Characterization of enamel caries lesions in rat molars using synchrotron X-ray microtomography.

Dental caries is a ubiquitous infectious disease with a nearly 100% lifetime prevalence. Rodent caries models are widely used to investigate the etiology, progression and potential prevention or treatment of the disease. To explore the suitability of these models for deeper investigations of intact surface zones during enamel caries, the structures of early-stage carious lesions in rats were characterized and compared with previous reports on white spot enamel lesions in humans. Synchrotron X-ray microcomputed tomography non-destructively mapped demineralization in carious rat molar specimens across a range of caries severity, identifying 52 lesions across the 30 teeth imaged. Of these lesions, 13 were shown to have intact surface zones. Depth profiles of fractional mineral density were qualitatively similar to lesions in human teeth. However, the thickness of the surface zone in the rat model ranges from 10 to 58 µm, and is therefore significantly thinner than in human enamel. These results indicate that a fraction of lesions in rat caries possess an intact surface zone and are qualitatively similar to human lesions at the micrometer scale. This suggests that rat caries models may be a suitable analog through which to investigate the structure of surface zone enamel and its role during dental caries.

Dental caries - not just holes in teeth! A perspective.

Cavitation in teeth results from a pathogenic process termed dental caries that has occurred on the tooth surface for weeks or even years. Accumulation of dental plaque (biofilm) on the tooth is usually the first manifestation of the disease. Although acid production is the immediate and proximal cause of dissolution of teeth; it is the milieu within which the acid is formed that should be of primary concern. Focusing on the 'critical pH' has detracted attention from the more biological aspects (biofilm formation) of dental caries. Dental caries is unique; it is a biological process occurring on essentially an inert surface. Investigation of the multitude of interactions occurring in plaque ranging from enamel interfaces to surfaces of bacteria and matrices poses challenges worthy of the best scientific minds. The mouth clearly offers unique opportunities to investigate the multi facets of biofilm formation in vivo, generating data that have relevance way beyond the mouth. Prevention of this ubiquitous disease, dental caries, continues to present serious challenges. The public health benefits of fluoride delivered in its various formats are well recognized. Nevertheless, additional preventive approaches are required. Overcoming the rapid clearance of agents from the mouth is particularly challenging. Building on the polymerizing capacity of glucosyltransferases it may be possible to incorporate a therapeutic agent into the matrix plaque, thereby delivering therapeutic agents precisely to where they are needed.

Fluorescence microscopic visualization of non cellular components during initial bioadhesion in situ.

OBJECTIVE: The formation of an intraoral biofilm is primarily determined by initial bioadhesion processes, including molecular interactions. Therefore, this study aimed to establish fluorescent labelling protocols to enable the simultaneous visualization of different pellicle enzymes, extracellular glucans and adherent bacteria throughout the initial phase of biofilm formation. DESIGN: In situ formed biofilm samples were collected on enamel and dentine slabs that were fixed on buccal sites of individual splints, being worn by 5 subjects. After an intraoral slab exposure from 30min to 8h, the following specially adapted fluorescent labelling assays were performed and analyzed by epifluorescent microscopy: pellicle-amylase, -lysozyme, -peroxidase and -glycosyltransferases B, C and D were marked with specific primary antibodies and then visualized by the aid of different fluorescently labelled secondary antibodies (Texas Red, DyLight 488, FITC). Afterwards the same samples were subjected to a combined DAPI-/Concanavalin A-staining to determine adherent bacteria and glucans. RESULTS: All fluorescence labelling assays were successfully established to visualize pellicle enzymes, glucans and adherent bacteria at different times of biofilm formation. The combination of the labelling protocols showed a characteristic agglomeration of glucans and bacteria as well as an increased concentration of the pellicle enzymes in the initial phase of bioadhesion. CONCLUSION: Fluorescent labelling techniques are a valuable supplement of dental research as they provide an insight into the mutual interactions of different biofilm determinants in situ. Based hereon, information could also be deduced about the influence of oral therapeutics on individual caries susceptibility.

Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms.

The importance of Streptococcus mutans in the etiology and pathogenesis of dental caries is certainly controversial, in part because excessive attention is paid to the numbers of S. mutans and acid production while the matrix within dental plaque has been neglected. S. mutans does not always dominate within plaque; many organisms are equally acidogenic and aciduric. It is also recognized that glucosyltransferases from S. mutans (Gtfs) play critical roles in the development of virulent dental plaque. Gtfs adsorb to enamel synthesizing glucans in situ, providing sites for avid colonization by microorganisms and an insoluble matrix for plaque. Gtfs also adsorb to surfaces of other oral microorganisms converting them to glucan producers. S. mutans expresses 3 genetically distinct Gtfs; each appears to play a different but overlapping role in the formation of virulent plaque. GtfC is adsorbed to enamel within pellicle whereas GtfB binds avidly to bacteria promoting tight cell clustering, and enhancing cohesion of plaque. GtfD forms a soluble, readily metabolizable polysaccharide and acts as a primer for GtfB. The behavior of soluble Gtfs does not mirror that observed with surface-adsorbed enzymes. Furthermore, the structure of polysaccharide matrix changes over time as a result of the action of mutanases and dextranases within plaque. Gtfs at distinct loci offer chemotherapeutic targets to prevent caries. Nevertheless, agents that inhibit Gtfs in solution frequently have a reduced or no effect on adsorbed enzymes. Clearly, conformational changes and reactions of Gtfs on surfaces are complex and modulate the pathogenesis of dental caries in situ, deserving further investigation.

Electron microscopic detection and activity of glucosyltransferase B, C, and D in the in situ formed pellicle.

OBJECTIVE: Glucosyltransferases (GTFs) represent a virulence factor of mutans streptococci. The aim of the present in situ study was to investigate the distribution of different GTF-isoforms in the pellicle. DESIGN: Bovine enamel slabs were fixed on buccal and palatal sites of individual splints worn by five subjects for 30 and 120 min to allow pellicle formation. Pellicle specimens were processed for transmission electron microscopy (TEM) and field emission in-lens scanning electron microscopy (FEI-SEM). Gold-immunolabelling was used for detection of GTF-isoforms B, C and D. Furthermore, glucosyltransferase activity of 3-, 30- and 120-min pellicles was tested via determination of fructose release. RESULTS: All isoforms of the enzyme were found to be randomly distributed within all layers of the pellicle. In cross-sections (TEM), GTF D was the most abundant isoform. More labelled molecules were detected on buccal sites compared with palatal surfaces, the number of molecules detected increased with time. The amount of GTF B, C and D found on the pellicle surface by FEI-SEM showed no correlation with pellicle formation time or localisation in the oral cavity. Overall, GTF D was detected more frequently on the surface than GTF B and C. All pellicles tested showed GTF-activity. CONCLUSION: The study shows for the first time the presence of the GTF-isoforms B, C and D within all layers of the in situ formed pellicle. This emphasises the impact of streptococcal products on the composition of the pellicle and illustrates a mechanism used by bacteria to colonize dental surfaces.

Influences of starch and sucrose on Streptococcus mutans biofilms.

