Dental Plaque Biofilm Research Articles

Virulence properties of Streptococcus mutans.

Streptococcus mutans is considered one of the primary causative agents of dental caries and can also be a source of infective endocarditis. The main virulence factors associated with cariogenicity include adhesion, acidogenicity, and acid tolerance. Each of these properties works coordinately to alter dental plaque ecology. The ecological changes are characterized by increased proportions of S. mutans and other species that are similarly acidogenic and aciduric. The selection for a cariogenic flora increases the magnitude of the drop in pH following the fermentation of available carbohydrate and increases the probability of enamel demineralization. This review focuses on the bacterial components that contribute to each of the major virulence properties. Further understanding of how these components work together in the development of dental caries will be aided by the recent completion of the sequence of the S. mutans genome and experimental designs that model the dental plaque biofilm.

Rheology of biofilms formed from the dental plaque pathogen Streptococcus mutans

Here we describe an experimental study of the mechanical properties of bacterial biofilms formed from the early dental plaque colonizer Streptococcus mutans. The S. mutans biofilms demonstrated the behavior of rheological fluids, with properties similar to those of organic polymers and other biological fluids. The time-dependent response of the biofilms was modeled on the basis of principles of viscoelasticity theory. The static and dynamic responses were defined in terms of the creep compliance, storage and loss moduli, and viscosity. The creep compliance and stress relaxation functions of S. mutans biofilms were characterized using the Burger model. Implications for developing more effective mechanical removal strategies of dental plaque biofilms are discussed.

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 (∼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 [ 14C]-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.

Acid tolerance response of biofilm cells of Streptococcus mutans

Streptococcus mutans, a major etiological agent of dental caries, is a component of the dental plaque biofilm and functions during caries progression in acidic lesions that may be at or below pH 4. In this study, we were interested in determining the acid tolerance of 1–7-day chemostat-grown biofilm cells of S. mutans BM71 growing in a semi-defined medium at a rate consistent with that of cells in dental plaque (dilution rate=0.1 h −1), as well as, assessing the capacity of 2- and 5-day biofilms to induce an acid tolerance response that would enhance survival at a killing pH (3.5). As expected, biofilm cell growth increased (2.5-fold) from day 1 to day 7 (10.6–25.7×10 6 cells cm − 2) with the percentage live cells over that period averaging 79.4%, slightly higher than that of planktonic cells (77.4%). Biofilms were highly resistant to acid killing at pH 3.5 for 2 h with survival ranging from 41.8 (1 day) to 63.9% (7 day), while the percentage of live cells averaged 43.4%. Planktonic and dispersed biofilm cells were very acid-sensitive with only 0.0009%– and 0.0002–0.2% survivors, respectively. Unlike the planktonic cells, the incubation of 2- and 5-day biofilms at pH 5.5 for periods of up to 6 h induced strong acid tolerance responses that enhanced survival during a subsequent exposure to acid killing at pH 3.5.

Are dental diseases examples of ecological catastrophes?

Dental diseases are among the most prevalent and costly diseases affecting industrialized societies, and yet are highly preventable. The microflora of dental plaque biofilms from diseased sites is distinct from that found in health, although the putative pathogens can often be detected in low numbers at normal sites. In dental caries, there is a shift towards community dominance by acidogenic and acid-tolerant Gram-positive bacteria (e.g. mutans streptococci and lactobacilli) at the expense of the acid-sensitive species associated with sound enamel. In contrast, the numbers and proportions of obligately anaerobic bacteria, including Gram-negative proteolytic species, increase in periodontal diseases. Modelling studies using defined consortia of oral bacteria grown in planktonic and biofilm systems have been undertaken to identify environmental factors responsible for driving these deleterious shifts in the plaque microflora. Repeated conditions of low pH (rather than sugar availability per se) selected for mutans streptococci and lactobacilli, while the introduction of novel host proteins and glycoproteins (as occurs during the inflammatory response to plaque), and the concomitant rise in local pH, enriched for Gram-negative anaerobic and asaccharolytic species. These studies emphasized (a). significant properties of dental plaque as both a biofilm and a microbial community, and (b). the dynamic relationship existing between the environment and the composition of the oral microflora. This research resulted in a novel hypothesis (the 'ecological plaque hypothesis') to better describe the relationship between plaque bacteria and the host in health and disease. Implicit in this hypothesis is the concept that disease can be prevented not only by directly inhibiting the putative pathogens, but also by interfering with the environmental factors driving the selection and enrichment of these bacteria. Thus, a more holistic approach can be taken in disease control and management strategies.

LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis.

Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-beta-D-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.

Application of carbon source utilization patterns to measure the metabolic similarity of complex dental plaque biofilm microcosms.

Biolog technology was applied to measure the metabolic similarity of plaque biofilm microcosms, which model the complex properties of dental plaque in vivo. The choice of Biolog plate, incubation time, and incubation conditions strongly influenced utilization profiles. For plaque biofilm microcosms, Biolog GP2 plates incubated anaerobically in an H2-free atmosphere gave the clearest profile. To test the application of the Biolog GP2 assay, plaque microcosms were developed under different nutrient conditions in which the frequency of sucrose application was varied. Cluster analysis of Biolog GP2 data from 10 microcosm biofilms correlated with sucrose frequency. Aciduric bacteria (Streptococcus mutans plus lactobacilli) predominated in the plaques receiving high-frequency sucrose applications. Agreement between the Biolog GP2 groupings with nutrient and compositional changes suggests that Biolog analysis is a valuable technique for analyzing the metabolic similarity of dental plaque biofilm microcosms and other high-nutrient or predominantly anaerobic ecosystems.

Calcium phosphate deposition in human dental plaque microcosm biofilms induced by a ureolytic pH-rise procedure

The objectives were to develop and characterize a procedure based on a ureolytic pH rise to deposit calcium phosphate into microcosm dental plaque biofilms and to test the importance of the plaque pH range. Plaque biofilms were cultured in a multiplaque culture system (‘artificial mouth’) with a continuous supply of a simulated oral fluid (basal medium mucin; BMM) with 146 mmol/l (5% w/v) sucrose periodically applied over 6 min every 8 h. After initial plaque growth, the biofilms were periodically exposed for up to 16 days to 6-min applications of calcium phosphate monofluorophosphate urea (CPMU) solution containing 20 mmol/l CaCl 2, 12 mmol/l NaH 2PO 4, 5 mmol/l monofluorophosphate and 500 mmol/l urea (pH 5.0). Three application regimes were examined, one included a sucrose-induced acidic pH fluctuation. Plaque hydrolysis of the urea in CPMU caused the pH to rise to between 8.2 and 8.8, depositing fluoridated and carbonated calcium phosphates, and possibly some calcium carbonate, into the plaque. Calcium, phosphate and fluoride deposition was rapid for about 4 days and then slowed. After 10 days’ treatment under standard conditions (BMM containing 1 mmol/l urea and 1 mmol/l arginine), plaque calcium and phosphate concentrations had increased up to 50-fold and 10-fold to approximately 2–4 and 1–2 mmol/g plaque protein, respectively. The calcium, phosphate and fluoride content increased steadily. Calcium phosphate deposition was proportional to the plaque resting pH, increasing over four-fold when the BMM urea concentration was increased from 0 to 20 mmol/l, which raised the resting pH from 6.4 to 7.2 and yielded a mean plaque calcium concentration of 14.3 mmol/g protein, one subsample reaching 20.8 mmol/g protein. Supplementation of BMM with 20% human serum inhibited deposition. These results support the hypothesis that an alkaline pH in plaque is critical in promoting plaque mineralization and that mineral deposition is modulated by serum. These factors are likely to be important in regulating calculus formation.

Induction and 3D reconstruction of caries-like lesions in an experimental dental plaque biofilm model.

