Acid tolerance properties of dental biofilms in vivo.

BACKGROUND: The ecological plaque hypothesis explains caries development as the result of the enrichment of acid tolerant bacteria in dental biofilms in response to prolonged periods of low pH. Acid production by an acid tolerant microflora causes demineralisation of tooth enamel and thus, individuals with a greater proportion of acid tolerant bacteria would be expected to be more prone to caries development. Biofilm acid tolerance could therefore be a possible biomarker for caries prediction. However, little is known about the stability of biofilm acid tolerance over time in vivo or the distribution throughout the oral cavity. Therefore the aim of this study was to assess intra-individual differences in biofilm acid-tolerance between different tooth surfaces and inter-individual variation as well as stability of acid tolerance over time. RESULTS: The majority of the adolescents showed low scores for biofilm acid tolerance. In 14 of 20 individuals no differences were seen between the three tooth sites examined. In the remaining six, acid-tolerance at the premolar site differed from one of the other sites. At 51 of 60 tooth sites, acid-tolerance at baseline was unchanged after 1 month. However, acid tolerance values changed over a 1-year period in 50% of the individuals. CONCLUSIONS: Biofilm acid tolerance showed short-term stability and low variation between different sites in the same individual suggesting that the acid tolerance could be a promising biological biomarker candidate for caries prediction. Further evaluation is however needed and prospective clinical trials are called for to evaluate the diagnostic accuracy.

Strains of Enterococcus faecalis differ in their ability to coexist in biofilms with other root canal bacteria.

AIM: To investigate the relationship between protease production and the ability of Enterococcus faecalis strains to coexist in biofilms with other bacteria commonly recovered from infected root canals. METHODOLOGY: Biofilms with bacteria in mono-, dual- and four-species communities were developed in flow chambers. The organisms used were Lactobacillus salivarius, Streptococcus gordonii and Actinomyces naeslundii and E. faecalis strains, GUL1 and OG1RF. Biovolume and species distribution were examined using 16S rRNA fluorescence in situ hybridization in combination with confocal microscopy and image analysis. The full proteome of the E. faecalis strains was studied using two-dimensional gel electrophoresis. Spots of interest were identified using tandem mass spectroscopy and quantified using Delta 2D software. RESULTS: All bacteria formed biofilms and an anova analysis revealed that the biofilm biomass increased significantly (P ≤ 0.01) between 6 and 24 h. L. salivarius, S. gordonii and A. naeslundii formed mutualistic biofilm communities, and this pattern was unchanged when E. faecalis GUL1 was included in the consortium. However, with OG1RF, L. salivarius and S. gordonii were outcompeted in a 24-h biofilm. Proteomic analysis revealed that OG1RF secreted higher levels of proteases, GelE (P = 0.02) and SprE (P = 0.002) and a previously unidentified serine protease (P = 0.05), than GUL1. CONCLUSIONS: Different strains of E. faecalis can interact synergistically or antagonistically with a consortium of root canal bacteria. A possible mechanism underlying this, as well as potential differences in virulence, is production of different levels of proteases, which can cause detachment of neighbouring bacteria and tissue damage.

Bacterial profiles and proteolytic activity in peri-implantitis versus healthy sites.

Peri-implantitis is a biofilm-induced destructive inflammatory process that, over time, results in loss of supporting bone around an osseointegrated dental implant. Biofilms at peri-implantitis sites have been reported to be dominated by Gram-negative anaerobic rods with a proteolytic metabolism such as, Fusobacterium, Porphyromonas, Prevotella and Tannerella, as well as anaerobic Gram-positive cocci. In this study, we hypothesized that protease activity is instrumental in driving bone destruction and we therefore compared the microbial composition and level of protease activity in samples of peri-implant biofluid (PIBF) from 25 healthy subjects (H group) and 25 subjects with peri-implantitis (PI group). Microbial composition was investigated using culture techniques and protease activity was determined using a FITC-labelled casein substrate. The microbial composition was highly variable in subjects both in the H and PI groups but one prominent difference was the prevalence of Porphyromonas/Prevotella and anaerobic Gram positive cocci which was significantly higher in the PI than in the H group. A subgroup of subjects with peri-implantitis displayed a high level of protease activity in the PIBF compared to healthy subjects. However, this activity could not be related to the presence of specific bacterial species. We propose that a high level of protease activity may be a predictive factor for disease progression in peri-implantitis. Further longitudinal studies are however required to determine whether assessment of protease activity could serve as a useful method to identify patients at risk for progressive tissue destruction.

