) or amino acid-degrading/proteolytic (alkalinogenic) stages, depending on the nature of the predominant substrates, leading to clinical diseases. Therefore, to maintain and restore the dynamic stability of the oral microbiota, it is necessary to control the drivers of disease, such as salivary flow and influx of bacterial nutrients into the oral cavity. Contrary to conventional wisdom, excessive intake of fermentable carbohydrates may contribute to inflammation in periodontal tissues resulting from hyperglycaemia. An integrated hypothesis emphasizes that both dental caries and periodontal diseases originate in the dynamic stability stage and emerge in response to nutritional imbalances in the microbiota. Periodontal diseases may belong to the sugar driven inflammatory diseases, similar to diabetes, obesity, and cardiovascular diseases.

Healthy food and meal habits can help promote and maintain good health throughout life. Only few 4-18-year-olds follow official Danish dietary recommendations, leaving room for improvement, notably among 13-18-year-old adolescents and children and adolescents of parents with short-term education. Specific focus areas for all families with children may help limit intake of sugary foods and beverages on weekends. Promotion of healthy food and meal habits among children and adolescents call for a comprehensive and well-coordinated approach with participation of main stakeholders in the field.

OBJECTIVES: The aim of this randomized, double-blind, crossover study was to measure fluoride in saliva and 7-day-old biofilm fluid and biofilm solids after rinsing three times per day for 3 weeks with 0, 1500, or 5000 ppm fluoride (NaF).MATERIALS AND METHODS: Following the 3-week wash-in/wash-out period, including 1 week of biofilm accumulation, saliva and biofilm samples were collected from 12 participants immediately before (background fluoride), and 10, 30, and 60 min after a single rinse. Biofilm samples were separated into fluid and solids, and samples were analyzed using a fluoride electrode (microanalysis).RESULTS: The background fluoride concentration was statistically significantly higher in the 5000 compared to the 1500 ppm F rinse group in all three compartments (22.3 and 8.1 μM in saliva, 126.8 and 58.5 μM in biofilm fluid, and 10,940 and 4837 μmol/kg in biofilm solids). The 1-h fluoride accumulation for the 5000 ppm F rinse was higher than for the 1500 ppm F rinse in all three compartments, although not statistically significant for saliva and biofilm solids.CONCLUSION: Regular exposure to 5000 ppm fluoride elevates background fluoride concentrations in saliva, biofilm fluid, and biofilm solids compared to 1500 ppm fluoride. Increasing the fluoride concentration almost 3.5 times (from 1500 to 5000 ppm) only elevates the background fluoride concentrations in saliva, biofilm fluid, and biofilm solids twofold.CLINICAL RELEVANCE: Even though fluoride toothpaste may be diluted by saliva, the results of the present study indicate that use of 5000 ppm fluoride toothpaste might lead to improved caries control.

Objectives The aim of this study was to investigate the occurrence and spatial localization of Candida species in intact in vivo biofilm from caries lesions in root and occlusal surfaces. Materials and methods Biofilm from 7 teeth with root caries and 9 teeth with occlusal caries were analyzed. The teeth were fixed, embedded, sectioned and decalcified before fluorescence in situ hybridization using oligonucleotide probes against all bacteria (EUB338), all yeast (PF2), streptococci (STR405, MUT590) and C. albicans (Du.al 1249). Sections were analyzed using fluorescence microscopy. Results Yeasts, most of which were C. albicans or C. dubliniensis, colonized occlusal and root surface sites and exhibited both yeast and hyphal forms. Two characteristic colonization patterns were apparent: In one pattern, the biofilm presented defined areas comprising Candida hyphal networks mixed with filamentous, rod-like and coccoid bacteria, often extending through the entire biofilm thickness. In the other pattern, Candida formed corncob configurations with non-mutans streptococci in the biofilm surface layer. Mutans streptococci formed isolated colonies and did not typically appear in close proximity with Candida. Conclusion For the first time, we showed that C. albicans and C. dubliniensis are integrated components of caries biofilm architecture. Co-localization of Candida and non-mutans streptococci indicates a beneficial interaction between these organisms. Diverse metabolic properties of Candida spp. suggest that these organisms may influence the ecology of cross-kingdom microbial communities in caries.