The aim of this study was to examine an in vitro dental plaque model for volumetric assessment and 3D reconstruction of experimentally bacterial induced caries-like lesions. Twenty-three extracted, impacted human molars were coated with a varnish leaving a 3 x 3 mm window on the smooth surface. The teeth were coated with human saliva and Streptococcus sobrinus 6715 adhered onto the enamel via a sucrose dependent mechanism. The coated teeth were incubated for 1, 7, 14 and 28 days. At the end of each incubation period microhardness was measured on three sectioned teeth of each incubation period at the enamel surface and at 450 and 750 microm depth perpendicular to the enamel surface. The enamel surface was investigated by scanning electron microscopy (SEM) prior to embedding and serial sectioning of 12 other teeth. The serial sections were studied with polarization light microscopy to investigate subsurface demineralization. The outlines of the caries-like lesions were then traced consecutively on transparencies, digitized and three-dimensionally reconstructed to demonstrate the expansion of the lesion into enamel for volumetric assessment. Microhardness was significantly reduced on the enamel surface after 7, 14 and 28 days incubation. The SEM investigation showed a surface demineralization with dissolution of the prism cores after 28 days incubation. Polarization light microscopy demonstrated caries-like lesions with an irregular expansion into enamel. The results demonstrate that the described method produces caries-like lesions which can be used for further experimental studies regarding demineralization and remineralization processes.

The role of fructans on dental biofilm formation by Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus

Dental plaque biofilm plays a pivotal role in the progression of dental diseases. Polysaccharides are of great importance in the ecology of the dental biofilm. We studied the effect of fructans, glucans and a mixture of both fructans and glucans, synthesized in situ by immobilized fructosyltransferase or glucosyltransferase, on the adhesion of Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus to hydroxyapatite beads coated with human saliva (sHA). The adhesion of A. viscosus to sHA was found to be fructan-dependent. Adhesion of both S. sobrinus and S. mutans was found to be mediated mainly by glucans, while the adhesion of S. gordonii was found to be both glucan- and fructan-dependent. Treatment with fructanase prior to A. viscosus adhesion resulted in a significant reduction in adhesion to sHA, while adhesion of S. sobrinus, S. mutans and S. gordonii was slightly influenced by fructanase treatment. Treatment with fructanase after adhesion of S. gordonii to sHA resulted in a significant reduction in their adhesion to sHA. Our results show that fructans may play a role in the adhesion and colonization of several cariogenic bacteria to sHA, thus contributing to the formation of dental plaque biofilm.

The role of fructans on dental biofilm formation by Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus.

Dental plaque biofilm plays a pivotal role in the progression of dental diseases. Polysaccharides are of great importance in the ecology of the dental biofilm. We studied the effect of fructans, glucans and a mixture of both fructans and glucans, synthesized in situ by immobilized fructosyltransferase or glucosyltransferase, on the adhesion of Streptococcus sobrinus, Streptococcus mutans, Streptococcus gordonii and Actinomyces viscosus to hydroxyapatite beads coated with human saliva (sHA). The adhesion of A. viscosus to sHA was found to be fructan-dependent. Adhesion of both S. sobrinus and S. mutans was found to be mediated mainly by glucans, while the adhesion of S. gordonii was found to be both glucan- and fructan-dependent. Treatment with fructanase prior to A. viscosus adhesion resulted in a significant reduction in adhesion to sHA, while adhesion of S. sobrinus, S. mutans and S. gordonii was slightly influenced by fructanase treatment. Treatment with fructanase after adhesion of S. gordonii to sHA resulted in a significant reduction in their adhesion to sHA. Our results show that fructans may play a role in the adhesion and colonization of several cariogenic bacteria to sHA, thus contributing to the formation of dental plaque biofilm.

Intergeneric communication in dental plaque biofilms.

Dental plaque is a complex biofilm that accretes in a series of discrete steps proceeding from a gram-positive streptococcus-rich biofilm to a structure rich in gram-negative anaerobes. This study investigated information flow between two unrelated plaque bacteria, Streptococcus cristatus and Porphyromonas gingivalis. A surface protein of S. cristatus caused repression of the P. gingivalis fimbrial gene (fimA), as determined by a chromosomal fimA promoter-lacZ reporter construct and by reverse transcription-PCR. Signaling activity was associated with a 59-kDa surface protein of S. cristatus and showed specificity for the fimA gene. Furthermore, P. gingivalis was unable to form biofilm microcolonies with S. cristatus. Thus, S. cristatus is capable of modulating virulence gene expression in P. gingivalis, consequently influencing the development of pathogenic plaque.