INTRODUCTION: The combination of starch and sucrose has been shown to be potentially more cariogenic than either alone. The aim of this study was to examine the influence of starch and sucrose, alone or in combinations, on formation, polysaccharide composition, gene expression, and acidogenicity of Streptococcus mutans biofilms. METHODS: S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite (sHA) discs in batch culture for 5 days in the presence of 1% (weight/volume) starch, 1% sucrose, 1% starch plus 1% sucrose, 1% starch plus 0.5% fructose plus 0.5% glucose, or 1% sucrose plus 1% glucose. RESULTS: Amylase activity from sHA disks was detected up to 48 h, thereby increasing the availability of reducing sugars and acidogenicity in the early stages of biofilm development. S. mutans grown in the presence of sucrose alone or in combinations formed well-defined and tightly adherent biofilms comprised of mostly water-insoluble polysaccharides (INS); in contrast, the presence of starch or starch + glucose + fructose resulted in little biofilm formation with minimal amounts of INS. However, the combination of starch + sucrose produced biofilms with more biomass and acidogenicity, and a higher content of INS than those grown in sucrose or sucrose + glucose (P < 0.05). The INS extracted from biofilms formed in the presence of starch + sucrose displayed a higher percentage of 3-linked branching (3,4-, 3,6-, and 3,4,6-linked glucose) compared to those from biofilms grown in sucrose or sucrose + glucose. Furthermore, biofilms grown in starch + sucrose expressed significantly higher levels of gtfB messenger RNA than sucrose-grown or sucrose + glucose-grown biofilms (P < 0.05). CONCLUSION: The combination of starch and sucrose has profound effects not only on the composition and structure of the polysaccharide matrix but also on gene expression of S. mutans within biofilms, which may enhance the cariogenic potential of dental biofilms.

Salivary glucosyltransferase B as a possible marker for caries activity.

Bacteria-derived glucosyltransferases (Gtf) (EC 2.4.1.5), through synthesizing glucan polymers from sucrose and starch hydrolysates, play an essential role in the etiology and pathogenesis of caries. We attempted to correlate the levels of Gtf in whole saliva with the prevalence of carious lesions in young children. We examined saliva from children who were either free of overt carious lesions, or had severe early childhood caries (mean dmfs = 18.72 +/- 9.0 SD), for Gtf by direct enzyme assay. The levels of GtfB, GtfC and GtfD from Streptococcus mutans in the saliva using monoclonal/specific antibodies in an enzyme-linked immunosorbent assay were determined. Multiple logistic regression analyses with model selection showed that GtfB levels correlated with dmfs values of the subjects (p = 0.006). There was no correlation between total Gtf activity as measured by direct enzyme assay and dmfs values. There was a strong correlation between mutans streptococci populations in saliva and caries activity. Collectively, these data show that GtfB levels in saliva correlate strongly with presence of clinical caries and with number of carious lesions in young children. It is also possible to measure different Gtfs, separately, in whole saliva. These observations may have important clinical implications, may lead to development of a chair side caries activity test and support the importance of GtfB in the pathogenesis of dental caries.

Influence of cranberry juice on glucan-mediated processes involved in Streptococcus mutans biofilm development.

Cranberry juice (CJ) has biological properties that may provide health benefits. In this study, we investigated the influence of CJ (pH 5.5) on several activities in vitro associated with the development of Streptococcus mutans UA159 biofilms. The ability of CJ to influence the adherence of S. mutans to either saliva- (sHA) or glucan-coated hydroxyapatite (gsHA), and to inhibit the glucan production by purified glucosyltransferases adsorbed to sHA was determined. For the adherence assays, we used both uncoated and saliva-coated bacterial cells. Furthermore, we examined whether CJ interferes with the viability, development, polysaccharide composition and acidogenicity of S. mutans biofilms. A solution containing equivalent amounts of glucose, fructose and organic acids at pH 5.5 was used as negative control. The adherence of S. mutans (uncoated and saliva-coated) to either sHA or gsHA treated with 25% CJ (v/v) was remarkably reduced (40-85% inhibition compared to control: p < 0.05), indicating that CJ effectively blocked the bacterial adherence to binding sites in salivary pellicle and in glucans. In contrast, when the bacterial cells alone were treated with CJ they adhered to the similar untreated surfaces. Cranberry juice (25%, v/v) also inhibited the activities of surface-adsorbed GTF B and C (70-80% inhibition compared to control, p < 0.05). The effect of CJ on the viability of microorganisms in biofilms was not significant. Biofilm formation and accumulation were significantly reduced by topical applications of 25% CJ (v/v) twice daily with 1-min exposures (p < 0.05). The biomass and insoluble glucan content of the biofilms in addition to its acidogenicity were significantly reduced by cranberry treatments (p < 0.05). Our data show that cranberry juice inhibited glucan-mediated biofilm development and acid production, and holds promise as a natural product to prevent biofilm-related oral diseases.