The Nyvad classification is a visual-tactile caries classification system devised to enable the detection of the activity and severity of caries lesions with special focus on low-caries populations. The criteria behind the classification reflect the entire continuum of caries, ranging from clinically sound surfaces through noncavitated and microcavitated caries lesions in enamel, to frank cavitation into the dentin. Lesion activity at each severity stage is discriminated by differences in surface topography and lesion texture. The reliability of the Nyvad criteria is high to excellent when used by trained examiners in the primary and permanent dentitions. The Nyvad criteria have construct validity for lesion activity assessments because of their ability to reflect the well-known caries-controlling effect of fluoride. Predictive validity was demonstrated by showing that active noncavitated lesions are at higher risk of progressing to a cavity or filled state than do inactive noncavitated lesions. Lesion activity assessment performed successfully as a screening tool to identify individuals with a poor caries prognosis. Because of their predictive validity, the Nyvad criteria are superior to other current caries lesion descriptors for the detection of changes in the lesion activity status over time. The Nyvad criteria fulfill all the formal requirements for a robust caries lesion classification and are recommended for evidence-based caries management in clinical practice and in research.

pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of in vivo -grown dental biofilms with unknown species composition. We then employed digital image analysis to remove the bacterial biomass from the microscopic images and adequately calculate extracellular pH values. As a proof of concept, we monitored the extracellular pH drop in in vivo -grown dental biofilms fermenting glucose. The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows the accurate monitoring of extracellular pH in bacterial biofilms in three dimensions in real time and represents a significant improvement to previously employed methods of biofilm pH measurement.

Root surfaces, which with increasing age become exposed to dental biofilms, will react to the intermittent pH fluctuations at the interface between the biofilm and the cementum/dentin surface. If dental biofilm is left undisturbed in stagnation sites in the dentition, the underlying mineral surfaces may gradually develop dental caries characterized by a subsurface loss of mineral. In root surfaces, the demineralization is accompanied by microbial invasion of the cementum and dentin resulting in a pulpo-dentinal defense reaction. Most lesions progress slowly and experimental in situ studies as well as clinical studies document that the daily removal of the biofilm using fluoride toothpaste can arrest lesion progression. By applying this caries control measure, caries lesions can thus be transformed from active lesions to inactive lesions. The diagnostic characteristics of these types of lesions are mandatory to apply in daily clinical practice to avoid unnecessary restorative and antimicrobial treatments.

Information on differences in biofilm fluoride concentration across intra-oral regions may help explain the distribution of caries within the dentition. The aim of this cross-sectional study was to describe the fluoride concentration in saliva and in biofilm fluid and biofilm solids across 6 intra-oral regions. Unstimulated whole saliva was collected from 42 participants and biofilm harvested from the buccal sites in the 4 molar and 2 anterior regions. Samples were collected at least 1 h after use of fluoride dentifrice. No attempt was made to control the participants' food consumption or use of other topical agents. Centrifuged saliva, biofilm fluid, and biofilm solids were analysed for fluoride using a fluoride ion-selective electrode, adapted for microanalysis. Fluoride in biofilm varied across intra-oral regions. The mean biofilm fluid fluoride concentrations across the oral cavity ranged from 11.6 to 16.8 µM, being statistically significantly higher in the upper anterior region than in any other region. In all regions the fluoride concentration in biofilm fluid was higher than in saliva. For biofilm solids the fluoride concentration was highest in the lower anterior region (2,461 μmol/kg) and lowest in the lower molar regions (388 and 406 μmol/kg, respectively). Within biofilm, the solids contained most of the fluoride (81 to >99%). The biofilm fluid fluoride concentration was significantly positively associated with salivary fluoride and only marginally associated with that of biofilm solids. In conclusion, this study has shown pronounced differences in fluoride distribution across intra-oral regions and compartments. This shows that the sampling site is a crucial factor for studies of biofilm fluoride.

