Salivary fluoride levels after daily brushing with 5000 ppm fluoride toothpaste: A randomised, controlled clinical trial.

This study explored salivary fluoride levels following toothbrushing with 5000 and 1450 ppm fluoride toothpaste and determined the decline in salivary fluoride levels following the return from 5000 to 1450 ppm fluoride toothpaste. The study was a randomised, controlled double-blind parallel clinical trial (n = 24/group) measuring salivary fluoride five times during a 3-week trial phase involving 2×/day use of 5000 or 1450 ppm fluoride toothpaste, and five times during an ensuing 2-week wash-out phase where all participants used 1450 ppm toothpaste. Salivary fluoride was measured using a fluoride electrode and data were analysed using multilevel mixed-effects linear regression. Baseline salivary fluoride geometric means were 0.014 and 0.016 ppm for the 1450 and 5000 ppm groups, while the values at the end of the trial phase were 0.023 and 0.044 ppm, respectively. During the trial phase, except at baseline, differences between groups were statistically significant. The salivary fluoride levels for the 5000 ppm group remained statistically significantly higher than for the 1450 ppm group only at the first measurement in the wash-out phase (≈30 h after the last 5000 ppm brushing), indicating that higher salivary fluoride levels resulting from use of 5000 ppm are sustained only as long as the brushing habit continues.

Candida species in intact in vivo biofilm from carious lesions.

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.

Non-operative control of cavitated approximal caries lesions in primary molars: a prospective evaluation of cases.

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.

Microbial geography of the oral cavity.

We aimed to determine the bacterial diversity of different oral micro-niches and to assess whether saliva and plaque samples are representative of oral microbial composition. We took minute samples from each surface of the individual teeth and gingival crevices of two healthy volunteers (112 samples per donor), as well as samples from the tongue dorsum and non-stimulated and stimulated saliva. DNA was extracted from 67 selected samples of each donor, and the 16S rRNA gene was amplified by PCR and pyrosequenced to obtain, on average, over 2,700 reads per sample, which were taxonomically assigned to obtain a geographic map of bacterial diversity at each tooth and sulcus location. Analysis of the data shows considerable differences in bacterial composition between teeth at different intra-oral locations and between surfaces of the same tooth. The most pronounced differences were observed in incisors and canines, where genera like Streptococcus were found at 40% to 70% on the vestibular surfaces but were almost absent on the lingual sides. Saliva samples, especially non-stimulated saliva, were not representative of supra-and subgingival plaque in the two individuals tested. We suggest that more precise sampling is required for the proper determination of oral microbial composition and to relate that diversity to epidemiological, clinical, and etiological parameters.

Saliva fluoride before and during 3 years of supervised use of fluoride toothpaste.

OBJECTIVES: The purpose of the study was to examine pre-brushing saliva fluoride concentrations before and during a large, 3-year, prospective toothpaste study on the effect of post-brushing rinsing on dental caries. The aims were to study saliva fluoride over time and the effect of rinsing on saliva fluoride and to relate saliva fluoride to caries increments and accumulation of plaque. MATERIALS AND METHODS: Saliva samples (baseline and 1, 2, and 3 years) were collected from 11-year-old children attending two schools (A and B) in Kaunas, Lithuania, who refrained from brushing the evening and morning before saliva collection. Numbers of saliva samples collected varied from 264 at baseline to 188 at the 3-year follow-up. Children in school A rinsed with water after daily brushing, while children in school B did not rinse. Total caries and visible plaque were registered at baseline and after 3 years. RESULTS: Mean saliva fluoride concentrations at baseline and after 1, 2, and 3 years from school A (rinsing) were 0.014, 0.026, 0.029, and 0.034 ppm and from school B (no rinsing) were 0.013, 0.028, 0.031, and 0.031 ppm, respectively. Increases in saliva fluoride from baseline were significant (Wilcoxon's test, p < 0.001), but the increase from baseline to year 1 was not statistically significantly different between schools. Saliva fluoride did not increase beyond year 1 and did at no time point differ between schools. Reductions in numbers of tooth surfaces with dental plaque were significantly positively related to the number of caries reversals over the 3 years. CONCLUSIONS: Background saliva fluoride concentration is increased by brushing at least once daily on schooldays, does not increase further over 3 years, and is not affected by rinsing after brushing. CLINICAL RELEVANCE: Continuous use of fluoride toothpaste produces ambient saliva fluoride levels similar to saliva fluoride in areas with fluoridated water.