A mouse caries model and evaluation of aqp5-/- knockout mice.

Current techniques to alter gene expression in mice allow direct analysis of the net role of a host factor in caries development. Towards this goal we first established protocols to induce and score caries in NFS/N mice and determined caries susceptibility in mice with targeted deletion of the gene encoding aquaporin-5 (Aqp5-/-), a water channel involved in the production of saliva. In the NFS/N strain of mice total sulcal caries and severity scores were consistent between experiments, whereas smooth surface caries scores were lower, more variable but distributed fairly evenly among the buccal, lingual and sulcal surfaces. In Black Swiss/129SvJ mice (genetic background of Aqp5-/- mice) caries scores were 50-75% lower compared to NFS/N mice, suggesting strain variation in caries susceptibility under our experimental conditions. In Aqp5-/- mice, in which the volume of total salivary secretion is reduced by 60-65%, there was a significant increase in caries, primarily on the buccal and sulcal surfaces. Results indicate that caries susceptibility increases with a reduced salivary flow that is associated with decreased water content of saliva.

Apigenin and tt-farnesol with fluoride effects on S. mutans biofilms and dental caries.

Apigenin (Api) and tt-farnesol (Far) are two naturally occurring agents that affect the development of cariogenic biofilms. Fluoride (F) interferes physicochemically with caries development and also exhibits antibacterial activity. We examined whether the association of Api and Far enhance the anti-caries properties of F by acting cooperatively on the expression of virulence of Streptococcus mutans. The biological effects of each of the agents were greatly enhanced when used in combination with F. In general, biofilms treated with Api and/or Far in combination with F displayed less biomass and fewer insoluble glucans and iodophilic polysaccharides than did those treated with the test agents alone (P < 0.05). The combination of the test agents with F was highly effective in preventing caries development in rats, especially Api+Far+F, and results were comparable with those observed with chlorhexidine + F (positive control). Results from these studies showed that apigenin and tt-farnesol may enhance the cariostatic effectiveness of fluoride.

Interactions of Streptococcus mutans glucosyltransferase B with lysozyme in solution and on the surface of hydroxyapatite.

Several active enzymes have been identified as components of acquired enamel pellicle. In the present study, the interactions of Streptococcus mutans glucosyltransferase B (GtfB) with lysozyme in solution and on the surface of hydroxyapatite (HA) beads were studied. Experiments were also performed to investigate whether structural differences exist between glucans formed by GtfB enzyme in the presence or absence of lysozyme in solution and on the surface of HA. Hen egg-white lysozyme (HEWL) and saliva were used as the sources of lysozyme; lysozyme-depleted saliva was used as control. Lysozyme activity was significantly reduced when adsorbed onto HA beads compared with that in solution. The GtfB enzyme did not affect the activity of lysozyme in solution or that of adsorbed lysozyme onto HA. The presence of HEWL increased GtfB activity; bovine serum albumin had an even greater enhancing effect. Depletion of lysozyme from whole saliva increased GtfB activity in solution, but not on the surface of saliva-coated HA. The presence of lysozyme affected the amount of glucan formation by GtfB, but not the structure of glucans formed in solution and on the surface. Therefore, the interaction of lysozyme and GtfB enzymes on HA surface may modulate the formation of glucan and dental plaque.

Effect of sucrose containing iron (II) on dental biofilm and enamel demineralization in situ.