The effect of non-operative caries control at cavity level is often questioned. This prospective study of cases aimed to evaluate the suitability of non-operative treatment of active cavitated approximal caries lesions in primary molars by assessing clinical changes of lesions over time. Further, we evaluate children's and parents' attitudes in response to non-operative cavity treatment. Thirty-nine children attending a community dental service, aged 5-11 years, joined the evaluation based on the following criteria: (i) the presence of at least one active cavitated caries lesion in the distal surface of first primary molar or mesial surface of the second primary molar extending up to two-thirds into dentin assessed radiographically, (ii) the absence of spontaneous pain, (iii) the absence of pulpal or periapical pathology. After informed consent, one randomly selected lesion per child had overhanging enamel margins adjusted and was treated non-operatively by the child/parent performing daily site-specific oral hygiene with a fluoridated toothpaste, supplemented by professional topical fluoride treatment and dietary advice. Nine lesions/children dropped out. Fifteen lesions survived without pulpal or periapical pathology for an average of 26 m (range 9-44). Lesion failure was associated with poor compliance or lesions not suited for the method. Children and parents were highly satisfied with the treatment. Our study identifies key factors in the performance of non-operative cavity treatment in a dental practice setting. It is concluded that non-operative cavity treatment can be a helpful method to control caries lesion progression in primary teeth and may familiarise the child with dental treatment.

Root surfaces, which with increasing age become exposed to dental biofilms, will react to the intermittent pH fluctuations at the interface between the biofilm and the cementum/dentin surface. If dental biofilm is left undisturbed in stagnation sites in the dentition, the underlying mineral surfaces may gradually develop dental caries characterized by a subsurface loss of mineral. In root surfaces, the demineralization is accompanied by microbial invasion of the cementum and dentin resulting in a pulpo-dentinal defense reaction. Most lesions progress slowly and experimental in situ studies as well as clinical studies document that the daily removal of the biofilm using fluoride toothpaste can arrest lesion progression. By applying this caries control measure, caries lesions can thus be transformed from active lesions to inactive lesions. The diagnostic characteristics of these types of lesions are mandatory to apply in daily clinical practice to avoid unnecessary restorative and antimicrobial treatments.

This study aimed at testing how active and inactive enamel caries lesions differ by their degree of resin infiltration, and whether the choice of acid pretreatment plays a crucial role. Four examiners assessed 104 human molars and premolars with noncavitated enamel lesions and classified them as ‘active’ or ‘inactive’ using the Nyvad criteria. Forty-five teeth were included in this study after independent unanimous lesion activity assessment. Lesions were cut perpendicularly into 2 halves. Each half lesion was pretreated with either 15% hydrochloric acid or 35% phosphoric acid. The lesions were infiltrated after staining with rhodamine isothiocyanate. Thin sections of 100 µm were prepared and the specimens were bleached with 30% hydrogen peroxide. The specimens were then counterstained with sodium fluorescein, subjected to confocal laser scanning microscopy and analyzed quantitatively. Outcome parameters were maximum and average infiltration depths as well as relative penetration depths and areas. In active lesions no significant difference of percentage maximum penetration depth and percentage average penetration depth between lesions pretreated with hydrochloric or phosphoric acid could be observed. In inactive lesions, however, phosphoric acid pretreatment resulted in significantly lower penetration compared to hydrochloric acid pretreatment. Surface conditioning with hydrochloric acid led to similar infiltration results in active and inactive lesions. Moreover, inactive lesions showed greater variability in all assessed infiltration parameters than did active lesions. In conclusion, caries lesion activity and acid pretreatment both influenced the infiltration. The use of phosphoric acid to increase permeability of the surface layer of active lesions should be further explored.

Dental caries results from an imbalance of the metabolic activity in the dental biofilm. The microbial communities of teeth have traditionally been studied by standard cultural approaches. More recently, cloning and sequencing of the 16S rRNA gene have been used to characterize the microbial composition of the oral biofilm, but the methodological limitations of this approach have now been recognized. Next-generation high-throughput sequencing methods have the potential to reveal the composition and functioning of the biofilm by means of metagenomic and metatranscriptomic analyses. Currently available high-throughput sequencing approaches are reviewed and discussed in relation to studying the biofilm associated with dental caries. Important in understanding the dynamic processes in caries is the metabolic activity of the biofilm; metabolome analysis is a new tool that might enable us to assess such activity. As caries is a localized disease, it is essential that biofilm samples are taken from precisely determined tooth sites; pooling samples is not appropriate. This paper presents the case that culture-based studies are important, but that the fullest understanding of the role of the biofilm in the caries process will only come from an integrated approach determining biological function and metabolic output.