Salivary proteins promote proteolytic activity in Streptococcus mitis biovar 2 and Streptococcus mutans.

A major function of the salivary pellicle on oral surfaces is to promote colonization of the commensal microbiota by providing binding sites for adherence. Streptococcus mitis is an early colonizer of the oral cavity whereas Streptococcus mutans represents a later colonizer. To survive and grow, oral bacteria produce enzymes, proteases and glycosidases, which allow them to exploit salivary proteins as a nutrient source. In this study, adherence and proteolytic activity of S. mitis biovar 2 and S. mutans were investigated in a flow-cell model in the presence of different populations of surface-associated salivary proteins. Streptococcus mitis biovar 2 adhered well to surfaces coated with both a MUC5B-enriched fraction and a pool of low-density proteins containing MUC7, amylase, cystatin, gp340, immunoglobulin A, lactoferrin, lysozyme and statherin, whereas adherence of S. mutans to these proteins was poor. In environments of MUC5B or the low-density proteins, both S. mitis biovar 2 and S. mutans showed high levels of proteolytic activity. For S. mitis in the MUC5B environment, most of this activity may be attributable to contact with the molecules in the fluid phase although activity was also enhanced by adherence to surface-associated MUC5B. These data suggest that although they differ in their capacity to adhere to surface-associated salivary proteins, in the natural environment exploitation of saliva as a nutrient source can contribute to survival and colonization of the oral cavity by both S. mitis biovar 2 and S. mutans.

Fluoride-supplemented milk inhibits acid tolerance in root caries biofilms.

In this study we investigated the effect of fluoride on plaque acid tolerance. The test group consumed 200 ml of milk supplemented with 5 mg F/l as NaF once a day, the milk control group drank 200 ml of unsupplemented milk, and the no-milk control group did not consume milk in this manner. Plaque samples were taken at baseline and after 15 months. The proportion of acid-tolerant bacteria in plaque was estimated using LIVE/DEAD® BacLight™ staining after exposure to pH 3.5 for 2 h. The fluoride group showed a statistically significant decrease in plaque acid tolerance compared to baseline. This study shows that daily intake of fluoride in milk reduces plaque acid tolerance.

Regulation of bacteriocin production and cell death by the VicRK signaling system in Streptococcus mutans.

The VicRK two-component signaling system modulates biofilm formation, genetic competence, and stress tolerance in Streptococcus mutans. We show here that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of competence-stimulating peptide (CSP) production in S. mutans. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including nlmAB, nlmC, and nlmD (P < 0.001), suggesting a role for the VicRK in producing mutacins IV, V, and VI. Bacteriocin overlay assays revealed an altered ability of the vic mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N(5)-TGTWAH) was identified within the comC coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the vic operon caused growth-phase-dependent repression of comC, comDE, and comX. In the vic mutants, transcription of nlmC/cipB encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by immB, were significantly affected relative to the wild type (P < 0.05). In contrast to previous reports that proposed a hyper-resistant phenotype for the VicK mutant in cell viability, the release of extracellular genomic DNA was significantly enhanced in SmuvicK (P < 0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using vic mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of S. mutans.

In situ analysis of multispecies biofilm formation on customized titanium surfaces.