Architecture of intact natural human plaque biofilms studied by confocal laser scanning microscopy.

Determination of the structure of human plaque will be of great benefit in the prediction of its formation and also the effects of treatment. However, a problem lies in the harvesting of undisturbed intact plaque samples from human volunteers and the viewing of the biofilms in their natural state. In this study, we used an in situ device for the in vivo generation of intact dental plaque biofilms on natural tooth surfaces in human subjects. Two devices were placed in the mouths of each of eight healthy volunteers and left to generate biofilm for 4 days. Immediately upon removal from the mouth, the intact, undisturbed biofilms were imaged by the non-invasive technique of confocal microscopy in both reflected light and fluorescence mode. Depth measurements indicated that the plaque formed in the devices was thicker round the edges at the enamel/nylon junction (range = 75-220 microm) than in the center of the devices (range = 35-215 microm). The reflected-light confocal images showed a heterogeneous structure in all of the plaque biofilms examined; channels and voids were clearly visible. This is in contrast to images generated previously by electron microscopy, suggesting a more compact structure. Staining of the biofilms with fluorescein in conjunction with fluorescence imaging suggested that the voids were fluid-filled. This more open architecture is consistent with recent models of biofilm structure from other habitats and has important implications for the delivery of therapeutics to desired targets within the plaque.

Development of sustained-release devices for modulation of dental plaque biofilm and treatment of oral infectious diseases

Dental diseases are among the most prevalent human illnesses. Dental biofilm plaque harboring cariogenic bacteria (caries-associated bacteria) is the major pathogenic factor leading to dental caries. Subgingival plaque harboring periopathogenic bacteria is an additional etiological factor associated with periodontal diseases. Many pharmaceutical dosage forms are used in the prevention and treatment of these oral diseases. Toothpastes and mouthwashes are among the most popular dental medicaments. The main disadvantage of these delivery systems is their low substantivity in the target site, resulting in a low penetration coefficiency into the biofilm. A local delivery application that prolongs the release of the active agent in the mouth offers great advantages in the prevention and treatment of caries and periodontal diseases. Sustained-release varnishes, containing fluorides or antibacterial agents, have been applied to hard surfaces in the oral cavity for prevention and treatment of dental caries. Sustained-release dosage forms such as films/chips, gels, or fibers containing antibacterial agents or antiinflammatory agents have been inserted into the periodontal pocket to treat periodontal diseases. These dosage forms have been shown to reduce periopathogenic bacteria along with clinical improvements in the periodontium. An additional advantage of sustained-release devices is high patient compliance, which is an important factor in dental treatment. Sustained-release devices are a relatively new concept in the dental field. It is predicted that the use of these devices at home and in the dental clinic will increase sharply in the next decade. Drug Dev. Res. 50:555–565, 2000. © 2000 Wiley-Liss, Inc.

The effect of extracellular polysaccharides from Streptococcus mutans on the bactericidal activity of human neutrophils

Extracellular polysaccharides (PS) synthesized by oral bacteria constitute one of their major virulence factors. The PS, synthesized from sucrose, facilitate adhesion and colonization by bacteria to tooth surfaces. The study was designed to test the effect of in situ production of extracellular PS by Streptococcus mutans on the bactericidal activity of human neutrophils. These effects were tested on bacteria pre-exposed to sucrose (PS-positive Strep. mutans) and compared to bacteria not exposed to sucrose (PS-negative Strep. mutans). The interactions between neutrophils and Strep. mutans were tested in suspension and on bacteria in an experimental model of dental plaque. Viability of Strep. mutans was measured by [ 3H]-thymidine incorporation into the bacteria. Degranulation of neutrophils was evaluated by the release of lysozyme, and the production of reactive oxygen products was measured by chemiluminescence. When neutrophils were incubated with suspended bacteria, the viability of PS-negative Strep. mutans was 20% of that of bacteria not incubated with neutrophils (control), while the viability of PS-positive Strep. mutans was 40% of the control. In the experimental dental-plaque model, 50% of the PS-negative Strep. mutans were killed by neutrophils while the viability of PS-positive Strep. mutans was not different than of the control. Degranulation of neutrophils was not affected by the presence of extracellular PS of Strep. mutans. Artificial stimulation of neutrophils with phorbol myristate acetate also did not enhance the bactericidal effect of neutrophils on PS-positive Strep. mutans. However, PS-positive Strep. mutans elicited oxygen-reactive products from neutrophils, 2-fold less than with PS-negative Strep. mutans. The results indicate that in situ production of bacterial extracellular polysaccharides might be a major virulence factor of Strep. mutans, enabling PS-positive Strep. mutans in the dental-plaque biofilm to evade killing by human neutrophils.

