J.M. ten Cate

Pyrosequencing analysis of the Oral Microflora of healthy adults

A good definition of commensal microflora and an understanding of its relation to health are essential in preventing and combating disease. We hypothesized that the species richness of human oral microflora is underestimated. Saliva and supragingival plaque were sampled from 71 and 98 healthy adults, respectively. Amplicons from the V6 hypervariable region of the small-subunit ribosomal RNA gene were generated by PCR, pooled into saliva and plaque pools, and sequenced by means of the Genome Sequencer 20 system at 454 Life Sciences. Data were evaluated by taxonomic and rarefaction analyses. The 197,600 sequences generated yielded about 29,000 unique sequences, representing 22 taxonomic phyla. Grouping the sequences in operational taxonomic units (6%) yielded 3621 and 6888 species-level phylotypes in saliva and plaque, respectively. This work gives a radically new insight into the diversity of human oral microflora, which, with an estimated number of 19,000 phylotypes, is considerably higher than previously reported.

Alternating Demineralization and Remineralization of Artificial Enamel Lesions

Mineral loss and uptake in artificial enamel lesions was investigated by pH cycling: samples were subjected to a sequence of de- and remineralizing solutions. The ratio between de- and remineralization per day and the presence of fluoride in the remineralizing solution were varied among different experimental groups. Calcium, phosphate and fluoride uptake and loss were measured. The lesions were also studied with a hardness tester and micro radiography. The results show a difference in remineralization mechanism between continuously remineralized and pH-cycled samples (with respect to Ca/P ratios). With fluoride present, after some time, ‘arrested’ carious lesions are formed, which can hardly be further demineralized or remineralized. This is in agreement with clinical findings of remineralization in areas with fluoridation of the drinking water.

Influence of Fluoride in Solution on Tooth Demineralization

In vitro enamel demineralization was studied in undersaturated calcium phosphate solutions containing fluoride in the concentration range 0–10 ppm. Depending on the fluoride concentration either erosi

Orthodontic appliances and enamel demineralization Part 2. Prevention and treatment of lesions

Clinical experiments were performed to investigate the effect of fluoride on carious lesion development and on lesions established during fixed orthodontic therapy. All presently available fluoride agents are developed from the concept of fluoridating the enamel in the form of fluorhydroxyapatite. Recent research has indicated, however, that calcium fluoride formation may be a major aspect of the mechanism of the cariostatic effect of topical fluoride. Therefore a fluoride solution with very low pH (1.9) that induced large amounts of calcium fluoride also was tested on lesion development underneath orthodontic bands. Daily fluoride mouth rinsing with a 0.2% solution sodium fluoride (NaF) retarded lesion development significantly, whereas the fluoride solution with low pH inhibited lesion formation completely. Fluoride applied as a mouth rinse to plaque-covered lesions underneath orthodontic bands retarded lesion progression. The remineralizing capacity of saliva was found to be rapid in the absence of any fluoride. Although white spot lesions may remineralize and even disappear, most of the emphasis should be directed against prevention of carious lesion development during treatment with fixed orthodontic appliances.

In vitro Studies on the Effects of Fluoride on De- and Remineralization

The recent literature extensively describes the role of ambient fluoride in the de- and remineralization of dental enamel. Fluoride in sub-ppm concentrations is effective in promoting mineral deposition and inhibiting mineral dissolution. The latter phenomenon is most likely attributable to the concomitant precipitation of a fluoride-rich mineral phase which inhibits further dissolution. These fundamental processes result in an inhibition of enamel demineralization and an enhancement of enamel lesion remineralization. Alternatively, fluoride may also induce the ‘arrestment’ of enamel lesions. For the in vivo patterns and the effects of caries-preventive substances to be studied, de- and remineralization can best be examined with a pH-cycling system in which the pH depressions occurring in the oral environment are mimicked in a laboratory model. Such an approach has proved useful in developing optimal fluoride schemes which can be tested in animal, intraoral, and clinical studies.