Many studies to identify surfaces that enhance the incorporation of dental implants into bone and soft-tissue have been undertaken previously. However, to succeed in the clinical situation, an implant surface must not support development of microbial biofilms with a pathogenic potential. As a first step in investigating this, we used two-species and three-species biofilm models with 16S ribosomal RNA fluorescence in situ hybridization and confocal laser scanning microscopy to examine the effect of surface characteristics on biofilm formation by species that can colonize titanium implants in vivo: Streptococcus sanguinis, Actinomyces naeslundii and Lactobacillus salivarius. Surfaces blasted with Al(2) O(3) (S(a) = 1.0-2.0 µm) showed a seven-fold higher bacterial adhesion after 2 h than turned surfaces (S(a) = 0.18 µm) whereas porous surfaces, generated by anodic oxidation (S(a) = 0.4 µm), showed four-fold greater adhesion than turned surfaces. Hence, increased roughness promoted adhesion, most likely through protection of bacteria from shear forces. Chemical modification of the blasted and oxidized surfaces by incorporation of Ca(2+) ions reduced adhesion compared with the corresponding non-modified surfaces. After 14 h, biofilm growth occurred in the three-species model but not in the two-species consortium (containing S. sanguinis and A. naeslundii only). The biofilm biovolume on all surfaces was similar, suggesting that the influence of surface characteristics on adhesion was compensated for by biofilm development.

Salivary gel-forming mucin MUC5B--a nutrient for dental plaque bacteria.

INTRODUCTION: Model systems with oral bacteria from dental plaque have demonstrated that the utilization of complex glycoproteins as a food source cannot be undertaken by single species but requires concerted degradation by a multi-species consortium, with each member contributing one or a few hydrolytic enzymes. Unlike previous studies, the aim of the present investigation was to explore the ability of fresh dental plaque to degrade salivary mucin, MUC5B, isolated by methods designed to retain intact the natural polymeric structure and physiological conformation, in an attempt to mimic the naturally occurring interaction between the oral microbiota and salivary mucins. METHODS: Human salivary MUC5B was isolated from whole saliva by density-gradient centrifugation and incubated with freshly isolated supragingival dental plaque with samples subjected to fluorescent staining for viability and metabolic activity. In addition, the degradation of MUC5B oligosaccharide side chains was studied using a lectin assay, recognizing three different carbohydrate epitopes commonly found on mucin oligosaccharide side chains. RESULTS: The addition of purified salivary MUC5B elicited a strong metabolic response from the biofilm cells, whereas individual strains of Streptococcus oralis and Streptococcus gordonii isolated from the same plaque were not able to utilize the MUC5B. The degradation of terminal saccharide moieties on the MUC5B was demonstrated by a marked decrease in both sialic acid and fucose reactivity. CONCLUSION: These results have shown that dental plaque is capable of utilizing human salivary MUC5B as a nutrient source, a process possibly requiring the synergistic degradation of the molecule by a consortium of oral bacteria in the plaque community.

Response to alkaline stress by root canal bacteria in biofilms.

AIM: To determine whether bacteria isolated from infected root canals survive alkaline shifts better in biofilms than in planktonic cultures. METHODOLOGY: Clinical isolates of Enterococcus faecalis, Lactobacillus paracasei, Olsenella uli, Streptococcus anginosus, S. gordonii, S. oralis and Fusobacterium nucleatum in biofilm and planktonic cultures were stressed at pH 10.5 for 4 h, and cell viability determined using the fluorescent staining LIVE/DEAD BacLight bacterial viability kit. In addition, proteins released into extracellular culture fluids were identified by Western blotting. RESULTS: Enterococcus faecalis, L. paracasei, O. uli and S. gordonii survived in high numbers in both planktonic cultures and in biofilms after alkaline challenge. S. anginosus, S. oralis and F. nucleatum showed increased viability in biofilms compared with planktonic cultures. Alkaline exposure caused all planktonic cultures to aggregate into clusters and resulted in a greater extrusion of cellular proteins compared with cells in biofilms. Increased levels of DnaK, HPr and fructose-1,6-bisphosphate aldolase were observed in culture fluids, especially amongst streptococci. CONCLUSIONS: In general, bacteria isolated from infected roots canals resisted alkaline stress better in biofilms than in planktonic cultures, however, planktonic cells appeared to use aggregation and the extracellular transport of specific proteins as survival mechanisms.

The oral ecosystem: implications for education.