Dental caries from a molecular microbiological perspective.

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.

Infiltration of natural caries lesions in relation to their activity status and acid pretreatment in vitro.

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 in Rome, 50-100 AD.

Scarce information exists on the clinical features of dental caries in the Imperial Roman population and no structural data on caries lesions from this period have so far been published. We report on the findings of 86 teeth (50-100 AD) found during archaeological excavations of the temple of Castor and Pollux in the Forum Romanum. We found that nearly all teeth had large carious cavities extending into the pulp. The distribution and size of the caries lesions were similar to those found in contemporary adult populations in Africa and China living without access to dental care. Most lesions had a hypermineralized zone in the dentin at the advancing front of the carious cavities as revealed by micro-computed tomography. This biological dentin reaction combined with the morphology of the cavities might indicate that some temporary topical pain relief and intervention treatment slowed down the rate of lesion progression. This is indirectly supported by examination of cavities of similar size and depth from a contemporary population without access to dental health care. In contrast to the lesions in the Roman teeth, these lesions did not exhibit a hypermineralized dentin reaction. We investigated whether the Pb isotopic composition of enamel and/or dentin of a single tooth matched that of a sample of an ancient Forum water lead pipe. The Pb isotopic composition of the tooth did not match that of the tube, suggesting that the subjects were exposed to different Pb sources during their lifetime other than the lead tubes.

Colonization of the oral cavity by probiotic bacteria.

The aim of this study was to investigate if three probiotic bacteria present in the milk product Cultura Dofilus® naturell could be detected in saliva and on oral mucosal surfaces, and if they colonized dental surfaces in situ in 8 caries-inactive individuals after 8 daily exposures to the milk product for up to 3 days. Bacteria were identified by fluorescence in situ hybridization and confocal laser scanning microscopy. While probiotic bacteria were present sporadically in the oral cavity on mucosal surfaces and in saliva after 3 days of frequent use of the probiotic milk, they were not detected on dental surfaces. Probiotic bacteria may thus contribute to general oral health, but their potential role in biofilm-induced dental diseases remains unclear.

Evaluation of perpendicular reflection intensity for assessment of caries lesion activity/inactivity.

The aim of this study was to evaluate, using visual assessment, an experimental optical sensor measuring perpendicular reflection intensity (PRI) as an indicator of enamel caries lesion activity/inactivity. Forty teeth with either an active or an inactive enamel lesion were selected from a pool of extracted teeth. Each tooth was cut into halves, with a clinically sound half and a half with a non-cavitated enamel lesion. After gentle plaque removal, the teeth were kept moistened. The lesions were then photographed and a defined measuring site per lesion was chosen and indicated with an arrow on a printout. Independently, the chosen site was visually assessed for lesion activity, and its glossiness was measured with PRI assessment. Surface roughness (SR) was assessed with optical profilometry using a confocal microscope. Visual assessment and PRI were repeated after several weeks and a reliability analysis was performed. For enamel lesions visually scored as active versus inactive, significantly different values were obtained with both PRI and SR. PRI values of the clinically sound control surfaces were significantly different only from active lesions. Generally, inactive lesions had the same glossiness and the same roughness as the sound control surfaces. The reliabilities for visual assessment (κ = 0.89) and for PRI (ICC = 0.86) were high. It is concluded that, within the limits of this study, PRI can be regarded as a promising tool for quantitative enamel lesion activity assessment. There is scope and potential for the PRI device to be considerably improved for in vivo use.