Aim To review evidence for the treatments of gingival recession and root caries in older populations. Materials & Methods A systematic approach was adopted to identify reviews and articles to allow us to evaluate the treatments for gingival recession and root caries. Searches were performed in PubMed, Medline and Embase, the Cochrane trials register and bibliographies of European and World Workshops. Observations Gingival recession: We identified no articles that focussed specifically on older populations. Conversely, no evidence suggested that Miller class I and II lesions should be managed differently in older patients when compared to younger cohorts. Six systematic reviews included older patients and suggested that connective tissue grafts are the treatment of choice, alone or in combination with enamel matrix derivative. Root caries can be controlled at the population level by daily brushing with fluoride-containing toothpastes, whilst active decay may be inactivated using professional application of fluoride varnishes/solutions or self-applied high-fluoride toothpaste. Active root caries lesions that cannot be cleaned properly by the patient may be restored by minimally invasive techniques. Conclusions Gingival recession and root caries will become more prevalent as patients retain their teeth for longer. Whilst surgical (gingival recession) and non-operative approaches (root caries) currently appear to be favoured, more evidence is needed to identify the most appropriate strategies for older people.

This 2-period crossover study investigated the effect of calcium-phosphate-osteopontin particles on biofilm formation and pH in 48-h biofilms grown in situ. Bovine milk osteopontin is a highly phosphorylated glycoprotein that has been shown to interfere with bacterial adhesion to salivary-coated surfaces. Calcium-phosphate-osteopontin particles have been shown to reduce biofilm formation and pH drops in a 5-species laboratory model of dental biofilm without affecting bacterial viability. Here, smooth surface biofilms from 10 individuals were treated ex vivo 6 times/day for 30 min with either calcium-phosphate-osteopontin particles or sterile saline. After growth, the amount of biofilm formed was determined by confocal microscopy, and pH drops upon exposure to glucose were monitored using confocal-microscopy-based pH ratiometry. A total of 160 biofilms were analysed. No adverse effects of repeated ex vivo treatment with calcium-phosphate-osteopontin particles were observed. Particle treatment resulted in a 32% lower amount of biofilm formed (p < 0.05), but large inter-individual differences could be observed. Biofilm pH was significantly higher upon particle treatment, both shortly after the addition of glucose and after 30 min of incubation with glucose (p < 0.05). Calcium-phosphate-osteopontin particles may represent a new therapeutic approach to caries control and aim at directly targeting virulence factors involved in the caries process. Further studies are required to determine the effect of particle treatment on more acidogenic/aciduric biofilms as well as the remineralizing potential of the particles.

Carious lesion activity assessment and caries risk assessment (CRA) are essential tools that assist professionals in proper caries management. This is to say that the management of carious lesions, the prescription of caries control measures, and the frequency with which these measures are applied vary according to the patient’s current caries activity and caries risk profile. Carious lesion activity should be assessed with the Nyvad criteria which have shown prognostic value in clinical trials and which guide the professional to the most appropriate treatment decision. Assessment of caries risk is not always straightforward. Although many attempts have been made to define which factor(s) should be taken into account to better predict new carious lesions, many of the CRA tools currently available have limited validity. When a single factor is used for predicting carious lesions, the patient’s past caries experience has been shown to be the most accurate predictor factor. The aim of this chapter is to summarize the evidence about CRA and carious lesion activity assessment and to discuss, through providing a few clinical cases as examples, the importance of these concepts in caries management.

Objectives: In clinical practice, a visual-tactile caries examination is frequently supplemented by bitewing radiography. This study evaluated strategies for combining visual-tactile and radiographic caries detection methods and determined their implications for clinical management decisions in a low-caries population. Methods: Each of four examiners independently examined preselected contacting interproximal surfaces in 53 dental students aged 20-37 years using a visual-tactile examination and bitewing radiography. The visual-tactile examination distinguished between noncavitated and cavitated lesions while the radiographic examination determined lesion depth. Direct inspection of the surfaces following tooth separation for the presence of cavitated or noncavitated lesions was the validation method. The true-positive rate (i.e. the sensitivity) and the false-positive rate (i.e. 1-specificity) were calculated for each diagnostic strategy. Results: Visual-tactile examination provided a true-positive rate of 34.2% and a false-positive rate of 1.5% for the detection of a cavity. The combination of a visual-tactile and a radiographic examination using the lesion in dentin threshold for assuming cavitation had a true-positive rate of 76.3% and a false-positive rate of 8.2%. When diagnostic observations were translated into clinical management decisions using the rule that a noncavitated lesion should be treated nonoperatively and a cavitated lesion operatively, our results showed that the visual-tactile method alone was the superior strategy, resulting in most correct clinical management decisions and most correct decisions regarding the choice of treatment.