Hypermineralization of Dentinal Lesions Adjacent to Glass-ionomer Cement Restorations

Previous reports have shown a release of fluoride from glass-ionomer cement (GIC) restorations into the oral fluids. Fluoride in the ambient fluids has a caries-preventive effect by enhancing remineralization and inhibiting demineralization of the dental hard tissues. Therefore, the current investigation was undertaken to determine whether GIC fillings could contribute to the remineralization of caries lesions in dentin. Small circular preparations were made in disks of dentin which had incipient caries-like lesions in the remaining tissue. The preparations were filled with amalgam or composite materials (as controls) or with GIC. The specimens thus contained a restoration close to a dentinal caries-like lesion. The specimens were placed contralaterally in the buccal surfaces of removable partial dentures and were worn intra-orally by volunteers for a 12-week experimental period, after which the specimens were sectioned and analyzed by microradiography. All specimens with GIC restorations exhibited hyperm...Previous reports have shown a release of fluoride from glass-ionomer cement (GIC) restorations into the oral fluids. Fluoride in the ambient fluids has a caries-preventive effect by enhancing remineralization and inhibiting demineralization of the dental hard tissues. Therefore, the current investigation was undertaken to determine whether GIC fillings could contribute to the remineralization of caries lesions in dentin. Small circular preparations were made in disks of dentin which had incipient caries-like lesions in the remaining tissue. The preparations were filled with amalgam or composite materials (as controls) or with GIC. The specimens thus contained a restoration close to a dentinal caries-like lesion. The specimens were placed contralaterally in the buccal surfaces of removable partial dentures and were worn intra-orally by volunteers for a 12-week experimental period, after which the specimens were sectioned and analyzed by microradiography. All specimens with GIC restorations exhibited hypermineralization in the tissue bordering the filling and in the wall of the preparation which had been in contact with the restoration. The (caries-like) lesions were remineralized, even under conditions of heavy plaque formation. In contrast, specimens with amalgam or composite restorations showed further extensive demineralization. This study demonstrates a significant remineralization potential exerted by the fluoride-releasing GIC restorative material. Consequently, the choice of the restorative material might be crucial for the occurrence or prevention of recurrent caries around restorations.

Confocal Microscopy Study of Undisturbed and Chlorhexidine-treated Dental Biofilm

Culturing of dispersed plaque samples and vitality staining of plaque smears are the most commonly used methods for evaluating the effects of antimicrobials on dental plaque. The visualization of the antimicrobial action on oral biofilm present on the substrate surface (in situ) would add valuable information to the existing knowledge about the treatment effects. This study aimed at combining the advantage of confocal laser scanning microscopy (CLSM) to visualize plaque non-destructively with a vitality staining technique to assess the immediate bactericidal effect of chlorhexidine (CHX) on biofilm. Three 200-μmwide grooves were cut into bovine dentin discs for plaque accumulation. The discs were worn by six subjects for 6, 24, and 48 hrs, then broken into halves, one of which received a one-minute extra-oral 0.2% CHX treatment, while the other served as control. Both halves were stained for vital fluorescence measurements and visualized by CLSM. Plaque vitality (in %) was quantified by image analysis in three plaque layers-outer, middle, and inner. The CHX effect was significant in six-hour samples (p < 0.001) and only in the outer layer of the 48-hour plaque (p < 0.001), demonstrating a resistant nature of dental biofilm to a single CHX treatment. With the present approach, we have shown that it is possible to visualize and quantitate the antimicrobial treatment effect on biofilm still present on the substrate on which it was grown.

