Caries lesion transition patterns of schoolchildren in a fluoridated community in Brazil.

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.

Integrated hypothesis of dental caries and periodontal diseases.

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.

Reliability and validity of a new classification of MIH based on severity.

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.

Terminology of Dental Caries and Dental Caries Management: Consensus Report of a Workshop Organized by ORCA and Cariology Research Group of IADR.

A 2-day workshop of ORCA and the IADR Cariology Research Group was organized to discuss and reach consensus on definitions of the most commonly used terms in cariology. The aims were to identify and to select the most commonly used terms of dental caries and dental caries management and to define them based on current concepts. Terms related to definition, diagnosis, risk assessment, and monitoring of dental caries were included. The Delphi process was used to establish terms to be considered using the nominal group method favored by consensus. Of 222 terms originally suggested by six cariologists from different countries, a total of 59 terms were reviewed after removing duplicates and unnecessary words. Sixteen experts in cariology took part in the process of reaching consensus about the definitions of the selected caries terms. Decisions were made following thorough "round table" discussions of each term and confirmed by secret electronic voting. Full agreement (100%) was reached on 17 terms, while the definitions of 6 terms were below the agreed 80% threshold of consensus. The suggested terminology is recommended for use in research, in public health, as well as in clinical practice.

[Improvement of Danish children and adolescents' food and meal habits].

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.

Fluoride in Saliva and Oral Mucosa after Brushing with 1,450 or 5,000 ppm Fluoride Toothpaste.

The aim was to measure and compare fluoride concentrations in oral mucosa and saliva following a single brushing with either 1,450 or 5,000 ppm fluoride toothpaste. Fourteen healthy participants provided saliva and oral mucosa samples in the morning before tooth brushing. Then participants brushed their teeth with 1,450 ppm fluoride toothpaste, and saliva and mucosa samples were collected after 1, 2, 4, and 6 h. The experiment was repeated 3-7 days later with 5,000 ppm fluoride toothpaste. All samples were analyzed for fluoride using an ion-selective electrode adapted for microanalysis. Pre-brushing fluoride concentrations were higher in mucosa (mean1,450 0.26 ppm and mean5,000 0.20 ppm) than in saliva (mean1,450 0.08 ppm and mean5,000 0.07 ppm). The mean fluoride concentrations increased in both mucosa and saliva following a single brushing with both 1,450 ppm (meanmuc1,450 (1 h) 1.15 ppm, meansal1,450 (1 h) 0.33 ppm) and 5,000 ppm fluoride toothpaste (meanmuc5,000 (1 h) 3.21 ppm and meansal5,000 (1 h) 0.90 ppm). At 6 h, the fluoride concentrations had returned to pre-brushing levels. Across the 6-h sampling period the fluoride concentration in saliva was statistically significantly 1.4 times higher following brushing with 5,000 ppm compared with 1,450 ppm fluoride toothpaste. For mucosa, this ratio was only 1.1 and not statistically significant. In conclusion, the fluoride level in oral buccal mucosa is higher than in saliva and follows the same fluoride clearance pattern as in saliva. Over the initial 6-h period following a single tooth brushing, the ratio of the fluoride concentration in mucosa to that in saliva is independent of the fluoride concentrations in the toothpastes used.

Nyvad Criteria for Caries Lesion Activity and Severity Assessment: A Validated Approach for Clinical Management and Research.

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.

Fluoride in saliva and dental biofilm after 1500 and 5000 ppm fluoride exposure.

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.

Root Surface Caries - Rationale Behind Good Diagnostic Practice.

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.

Fluoride in Dental Biofilm Varies across Intra-Oral Regions.

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.

Dental caries and periodontal diseases in the ageing population: call to action to protect and enhance oral health and well-being as an essential component of healthy ageing - Consensus report of group 4 of the joint EFP/ORCA workshop on the boundaries between caries and periodontal diseases.

BACKGROUND: Over the last two decades, progress in prevention and treatment of caries and periodontal diseases has been translated to better oral health and improved tooth retention in the adult population. The ageing population and the increasing expectations of good oral health-related quality of life in older age pose formidable challenges to clinical care and healthcare systems. AIMS: The objective of this workshop was to critically review scientific evidence and develop specific recommendations to: (i) prevent tooth loss and retain oral function through prevention and treatment of caries and periodontal diseases later in life and (ii) increase awareness of the health benefits of oral health as an essential component of healthy ageing. METHODS: Discussions were initiated by three systematic reviews covering aspects of epidemiology of caries and periodontal diseases in elders, the impact of senescence on caries and periodontal diseases and the effectiveness of interventions. Recommendations were developed based on evidence from the systematic reviews and expert opinion. RESULTS: Key messages included: (i) the ageing population, trends in risk factors and improved tooth retention point towards an expected increase in the total burden of disease posed by caries and periodontal diseases in the older population; (ii) specific surveillance is required to monitor changes in oral health in the older population; (iii) senescence impacts oral health including periodontitis and possibly caries susceptibility; (iv) evidence indicates that caries and periodontal diseases can be prevented and treated also in older adults; (v) oral health and functional tooth retention later in life provides benefits both in terms of oral and general quality of life and in terms of preventing physical decline and dependency by fostering a healthy diet; (vi) oral healthcare professionals and individuals should not base decisions impacting tooth retention on chronological age but on level of dependency, life expectancy, frailty, comfort and quality of life; and (vii) health policy should remove barriers to oral health care for vulnerable elders. CONCLUSIONS: Consensus was reached on specific actionable priorities for public health officials, oral healthcare professionals, educators and workforce planners, caregivers and relatives as well as for the public and ageing patients. Some priorities have major implications for policymakers as health systems need to adapt to the challenge by systemwide changes to enable (promote) tooth retention later in life and management of deteriorating oral health in increasingly dependent elders.