It has become evident that aggregation or biofilm formation is an important survival mechanism for bacteria in almost any environment. In this review, we summarize recent visualizations of bacterial aggregates in several chronic infections (chronic otitis media, cystic fibrosis, infection due to permanent tissue fillers and chronic wounds) both as to distribution (such as where in the wound bed) and organization (monospecies or multispecies microcolonies). We correlate these biofilm observations to observations of commensal biofilms (dental and intestine) and biofilms in natural ecosystems (soil). The observations of the chronic biofilm infections point toward a trend of low bacterial diversity and sovereign monospecies biofilm aggregates even though the infection in which they reside are multispecies. In contrast to this, commensal and natural biofilm aggregates contain multiple species that are believed to coexist, interact and form biofilms with high bacterial and niche diversity. We discuss these differences from both the diagnostic and the scientific point of view.

Less invasive excavation methods have been suggested for deep caries lesions. We tested the effects of stepwise vs. direct complete excavation, 1 yr after the procedure had been carried out, in 314 adults (from six centres) who had received treatment of a tooth with deep caries. The teeth had caries lesions involving 75% or more of the dentin and were centrally randomized to stepwise or direct complete excavation. Stepwise excavation resulted in fewer pulp exposures compared with direct complete excavation [difference: 11.4%, 95% confidence interval (CI) (1.2; 21.3)]. At 1 yr of follow-up, there was a statistically significantly higher success rate with stepwise excavation, with success being defined as an unexposed pulp with sustained pulp vitality without apical radiolucency [difference: 11.7%, 95% CI (0.5; 22.5)]. In a subsequent nested trial, 58 patients with exposed pulps were randomized to direct capping or partial pulpotomy. We found no significant difference in pulp vitality without apical radiolucency between the two capping procedures after more than 1 yr [31.8% and 34.5%; difference: 2.7%, 95% CI (-22.7; 26.6)]. In conclusion, stepwise excavation decreases the risk of pulp exposure compared with direct complete excavation. In view of the poor prognosis of vital pulp treatment, a stepwise excavation approach for managing deep caries lesions is recommended.

The combined use of confocal laser scanning microscopy (CLSM) and fluorescentin situhybridization (FISH) offers new opportunities for analysis of the spatial relationships and temporal changes of specific members of the microbiota of intact dental biofilms. The purpose of this study was to analyse the patterns of colonization and population dynamics ofActinomyces naeslundiicompared to streptococci and other bacteria during the initial 48 h of biofilm formation in the oral cavity. Biofilms developed on standardized glass slabs mounted in intra-oral appliances worn by ten individuals for 6, 12, 24 and 48 h. The biofilms were subsequently labelled with probes againstA. naeslundii(ACT476), streptococci (STR405) or all bacteria (EUB338), and were analysed by CLSM. Labelled bacteria were quantified by stereological tools. The results showed a notable increase in the number of streptococci andA. naeslundiiover time, with a tendency towards a slower growth rate forA. naeslundiicompared with streptococci.A. naeslundiiwas located mainly in the inner part of the multilayered biofilm, indicating that it is one of the species that attaches directly to the acquired pellicle. The participation ofA. naeslundiiin the initial stages of dental biofilm formation may have important ecological consequences.

Udgivelsesdato: 2009-Apr The aim of this study was to assess the reliability of the Nyvad visual-tactile caries-diagnostic criteria when used among children who have been lifelong residents in areas with 'optimal' or low concentrations of fluoride in the drinking water. In each of two areas with drinking water fluoride concentrations of 0.3 and 1.1 ppm (0.3 and 1.1 mg/l) fluoride, respectively, 150 children were clinically examined twice, 2 wk apart, for dental fluorosis, using the Thylstrup-Fejerskov index (TF index), and for dental caries using the Nyvad visual-tactile caries criteria. The prevalence of dental fluorosis was 45% in the 1.1 ppm fluoride area and 21% in the 0.3 ppm fluoride area. When the results of the duplicate caries recordings were compared at the surface level, only minute differences were observed in the percentage agreement (91.7 and 90.7%, respectively) and in the kappa values (0.73 and 0.72, respectively). When individual DFS counts were compared across examinations using Bland-Altman plots and estimation of prediction intervals for the differences, we observed a greater variability of the differences between recordings among children from the low-fluoride area. Contrary to our expectations, a pronounced dental fluorosis background did not reduce the reliability of the caries recordings, which appeared to be slightly less reliable at very low levels of dental fluorosis.