The role of bacteria in the caries process: ecological perspectives.

Dental biofilms produce acids from carbohydrates that result in caries. According to the extended caries ecological hypothesis, the caries process consists of 3 reversible stages. The microflora on clinically sound enamel surfaces contains mainly non-mutans streptococci and Actinomyces, in which acidification is mild and infrequent. This is compatible with equilibrium of the demineralization/remineralization balance or shifts the mineral balance toward net mineral gain (dynamic stability stage). When sugar is supplied frequently, acidification becomes moderate and frequent. This may enhance the acidogenicity and acidurance of the non-mutans bacteria adaptively. In addition, more aciduric strains, such as 'low-pH' non-mutans streptococci, may increase selectively. These microbial acid-induced adaptation and selection processes may, over time, shift the demineralization/remineralization balance toward net mineral loss, leading to initiation/progression of dental caries (acidogenic stage). Under severe and prolonged acidic conditions, more aciduric bacteria become dominant through acid-induced selection by temporary acid-impairment and acid-inhibition of growth (aciduric stage). At this stage, mutans streptococci and lactobacilli as well as aciduric strains of non-mutans streptococci, Actinomyces, bifidobacteria, and yeasts may become dominant. Many acidogenic and aciduric bacteria are involved in caries. Environmental acidification is the main determinant of the phenotypic and genotypic changes that occur in the microflora during caries.

Actinomyces naeslundii in initial dental biofilm formation.

The combined use of confocal laser scanning microscopy (CLSM) and fluorescent in situ hybridization (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 of Actinomyces naeslundii compared 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 against A. 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 and A. naeslundii over time, with a tendency towards a slower growth rate for A. naeslundii compared with streptococci. A. naeslundii was 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 of A. naeslundii in the initial stages of dental biofilm formation may have important ecological consequences.

Application of stereological principles for quantification of bacteria in intact dental biofilms.

INTRODUCTION: Quantative confocal laser scanning microscopy (CLSM) in combination with fluorescent in situ hybridization (FISH) may help to increase our knowledge about biofilm formation. The purpose of this study was to develop and evaluate a stereological method for quantification of bacteria in intact biofilm. The method was applied in a quantitative study of the proportion of streptococci relative to other bacteria in initial in-situ-grown dental biofilms as a function of time. METHODS: Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by 10 individuals for 6, 12, 24, and 48 h. Biofilms were analysed using CLSM. Quantification of bacteria labelled with 16S ribosomal RNA oligonucleotide probes was performed with stereological tools: the unbiased counting frame and the two-dimensional fractionator. RESULTS: Results showed a notable increase in the total number of bacteria and streptococci over time, with a considerable inter-individual variation at each time-point. After 48 h there was a 12.5-fold difference between individuals in the total number of bacteria and a 12.6-fold difference in the number of streptococci. The number of streptococci exceeded that of other bacteria and over the examination period there was a relatively constant relationship between the number of streptococci and other bacteria (streptococci vs. non-streptococci: median 15.2; minimum 1.0; maximum 89.3). CONCLUSION: The study demonstrates that the combined use of FISH and stereology is a relevant and reliable tool for obtaining unbiased information about the numerical contributions of specific bacterial populations during early biofilm formation.

Caries ecology revisited: microbial dynamics and the caries process.

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.

Dental caries increments and related factors in children with type 1 diabetes mellitus.