We propose a model that is applicable to oral health education. The model describes the oral cavity in a complexity-based ecological context. This concept includes the premise that factors from different organisational levels (biological, individual, community, society) interact in a complex way with the potential to 'stress' the ecosystem and thereby provoke changes. This mode of action complies with the understanding of the oral cavity as a complex adaptive system. An ecological model is actively used in the undergraduate problem-based curriculum at the Faculty of Odontology, Malmö University, Sweden and has recently been applied as a conceptual basis for the new dental curriculum being established at the University of Tromsø in Northern Norway. The purpose is to encourage and promote an ecological, health-oriented view and to stimulate reflections on premises for oral health and diseases in an integrated context.

Protein expression by Streptococcus mutans during initial stage of biofilm formation.

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a (14)C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished > or =1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.

Effect of acid shock on protein expression by biofilm cells of Streptococcus mutans.

Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries. Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions. In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negligible induction of an ATR. Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein synthesis by the two cell types. For this, cells were pulse labeled with [(14)C]-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis followed by autoradiography and computer-assisted image analysis. A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis. While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observation that acidification of biofilm cells induced a negligible ATR. Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the glycolytic enzymes in control biofilm cells were significantly less downregulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux.

The acid-tolerant microbiota associated with plaque from initial caries and healthy tooth surfaces.

The intent of this study was to compare the inherent acid tolerance of bacteria in samples of dental plaque from tooth sites in subjects with and without initial caries. Plaque was collected from approximal surfaces showing early enamel caries and from healthy tooth surfaces in the same subjects, as well as from enamel surfaces of caries-free individuals. In addition to plating on blood agar, the plaque samples were plated directly on non-selective solid agar medium buffered to pH 7.0, 6.0, 5.5, 5.0, 4.5 and 4.0 to avoid any loss of adaptation to acid during primary isolation of plaque bacteria. The results showed that approximately 50% of the total cultivable plaque microbiota from caries, as well as healthy tooth sites, was able to grow at pH 5.5 and 1% at pH 5.0, pH values regarded as critical for the demineralization of tooth enamel. At pH 5.0, members of the genus Streptococcus were the dominant group, but mutans streptococci accounted for less than half of the streptococcal viable count. The other acid-tolerant streptococcal isolates included Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordinii, Streptococcus intermedius, Streptococcus mitis, Streptococcus oralis, Streptococcus salivarius and SStreptococcus sanguis. Analysis of the results indicated that the mutans streptococci in dental plaque were highly variable with respect to acid tolerance, and that both caries and healthy sites harboured significant numbers of mutans streptococci that were not acid-tolerant.

Protein expression by planktonic and biofilm cells of Streptococcus mutans.

Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities. As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h). Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis. Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells. Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells. The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced. The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.

Effect of carbon starvation and proteolytic activity on stationary-phase acid tolerance of Streptococcus mutans.

Previous research with Streptococcus mutans and other oral streptococci has demonstrated that the acid shock of exponential-phase cells (pH 7.5 to 5.5) resulted in the induction of an acid tolerance response (ATR) increasing survival at low pH (3.5-3.0). The current study was designed to determine whether two fresh isolates, H7 and BM71, and two laboratory strains, Ingbritt and LT11, were capable of a stationary-phase ATR as estimated by a survival test at pH 3.5 for 3 h. All four strains were unable to generate a stationary-phase ATR under control conditions at pH 7.5, with the exception of a burst of survivors in the transition between the exponential and stationary phases when the carbon source (glucose) was depleted. Adaptation at pH 5.5 resulted in the expected pH-dependent exponential-phase ATR, but only the fresh isolates exhibited a stationary-phase ATR at this pH. Glucose starvation of cells in complex medium was shown to enhance acid tolerance for the fresh isolates, but not the laboratory strains. This tolerance was, however, greatly diminished for all strains in a defined medium with a low concentration of amino acids. Growth of strain H7 in complex medium resulted in the formation of at least 56 extracellular proteins, nine of which were degraded in the early stationary phase following the induction of proteolytic activity during the transition period. No proteolytic activity was observed with strain LT11 and only 19 extracellular proteins/peptides were apparent in the medium with only one being degraded in the early stationary phase. Strain H7 was also shown to have two- to fourfold higher levels of intracellular glycogen in the stationary phase than strain LT11. These results suggest that S. mutans H7 possessed the required endogenous metabolism to support amino acid/peptide uptake in the early-stationary phase, which resulted in the formation of basic end products that, in turn, contributed to enhanced intracellular pH homeostasis.