22] Use of continuous flow techniques in modeling dental plaque biofilms

This chapter focuses on the use of chemostat models that incorporate surfaces for biofilm formation. A number of other systems have been described for investigating the physiology and metabolism of dental plaque using chemostat systems and other continuous flow techniques. The chapter tabulates the evolution and applications of some of these models. The formation of oral microbial biofilms in chemostat systems has permitted greater insights into the ecology of dental plaque in health and disease. The chemostat permits the modeling, under highly controlled and reproducible conditions, of specific events that occur in vivo . Some of the key practical issues for modeling dental plaque using this approach are described in this chapter. These approaches allow the identification of key determinants of oral microbial biofilm formation and development.

An in vitro System for the Analysis of Changes in Depth Distribution of Diffusates in Bacterial Films

No suitable technique exists which allows simultaneous analysis of time- and depth-dependent concentrations of all components of interest in single samples of intercellular fluid from dental plaque biofilms. We have developed an in vitro model which allows detailed study of these interactions by analysis of intercellular fluid and pH measurement at the film base. Compact, defined-depth films of Streptococcus mutans were formed in a micrometer-controlled, variable-depth well and bathed in synthetic saliva. Films exposed to synthetic saliva containing glucose for 2 min followed by clearance with glucose-free synthetic saliva gave typical ‘Stephan-type’ pH profiles. Intercellular fluid isolated from successive 200-μm-thick sections of 600-μm-deep films was analysed by ion chromatography. A concentration gradient of lactate, falling with depth, was measured. The experimental system described here, the first of its type to be described, can easily be used to analyse pH changes and the depth-dependent distribution of diffusates in a model bacterial film. Although this bacterial film is far removed from a natural biofilm, the apparatus has potential for the study of grown biofilms and is an important advance towards position-dependence analysis of diffusates in biofilms.

Factors affecting the resting pH of in vitro human microcosm dental plaque and Streptococcus mutans biofilms

The aim was to examine factors that potentially control the resting pH, defined as the pH unaffected by meals, of microcosm dental plaques and Streptococcus mutans biofilms under standard conditions, and to examine the effect of supplying urea at concentrations found intraorally. Microcosm plaques were cultured from plaque bacteria-enriched saliva in an ‘artificial mouth’ with a continuous supply of a medium including 0.25% mucin [Basal Medium Mucin, (BMM), 3.6 ml/hr per plaque] and a periodic supply of sucrose. The steady-state resting pH was 6.4 (range±0.1) in BMM containing no urea and supplied at the standard flowrate. This is a robust property of the ecosystem. In one experiment with a replicated ( n=9) set of measurements, the resting pH was approx. pH 6.3, 6.4, 6.7 and 7.3 with 0, 1, 5 and 20 mmol/l urea in the BMM. The magnitude of sucrose- and urea-induced pH responses was unaffected by elevating the resting pH to produce parallel pH curves. The sucrose-induced pH curves were analogous to those classically reported by Stephan that showed an association between caries activity and increasingly acidic plaque pH responses to glucose. Stopping the BMM flow caused a pH rise, indicating continuing net alkali generation from BMM components in the absence of a fluid flow. Strep. mutans monoculture biofilms had an acidic resting pH of 5.0 to 5.3, which increased to 6.8 following an adventitious superinfection by Bacillus cereus. It was concluded that the resting pH in plaque results from a delicate balance between alkali and acid generation, which is in turn dependent both on the bacterial composition of the plaque and on the supply of substrates and buffers from, and metabolite clearance into, flowing oral fluid. In vivo the resting pH will vary with site-specific changing saliva flows. Urea continuously supplied at concentrations normal for saliva and gingival crevicular fluid can raise the resting pH of microcosm plaque by an amount that in vivo would probably be significant in reducing dental caries.