Gingival recession and root caries in the ageing population: a critical evaluation of treatments.

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.

Calcium-Phosphate-Osteopontin Particles Reduce Biofilm Formation and pH Drops in in situ Grown Dental Biofilms.

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.

Ecological Hypothesis of Dentin and Root Caries.

Recent advances regarding the caries process indicate that ecological phenomena induced by bacterial acid production tilt the de- and remineralization balance of the dental hard tissues towards demineralization through bacterial acid-induced adaptation and selection within the microbiota - from the dynamic stability stage to the aciduric stage via the acidogenic stage [Takahashi and Nyvad, 2008]. Dentin and root caries can also be partly explained by this hypothesis; however, the fact that these tissues contain a considerable amount of organic material suggests that protein degradation is involved in caries formation. In this review, we compiled relevant histological, biochemical, and microbiological information about dentin/root caries and refined the hypothesis by adding degradation of the organic matrix (the proteolytic stage) to the abovementioned stages. Bacterial acidification not only induces demineralization and exposure of the organic matrix in dentin/root surfaces but also activation of dentin-embedded and salivary matrix metalloproteinases and cathepsins. These phenomena initiate degradation of the demineralized organic matrix in dentin/root surfaces. While a bacterial involvement has never been confirmed in the initial degradation of organic material, the detection of proteolytic/amino acid-degrading bacteria and bacterial metabolites in dentin and root caries suggests a bacterial digestion and metabolism of partly degraded matrix. Moreover, bacterial metabolites might induce pulpitis as an inflammatory/immunomodulatory factor. Root and dentin surfaces are always at risk of becoming demineralized in the oral cavity, and exposed organic materials can be degraded by host-derived proteases contained in saliva and dentin itself. New approaches to the prevention and treatment of root/dentin caries are required.

Calcium-phosphate-osteopontin particles for caries control.

Caries is caused by acid production in biofilms on dental surfaces. Preventing caries therefore involves control of microorganisms and/or the acid produced. Here, calcium-phosphate-osteopontin particles are presented as a new approach to caries control. The particles are made by co-precipitation and designed to bind to bacteria in biofilms, impede biofilm build-up without killing the microflora, and release phosphate ions to buffer bacterial acid production if the pH decreases below 6. Analysis of biofilm formation and pH in a five-species biofilm model for dental caries showed that treatment with particles or pure osteopontin led to less biofilm formation compared to untreated controls or biofilms treated with osteopontin-free particles. The anti-biofilm effect can thus be ascribed to osteopontin. The particles also led to a slower acidification of the biofilm after exposure to glucose, and the pH always remained above 5.5. Hence, calcium-phosphate-osteopontin particles show potential for applications in caries control.

Monitoring of extracellular pH in young dental biofilms grown in vivo in the presence and absence of sucrose.

BACKGROUND AND OBJECTIVE: pH in dental biofilms is of central importance for the development of caries. We used the ratiometric pH-sensitive dye C-SNARF-4 in combination with digital image analysis to monitor extracellular pH in dental biofilms grown in situ with and without sucrose supply. DESIGN: Dental biofilms (48 h) from 10 individuals were collected on glass slabs mounted on intra-oral appliances. During growth, appliances were immersed extra-orally in either physiological saline or 4% sucrose for 2 min, eight times per day. Fluorescence emissions of C-SNARF-4 in deep layers of the biofilms were recorded ex vivo with confocal microscopy for 15 min or for 1 h after exposure to 0.4% glucose. Extracellular pH was determined ratiometrically using digital image analysis. RESULTS: Extracellular pH dropped rapidly in most examined sites after addition of glucose. Distinct pH microenvironments were observed within single biofilms. The variation in pH was similar between sites within the same biofilm and sites from different individuals. pH drop patterns did not differ between biofilms exposed to sucrose-free and sucrose-rich environments. CONCLUSION: The present study is the first of its kind to apply the combination of pH ratiometry and digital image analysis to systematically record extracellular pH in intact dental biofilms from several individuals for up to 1 h. We observed highly heterogeneous pH landscapes and the presence of acidogenic microenvironments - 'acidogenic hotspots' - within the biofilms. The data suggest that pH drops in young (48 h) dental biofilms are independent of the sucrose supply during growth.

Infected Dentine Revisited.

Dentine becomes infected as a result of caries lesion formation on root surfaces and when lesions progress following cavitation of enamel lesions. However, this infection is unimportant because the driving force for lesion formation and progression is the overlying biofilm. This explains why root surface caries can be controlled by mechanical plaque control and fluoride, and restorations are not needed to arrest these lesions. Similarly, the infected dentine in cavitated coronal lesions does not have to be removed to arrest the lesion. If the lesion is either accessible or opened for cleaning by the patient or parent, the lesion can be arrested. Sealing of infected dentine within the tooth, either by a Hall crown in the primary dentition or by partial caries removal prior to placing a well-sealed filling, will also arrest the lesion. When restoring deep lesions in symptomless, vital teeth, vigorous excavation of infected dentine is likely to expose the pulp and make root canal treatment necessary. Thus complete excavation'is not needed and should be avoided. CPD/CLINICAL RELEVANCE: Root surface caries can be arrested by cleaning and fluoride application. Restorations are not essential. Vigorous excavation of softened dentine in deep cavities of symptomless, vital teeth is contra-indicated. It is not needed and increases the risk of pulp exposure.

Ratiometric imaging of extracellular pH in bacterial biofilms with C-SNARF-4.

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 <6, C-SNARF-4 stained 15 bacterial species frequently isolated from dental biofilm and visualized the entire bacterial biomass in 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.

Molecular studies of the structural ecology of natural occlusal caries.

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.