The aim of this study was to analyse possible associations between caries increments and selected caries determinants in children with type 1 diabetes mellitus and their age- and sex-matched non-diabetic controls, over 2 years. A total of 63 (10-15 years old) diabetic and non-diabetic pairs were examined for dental caries, oral hygiene and salivary factors. Salivary flow rates, buffer effect, concentrations of mutans streptococci, lactobacilli, yeasts, total IgA and IgG, protein, albumin, amylase and glucose were analysed. Means of 2-year decayed/missing/filled surface (DMFS) increments were similar in diabetics and their controls. Over the study period, both unstimulated and stimulated salivary flow rates remained significantly lower in diabetic children compared to controls. No differences were observed in the counts of lactobacilli, mutans streptococci or yeast growth during follow-up, whereas salivary IgA, protein and glucose concentrations were higher in diabetics than in controls throughout the 2-year period. Multivariable linear regression analysis showed that children with higher 2-year DMFS increments were older at baseline and had higher salivary glucose concentrations than children with lower 2-year DMFS increments. Likewise, higher 2-year DMFS increments in diabetics versus controls were associated with greater increments in salivary glucose concentrations in diabetics. Higher increments in active caries lesions in diabetics versus controls were associated with greater increments of dental plaque and greater increments of salivary albumin. Our results suggest that, in addition to dental plaque as a common caries risk factor, diabetes-induced changes in salivary glucose and albumin concentrations are indicative of caries development among diabetics.

Effect of saliva composition on experimental root caries.

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 (DeltaZ; r(s) = -0.72, p < 0.001) and UWS total protein concentration and DeltaZ (r(s) = -0.70, p < 0.01). SWS and its constituents had only limited or no effect on DeltaZ. Qualitative UWS protein analysis (SDS-PAGE) revealed that subjects with low DeltaZ values had broader and more stained amylase bands than subjects with high DeltaZ 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.

Comparison of diagnostic yields of clinical and radiographic caries examinations in children of different age.

AIM: This was to investigate if the pattern of distribution of caries lesions detected by clinical and radiographic examinations at different diagnostic thresholds changed over a 3-year period during the course of eruption of the permanent dentition. It has been hypothesized that the contribution of bitewing radiography to caries detection in posterior teeth may increase when approximal contacts are established during maturation of the dentition. METHODS: Clinical and radiographic caries recordings were made using the non- cavitated/enamel and cavity/dentine thresholds for caries detection among a group of 12-year-old Lithuanian children with a high caries experience. Examinations were repeated in the same children after 3 years. The bitewing radiographs from both examination sessions were coded to ensure unbiased recordings and were read by a single examiner. RESULTS: The diagnostic yields of the clinical and radiographic examination methods did not change after the permanent teeth had fully erupted. At both examination sessions the clinical examination resulted in the detection of significantly more lesions than did the radiographic method at the non-cavitated level (43- 47% lesions detected by clinical means only and 2-26% by radiographic means only). Only for approximal surfaces at the cavity/dentine diagnostic threshold did radiographs contribute to more lesions (15-16% lesions detected by clinical means only and 38-41% by radiographic means only). Establishment of approximal contacts in the permanent dentition did not increase the relative diagnostic yields of bite-wing radiography in this study group. CONCLUSION: The contribution of the two methods to caries diagnosis depends more on the diagnostic threshold selected than on the stage of maturation of the dentition.

Diagnosis versus detection of caries.

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.

Construct and predictive validity of clinical caries diagnostic criteria assessing lesion activity.

Even though there is no "gold standard" for determining caries lesion activity, it is nonetheless possible to evaluate the validity of such diagnostic measures. The aim of this study was to estimate the construct and predictive validity of caries lesion activity assessments by means of their ability to reflect known effects of fluoride on caries. A three-year trial of the effect of daily supervised brushing with fluoride toothpaste was carried out among 273 12-year-old children. All children were examined clinically according to diagnostic criteria for activity assessment. The relative risk (fluoride vs. control) for caries lesion transitions among diagnostic categories was calculated. Fluoride inhibited progression of caries at all stages of lesion formation while at the same time enhancing lesion regression. The effects were most pronounced for active non-cavitated lesions. It is concluded that the clinical diagnostic criteria have construct and predictive validity for the assessment of caries lesion activity.