Cell density modulates acid adaptation in Streptococcus mutans: implications for survival in biofilms.

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.

Dental plaque mass and acid production activity of the microbiota on teeth.

With the purpose of elucidating the effect of dental plaque thickness on the acid production activity in dental plaque, we studied the prevalence of acid anions in dental plaque of children harbouring different amount of plaque on their teeth. On two occasions, the occurrence of plaque on the dentition was scored (Silness-Löe's index), and plaque on available smooth surfaces was collected and the wet weight determined. On the first occasion, the amount of acid anions in resting plaque, and on the second occasion, the acid anions of sucrose-exposed plaque were analysed with isotachophoresis. Mean value of plaque wet weights per individual dentition was 11.7 +/- 9.0 mg (first occasion) and 11.1 +/- 8.9 mg (second occasion). Dominating anions in resting plaque were acetate < propionate < lactate and in sucrose-exposed plaque lactate < acetate < propionate. The microbial acid production activity (acid anion per mg wet plaque weight) decreased with increasing weights of the plaque mass. The findings illustrate the cellular glycolytic metabolism of plaque microorganisms in thin and thick plaque, being a consequence of the diverse environmental condition existing in these ecosystems.

Acid formation in sucrose-exposed dental plaque in relation to caries incidence in schoolchildren.

The aim was to investigate the composition and concentration of organic acids produced by plaque bacteria in vivo and its possible relation to caries development in schoolchildren. Sucrose-exposed pooled plaque from 25 healthy teenagers was collected and the levels of Streptococcus mutans and lactobacilli were estimated. The acid anions were analysed with isotachophoresis. The prevalence and incidence of dental caries during a 2-year period was recorded. Depending on the amount of plaque collected, the subjects were divided into a low (n = 12) and a high (n = 13) plaque weight group. The dominating anions in sucrose exposed plaque were lactate, acetate and propionate. For the entire study group, no association was found between the total acid concentration and caries development but for the participants with low plaque weight a positive relationship (P < 0.01) was disclosed. No correlation was found between the concentrations of acids and the number of lactobacilli or Streptococcus mutans. The present results did not suggest that the acidogenicity of sugar-exposed pooled plaque samples was a suitable indicator of caries activity in teenagers.

The Malmö model: a problem-based learning curriculum in undergraduate dental education.

A new undergraduate dental curriculum was introduced in Malmö in 1990 which is based on problem-based learning (PBL). The principles of PBL are strongly influenced by evidence from cognitive psychology and they form the educational strategy throughout the whole curriculum. Two further essential principles underpin the curriculum: a holistic attitude to patient care, and the promotion of oral health. Basic sciences and clinical dentistry are integrated within a structure based on oral conditions prevalent in the community. Students are encouraged to build their new knowledge, understanding and skills into the context of what they have already learned. This approach is facilitated by the presentation of conceptual models, one of which, The Oral Ecosystem is described in detail. The educational programme also gives students opportunities to learn in the clinical context from an early stage, and we endeavour to promote a scientific attitude from the very beginning of the programme. This paper describes the curriculum at three organisational levels (whole curriculum, single course, individual week).

Presence of cortisol in gingival crevicular fluid. A pilot study.

Cortisol is one of the primary mediators of the stress response, in the main having immunosuppressive effects. An important component of the host response in periodontal inflammation is gingival crevicular fluid (GCF), with constituents mainly derived from serum. Cortisol, like many other steroids, is present in saliva but its occurrence in GCF does not seem to be documented. Unstimulated whole saliva was collected and GCF was sampled on filter disks. The samples were analysed by a modified RIA method for serum in such a way that small volumes and low concentrations could be measured. Our findings suggest that the total concentration of cortisol in GCF might be estimated to levels below 1/10 of that in serum. However, what appears as a distinctive feature is the considerable variation of the cortisol concentrations for individual teeth. To our knowledge, this is the first time cortisol has been measured in gingival crevicular fluid, and this opens the prospects for further in vivo research.