In this essay we propose an extension of the caries ecological hypothesis to explain the relation between dynamic changes in the phenotypic/genotypic properties of plaque bacteria and the demineralization/remineralization balance of the caries process. Dental plaque represents a microbial ecosystem in which non-mutans bacteria (mainly non-mutans streptococci and Actinomyces) are the key microorganisms responsible for maintaining dynamic stability on the tooth surface (dynamic stability stage). Microbial acid adaptation and subsequent acid selection of ‘low-pH’ non-mutans bacteria play a critical role for destabilizing the homeostasis of the plaque by facilitating a shift of the demineralization/remineralization balance from ‘net mineral gain’ to ‘net mineral loss’ (acidogenic stage). Once the acidic environment has been established, mutans streptococci and other aciduric bacteria may increase and promote lesion development by sustaining an environment characterized by ‘net mineral loss’ (aciduric stage). Hence, high proportions of mutans streptococci and/or other aciduric bacteria may be considered biomarkers of sites of particularly rapid caries progression. This cascade of events may change the surface texture of caries lesions from smooth to rough (enamel) or hard to soft (dentin). These clinical surface features can be reversed at any stage of lesion development provided that the acidogenic/aciduric properties of the biofilm are resolved. From an ecological point of view it is therefore not only important to describe which bacteria are involved in caries, but also to know what the bacteria are doing.

Confocal laser scanning microscopy (CLSM) has been employed as a method for studying intact natural biofilm. When combined with fluorescence in situ hybridization (FISH) it is possible to analyze spatial relationships and changes of specific members of microbial populations over time. The aim of this study was to perform a systematic description of the pattern of initial dental biofilm formation by applying 16S rRNA-targeted oligonucleotide probes to the identification of streptococci and other bacteria, and to evaluate the usefulness of the combination of CLSM and FISH for structural studies of bacterial populations in dental biofilm. Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by 10 individuals for 6, 12, 24 or 48 h. After intra-oral exposure the biofilms were labelled with probes against either streptococci (STR405) or all bacteria (EUB338) and analysed by CLSM. The current approach of using FISH techniques enabled differentiation of streptococci from other bacteria and determination of their spatio-temporal organization. The presence of chimney-like multilayered microcolonies with different microbial compositions demonstrated by this methodology provided information supplementary to our previous knowledge obtained by classical electron microscopic methods and increased our understanding of the structure of developing biofilms.

Objectives: pH in dental biofilms plays a central role for the development of caries lesions. For decades, pH measurements in biofilms have been limited to recording pH with electrodes/microelectrodes that do not permit monitoring horizontal pH gradients in biofilms in real-time. Quantitative fluorescent microscopy can overcome these problems. The aim of this demonstration study was to monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4 in in-situ-grown dental biofilms.Methods: Using confocal microscopy, the dye C-SNARF-4 was employed both as pH-sensitive ratiometric dye and as a bacterial stain. We tested the method on natural 48-h in-situ-grown dental biofilms from two individuals. Four biofilms per person were collected on standardized glass slabs mounted in intra-oral appliances. Digital image analysis was employed to remove the bacterial biomass from the microscopic images in order to exclusively determine extracellular pH. We monitored the pH drop at the biofilm-substratum interface in six microscopic fields of view per biofilm for 1h after exposure to 0.4% glucose.Results: Extracellular pH dropped rapidly in all specimens. In both individuals, analysis of variance showed that terminal-pH varied more between specimens (F=6.71, p=0.001) than between microscopic fields of view within single specimens (F=1.93, p=0.112). The rate of the pH drop differed significantly between the two individuals (F=6.25, p=0.017).Conclusions: The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows monitoring of extracellular pH in in-situ biofilms without destroying the complex three-dimensional biofilm architecture. Within the limitations of this study using young biofilms and only two individuals the data suggest that biofilm pH differs more between individual specimens than between different microenvironments within one biofilm. Dent Res 94 (Spec Iss A), abstract number 4072, 2015

In this article, the fundamentals of caries diagnosis are reviewed from the three component perspectives, namely the strategy, the logics, and the tactics. Strategy concerns the objectives of the diagnostic process (i.e. why we diagnose caries). The logics describe how we assemble and evaluate the information collected and how this leads to an assessment of diagnostic value. Finally, tactics are about how we collect the information necessary to arrive at a correct diagnosis. We argue that the hitherto-dominant essentialistic caries paradigm should be replaced by a nominalistic caries concept. This allows us to circumvent the problem of a lack of a caries gold standard and to proceed in caries-diagnostic research to find the diagnostic methods that result in the best health outcomes for our patients. We also demonstrate the limitations of the medical model when attempting to understand caries diagnosis, and adhere to the Bader & Shugars caries script model. It is concluded that the current caries profile, characterized by lower prevalence and extent, and slower progression, has increased the need for an academic strengthening of the dental curriculum with respect to diagnostic reasoning and clinical decision-making processes.