Since the effect of iron (Fe) on the cariogenicity of sucrose in humans is unexplored, this study assessed in situ the effect of Fe co-crystallized with sucrose (Fe-sucrose) topically applied in vitro on the acidogenicity, biochemical and microbiological composition of the dental biofilm formed in vivo and on the demineralization of the enamel. During two phases of 14 days each, 16 volunteers wore palatal appliances containing blocks of human enamel, which were submitted to four groups of separate treatments: (1) water; (2) 20% sucrose; (3) 20% (w/v) sucrose plus 18 microg Fe/ml, and (4) 20% (w/v) sucrose plus 70 microg Fe/ml. The solutions were dripped onto the blocks 8 times per day. The biofilms formed on the blocks were analyzed with respect to acidogenicity, biochemical and microbiological composition. Mineral loss was determined on enamel by surface and cross-sectional microhardness. Lower demineralization was found in the blocks subjected to Fe-sucrose (70 microg Fe/ml) than in those treated with sucrose (p < 0.05). This concentration of Fe also reduced significantly the populations of mutans streptococci in the biofilm formed on the blocks. In conclusion, our data suggest that Fe may reduce in situ the cariogenic potential of sucrose and the effect seems to be related to the reduction in the populations of mutans streptococci in the dental biofilm formed.

The effects of egg-derived antibodies to glucosyltransferases on dental caries in rats.

The role of Streptococcus mutans in the development of dental caries is well recognized. Important virulence factors include the glucosyltransferases (gtf), essential for production of glucans. We evaluated the anticariogenic effects of orally administered chicken anti-cell-associated (CA) Gtf antibodies in desalivated rats. The animals were infected with S. mutans MT8148R and treated with chicken anti-CA-Gtf egg yolk antibodies (IgY) or nonimmune egg yolk powder. Smooth surface lesions were significantly lower in the anti-CA-Gtf-treated group in comparison to the control groups. Sulcal surface caries was also decreased and of less severity. Our study suggests that chicken anti-CA-Gtf antibodies may have promise as a prophylaxis for high caries risk patients.

Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol.

OBJECTIVES: Apigenin is a potent inhibitor of glucosyltransferases and tt-farnesol affects the membrane integrity of Streptococcus mutans. We investigated the influence of apigenin and tt-farnesol, alone and in combination, on the accumulation, polysaccharide composition and viability of S. mutans UA159 biofilms. METHODS: Initially, biofilms were grown for 54 h; then, the early-formed biofilms were treated for 1 min twice daily with one of the following: (i). 1.33 mM tt-farnesol; (ii). 1.33 mM apigenin; (iii). apigenin + tt-farnesol (1.33 mM each); (iv). vehicle control (20% ethanol with 0.75% dimethyl sulphoxide); (v). 0.12% chlorhexidine (1.33 mM); or (vi). physiological saline (145 mM NaCl). The procedure was repeated at biofilm ages of 78 and 102 h, and biofilms were harvested at 126 h. The dry weight, protein concentration, number of cfu, and polysaccharide composition per biofilm were determined. RESULTS: The dry weights of the biofilms treated with the test agents were significantly less (30-50%) than those treated with vehicle control (P < 0.05). Biofilms treated with the test agents also resulted in lower amounts of extracellular alkali-soluble glucans, intracellular iodophilic polysaccharides and, to a lesser extent, fructans. The fructosyltransferase activity was affected only by apigenin and apigenin + tt-farnesol. The recoverable viable counts of S. mutans were slightly lower (0.5 to 1 log10 decrease in cfu/biofilm) after apigenin and tt-farnesol treatments compared with the vehicle control. Chlorhexidine displayed potent bactericidal activity, and virtually halted the further accumulation of early-formed (54 h old) biofilms. CONCLUSIONS: Apigenin and tt-farnesol affected the accumulation and polysaccharide content of S. mutans biofilms without major impact on the bacterial viability.