Dental calculus: recent insights into occurrence, formation, prevention, removal and oral health effects of supragingival and subgingival deposits.

Dental calculus, both supra- and subgingival occurs in the majority of adults worldwide. Dental calculus is calcified dental plaque, composed primarily of calcium phosphate mineral salts deposited between and within remnants of formerly viable microorganisms. A viable dental plaque covers mineralized calculus deposits. Levels of calculus and location of formation are population specific and are affected by oral hygiene habits, access to professional care, diet, age, ethnic origin, time since last dental cleaning, systemic disease and the use of prescription medications. In populations that practice regular oral hygiene and with access to regular professional care, supragingival dental calculus formation is restricted to tooth surfaces adjacent to the salivary ducts. Levels of supragingival calculus in these populations is minor and the calculus has little if any impact on oral-health. Subgingival calculus formation in these populations occurs coincident with periodontal disease (although the calculus itself appears to have little impact on attachment loss), the latter being correlated with dental plaque. In populations that do not practice regular hygiene and that do not have access to professional care, supragingival calculus occurs throughout the dentition and the extent of calculus formation can be extreme. In these populations, supragingival calculus is associated with the promotion of gingival recession. Subgingival calculus, in "low hygiene" populations, is extensive and is directly correlated with enhanced periodontal attachment loss. Despite extensive research, a complete understanding of the etiologic significance of subgingival calculus to periodontal disease remains elusive, due to inability to clearly differentiate effects of calculus versus "plaque on calculus". As a result, we are not entirely sure whether subgingival calculus is the cause or result of periodontal inflammation. Research suggests that subgingival calculus, at a minimum, may expand the radius of plaque induced periodontal injury. Removal of subgingival plaque and calculus remains the cornerstone of periodontal therapy. Calculus formation is the result of petrification of dental plaque biofilm, with mineral ions provided by bathing saliva or crevicular fluids. Supragingival calculus formation can be controlled by chemical mineralization inhibitors, applied in toothpastes or mouthrinses. These agents act to delay plaque calcification, keeping deposits in an amorphous non-hardened state to facilitate removal with regular hygiene. Clinical efficacy for these agents is typically assessed as the reduction in tartar area coverage on the teeth between dental cleaning. Research shows that topically applied mineralization inhibitors can also influence adhesion and hardness of calculus deposits on the tooth surface, facilitating removal. Future research in calculus may include the development of improved supragingival tartar control formulations, the development of treatments for the prevention of subgingival calculus formation, the development of improved methods for root detoxification and debridement and the development and application of sensitive diagnostic methods to assess subgingival debridement efficacy.

A method for the quantitative site-specific study of the biochemistry within dental plaque biofilms formed in vivo.

The study of plaque biofilms in the oral cavity is difficult as plaque removal inevitably disrupts biofilm integrity precluding kinetic studies involving the penetration of components and metabolism of substrates in situ. A method is described here in which plaque is formed in vivo under normal (or experimental) conditions using a collection device which can be removed from the mouth after a specified time without physical disturbance to the plaque biofilm, permitting site-specific analysis or exposure of the undisturbed plaque to experimental conditions in vitro. Microbiological analysis revealed plaque flora which was similar to that reported from many natural sources. Analytical data can be related to plaque volume rather than weight. Using this device, plaque fluoride concentrations have been shown to vary with plaque depth and in vitro short-term exposure to radiolabelled components may be carried out, permitting important conclusions to be drawn regarding the site-specific composition and dynamics of dental plaque.