The aim of this study was to determine the effect of saliva composition on caries lesion development independently of the flow rate of unstimulated whole saliva (UWS) and other caries-related variables such as lesion progression time, oral hygiene level, and fluoride exposure. We hypothesized that this could be done by developing experimental root caries under carefully controlled conditions in situ in test subjects with UWS flow rates within a narrow window of normalcy. Fifteen female and 5 male subjects (66 ± 6 years) were selected for the study according to their UWS flow rates between 0.2 and 0.4 ml/min. All subjects developed experimental root caries lesions during a 62-day period in which UWS as well as stimulated whole saliva (SWS) were repeatedly collected and analysed for flow rate, pH, buffer capacity, inorganic, and organic composition. Caries lesion development was determined by quantitative microradiography. The mean UWS flow rate was 0.30 ± 0.05 ml/min. Significant negative correlations were obtained between UWS total phosphate concentration and mineral loss (ΔZ; rs = –0.72, p < 0.001) and UWS total protein concentration and ΔZ (rs = –0.70, p < 0.01). SWS and its constituents had only limited or no effect on ΔZ. Qualitative UWS protein analysis (SDS-PAGE) revealed that subjects with low ΔZ values had broader and more stained amylase bands than subjects with high ΔZ values. These findings were confirmed quantitatively by HPLC. We conclude that, within a group of subjects with normal UWS flow rates, the UWS composition was more important for caries lesion development than the SWS composition. Furthermore, high UWS concentrations of phosphate, protein, and amylase were caries-protective.

Caries diagnosis is the art or act of identifying a disease from its signs and symptoms. This is distinct from the detection of the signs and symptoms themselves. The diagnosis forms the basis for making informed treatment decisions. Hence, if there is no diagnostic step expressed in terms of the probability of present and future occurrence of disease, practitioners may resort to treatments guided by previous experiences with similar clinical manifestations. This paper reviews various methodological aspects of caries diagnostic testing. It is concluded that rather than continuing to search for the truth of the diagnosis, it may be more informative to consider the consequences of the diagnosis. This view is supported by results from caries-preventive trials in which the activity of carious lesions has been monitored longitudinally over years.

Objective: To demonstrate the usefulness of a survival-time regression model for the analysis of data from two 3-year trials of the caries-preventive effect of sugar-substituted chewing gums and fluoride toothpaste, carried out among 892 Lithuanian children. Methods: A caries onset was defined as a transition from sound to carious and a caries recovery was defined as a transition from carious to sound. The time at risk for each type of transition was calculated. Using an exponential survival-time regression model, the hazard ratios for the covariates experimental group (control, sugar substitute, fluoride), age, gender, surface type and posteruptive surface age was estimated. This analysis was repeated using two alternative definitions of the caries transitions. Results: The analyses confirmed that caries rates are higher in occlusal surfaces, and that posteruptive surface age influences caries rates. Moreover, it also confirmed that fluoride affects the outcome of ongoing caries activity more than the initiation of caries. Conclusions: Survival-time analysis of caries transitions allows for the extraction of much more information from caries trials than does the traditional DMF-based analysis, and traditional DMF incremental values may easily be derived from the models.