Effects of apigenin and tt-farnesol on glucosyltransferase activity, biofilm viability and caries development in rats.

Propolis, a resinous hive product secreted by Apis mellifera bees, has been shown to reduce the incidence of dental caries in rats. Several compounds, mainly polyphenolics, have been identified in propolis. Apigenin and tt-farnesol demonstrated biological activity against mutans streptococci. We determined here their effects, alone or in combination, on glucosyltransferase activity, biofilm viability, and development of caries in rats. Sprague-Dawley rats were infected with Streptococcus sobrinus 6715 and treated topically twice daily as follows: (1) tt-farnesol, (2) apigenin, (3) vehicle control, (4) fluoride, (5) apigenin +tt-farnesol, and (6) chlorhexidine. Apigenin (1.33 mM) inhibited the activity of glucosyltransferases in solution (90-95%) and on the surface of saliva-coated hydroxyapatite beads (35-58%); it was devoid of antibacterial activity. tt-Farnesol (1.33 mM) showed modest antibacterial activity against biofilms and its effects on glucosyltransferases were minimal. The incidence of smooth-surface caries was significantly reduced by apigenin +tt-farnesol (60%), fluoride (70%), and chlorhexidine (72%) treatments compared to control (P < 0.05).

Lactoperoxidase inhibits glucosyltransferases from Streptococcus mutans in vitro.

This study examines the possible effect of the antimicrobial peroxidase system on the activity of streptococcal glucosyltransferases B, C and D (GtfB, GtfC and GtfD), either in solution (GtfB and GtfC) or when adsorbed to hydroxyapatite (GtfC and GtfD) at pH 6.5. The lactoperoxidase (LP) system (LP, H(2)O(2), SCN(-)) had no effect on the activity of dissolved GtfC, but the activity of dissolved GtfB was enhanced. The LP system, however, strongly inhibited the activities of both GtfC and GtfD in their adsorbed form. LP enzyme, without its substrates, inhibited all three Gtf enzymes: GtfB and GtfC in concentrations between 10 and 100 microg/ml in liquid phase and adsorbed GtfC and GtfD in concentrations between 25 and 50 microg/ml. This inhibition was in part abolished in liquid phase, but not in solid phase, if the substrates of LP were added. This study shows that the lactoperoxidase system can exert inhibitory activity against streptococcal Gtfs without generating oxidizing agents.

Exposure to metal ions and susceptibility to dental caries.

Results from several epidemiologic studies have shown that there are large differences in the prevalence of dental caries from one region to another within the United States as well as in other countries. It has been postulated that the observed differences may be attributed in part at least to exposure to trace elements such as selenium, vanadium, molybdenum, strontium, and lead. Although data from epidemiologic studies usually support this hypothesis, direct evidence is sparse with the possible exception of exposure to lead. Data from several epidemiologic studies and animal-based research support the concept that lead is a caries-promoting element. Lead mimics calcium in several respects and may affect development of teeth and salivary glands, clearly enhancing susceptibility to dental caries. Elevated blood levels are found most commonly in persons residing in inner cities, particularly among the poor. Many states require blood lead level to be monitored in young children. Where feasible, these records should form part of health history and be available to the treating dental practitioner to ensure that extra preventive procedures may be implemented.

The effects of benzoate and fluoride on dental caries in intact and desalivated rats.

The decline in prevalence of dental caries in some segments of the population has been attributed mainly to extensive exposure to fluoride. Over the past decades, the use of fluoridated products has increased. During the same period, the consumption of food preservatives such as benzoates and sorbates has also increased substantially. Benzoates, in vitro, possess antibacterial properties similar to those of fluoride and in combination with fluoride could affect caries development. In the present study we explored the effects of sodium benzoate and fluoride in combination and alone on dental caries in our animal model. The results showed a combination of benzoate and fluoride reduced caries activity more effectively in rodents fed a cariogenic diet ad libitum than fluoride alone (p = 0.038).