Host-Derived Proteinases and Degradation of Dentine Collagen in situ

Dentine root caries is a process of demineralization and degradation of the organic matrix by proteinases. In this in situ study, the presence and activity of the matrix metalloproteinases 1, 2 and 9 (MMP-1, MMP-2, MMP-9) in saliva and in completely demineralized dentine specimens were investigated. Furthermore, the activity of cathepsin B was determined in saliva. A correlation between these enzymes and the level of degraded collagen was investigated. Demineralized dentine specimens were mounted in the partial prosthesis of 17 volunteers. Saliva samples were taken at 0, 2 and 4 weeks. After 4 weeks, the enzymes were extracted from the dentine specimens and the collagen loss was assessed. The collagen loss varied between 0 and 40.3%. Zymography of the saliva and the dentine extract samples showed that (pro-)MMP-2 and (pro-)MMP-9 were present. The levels of active MMPs were assessed, using fluorogenic MMP-specific substrates. All but 3 of the 51 saliva samples showed MMP-1 activity ranging from 1.5 to 101.1 relative fluorescence units (RFU)/s. Forty-eight saliva samples showed gelatinolytic MMP-2/MMP-9 activity (1.7–141.1 RFU/s). MMP-1 activity was shown in all dentine extracts varying between 3.5 and 295.0 RFU/s. From the dentine extracts, 15 showed MMP-2/MMP-9 activity (0.2–13.7 RFU/s). The MMP activity from both saliva and dentine extracts did not correlate with the collagen loss. The activity of salivary cathepsin B varied from 4.8 to 42.2 arbitrary units/min. A positive correlation was found between salivary MMP activity and cathepsin B activity. This study revealed that gelatinolytic enzyme activity was present both in saliva and dentine collagen. No correlation could be observed, however, between the level of enzyme activity and the collagen loss of the dentine specimens.

Remineralization of Caries Lesions Extending into Dentin

Remineralization is one aspect of the overall process of tooth decay. However, it is primarily studied in shallow lesions. The aim of this study was to explore whether caries lesions in enamel and extending into the dentin can be remineralized. A single-section model was developed for the longitudinal and non-destructive monitoring of changes in enamel and dentin. Lesions at least 200 microm into dentin were formed in undersaturated acetate buffers. Next, the lesions were divided into groups (three treatment and one control) and remineralized. The treatments were: weekly immersion in 1,000 ppm fluoride, single treatment with methanehydroxybisphosphonate, and a constant level of 1 ppm fluoride. De- and remineralization was assessed by transverse microradiography. Remineralization was observed in enamel, but also in dentin, indicating that, deep into dentin, the pores become supersaturated to apatite formation. Treatments affected remineralization only in the outer part of enamel. Both findings are explained by a relatively fast diffusion of mineral ions, with precipitation being rate-limiting. The results suggest that dentin remineralization, underneath enamel, can be achieved and could possibly be used in clinical treatment strategies.Remineralization is one aspect of the overall process of tooth decay. However, it is primarily studied in shallow lesions. The aim of this study was to explore whether caries lesions in enamel and extending into the dentin can be remineralized. A single-section model was developed for the longitudinal and non-destructive monitoring of changes in enamel and dentin. Lesions at least 200 μm into dentin were formed in undersaturated acetate buffers. Next, the lesions were divided into groups (three treatment and one control) and remineralized. The treatments were: weekly immersion in 1000 ppm fluoride, single treatment with methanehydroxybisphosphonate, and a constant level of 1 ppm fluoride. De- and remineralization was assessed by transverse microradiography. Remineralization was observed in enamel, but also in dentin, indicating that, deep into dentin, the pores become supersaturated to apatite formation. Treatments affected remineralization only in the outer part of enamel. Both findings are explained by a relatively fast diffusion of mineral ions, with precipitation being rate-limiting. The results suggest that dentin remineralization, underneath enamel, can be achieved and could possibly be used in clinical treatment strategies.

Molecular and Cellular Mechanisms That Lead to Candida Biofilm Formation

Fungal infections in the oral cavity are mainly caused by C. albicans, but other Candida species are also frequently identified. They are increasing in prevalence, especially in denture-wearers and aging people, and may lead to invasive infections, which have a high mortality rate. Attachment to mucosal tissues and to abiotic surfaces and the formation of biofilms are crucial steps for Candida survival and proliferation in the oral cavity. Candida species possess a wide arsenal of glycoproteins located at the exterior side of the cell wall, many of which play a determining role in these steps. In addition, C. albicans secretes signaling molecules that inhibit the yeast-to-hypha transition and biofilm formation. In vivo, Candida species are members of mixed biofilms, and subject to various antagonistic and synergistic interactions, which are beginning to be explored. We believe that these new insights will allow for more efficacious treatments of fungal oral infections. For example, the use of signaling molecules that inhibit biofilm formation should be considered. In addition, cell-wall biosynthetic enzymes, wall cross-linking enzymes, and wall proteins, which include adhesins, proteins involved in biofilm formation, fungal-bacterial interactions, and competition for surface colonization sites, offer a wide range of potential targets for therapeutic intervention.