Microbiological studies of occlusal dental biofilms have hitherto been hampered by inaccessibility to the sampling site and demolition of the original biofilm architecture. This study shows for the first time the spatial distribution of bacterial taxa in vivo at various stages of occlusal caries, applying a molecular methodology involving preparation of embedded hard dental tissue slices for fluorescence in situ hybridization (FISH) and confocal microscopy. Eleven freshly extracted teeth were classified according to their occlusal caries status. The teeth were fixed, embedded, sectioned and decalcified before FISH was performed using oligonucleotide probes for selected abundant species/genera associated with occlusal caries including Streptococcus, Actinomyces, Veillonella, Fusobacterium, Lactobacillus and Bifidobacterium. The sites showed distinct differences in the bacterial composition between different ecological niches in occlusal caries. Biofilm observed along the entrance of fissures showed an inner layer of microorganisms organized in palisades often identified as Actinomyces, covered by a more loosely structured bacterial layer consisting of diverse genera, similar to supragingival biofilm. Biofilm within the fissure proper seemed less metabolically active, as judged by low fluorescence signal intensity and presence of material of non-bacterial origin. Bacterial invasion (often Lactobacillus and Bifidobacterium spp.) into the dentinal tubules was seen only at advanced stages of caries with manifest cavity formation. It is concluded that the molecular methodology represents a valuable supplement to previous methods for the study of microbial ecology in caries by allowing analysis of the structural composition of the undisturbed biofilm in caries lesions in vivo.

–Objectives: The aim of this 3-year community intervention trial was to determine the caries preventive effect of sugar-substituted chewing gum among Lithuanian school children, and to assess compliance with the instructions for gum use. Methods: A total of 602 children, aged 9–14 years, from 28 school classes in five secondary schools in Kaunas, Lithuania, were given a baseline clinical and radiographic caries examination. The schools were randomly allocated to receive one of the following interventions: sorbitol/carbamide gum; sorbitol gum; xylitol gum; control gum; and no gum. Children in the four active intervention groups were asked to chew at least five pieces of gum per day, preferably after meals. The children were reexamined clinically after 1, 2 and 3 years, and radiographically after 3 years. Self-reported compliance was monitored anonymously four times during the study. Results: A total of 432 children were available at the 3-year clinical follow-up examination. The crude mean 3-year caries increments (DMFS – all stages of lesion formation) were 11.8 for the sorbitol/carbamide gum group; 9.0 for the sorbitol gum group; 8.1 for the xylitol gum group; 8.3 for the control gum group; and 12.4 for the no gum group. The adjusted 3-year caries increments were statistically significantly lower in the sorbitol gum group, the xylitol gum group and the control gum group than in the no gum group, whereas no statistically significant difference was seen between the no gum group and the sorbitol/carbamide gum group. Adjusted 3-year caries increments in the xylitol gum group and the sorbitol gum group did not differ statistically significantly from the caries increments in the control gum group. Compliance with the study protocol was better in School C (xylitol gum) than in the other schools. In all schools, compliance decreased over time. Conclusion: The results indicate that the caries preventive effect of chewing sugar-free gum is related to the chewing process itself rather than being an effect of gum sweeteners or additives, such as polyols and carbamide.

The buffer capacity of unstimulated (UWS) and stimulated (SWS) whole-mouth saliva involves three major buffer systems. The aim was to determine the buffer capacity of UWS and SWS at specific pH in the interval from pH 7.5 down to pH 3.0. The contribution of each of the buffer systems was also determined under conditions resembling those in the mouth. UWS and SWS were collected from 20 healthy volunteers; the saliva was collected under paraffin oil in order to avoid loss of CO2. The buffer capacity of UWS and SWS in samples with and without bicarbonate (HCO3-) and CO2 were measured at various pH by acid titration in a closed system at 36 C. The mean concentrations of the buffer systems in UWS (mean flow rate 0.55 ml/min) were 4.4 mmol/l HCO3-, 4.5 mmol/l phosphate (of which 1.3 mmol/l was present in the form of HPO4(2-)), 1876 microg/ml protein; the saliva pH was 6.8 and the P(CO2) 29.3 mmHg. The corresponding mean concentrations in SWS (mean flow rate 1.66 ml/min) were 9.7 mmol/l HCO3-, 3.8 mmol/l phosphate (of which 1.9 mmol/l was present in the form of HPO4(2-)), 1955 microg/ml protein; pH 7.2 and P(CO2) 25.7 mmHg, The highest buffer capacity of UWS and SWS was 6.0 and 8.5 mmol H+ /(1 saliva*pH unit) at pH 6.25, respectively. At saliva pH in the range from pH 7 down to pH 5, the following had significant impact on buffer capacity: the HCO3- concentration (p < 0.001), the flow rate (p < 0.01), and the pH of the saliva (p < 0.05). At acidic pH in the range from pH 5 down to pH 4, however, only the protein concentration had a significant impact on buffer capacity (p < 0.01).