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This study evaluated the effect of chlorhexidine/thymol (CHX/T) and fluoride (F) varnishes on biofilm formation in vitro. Hydroxyapatite discs coated with varnish were first immersed in saline for 0, 3, 7 or 14 d, then immersed in pasteurized saliva. The discs were incubated for 20 h with a bacterial suspension containing Actinomyces naeslundii, Fusobacterium nucleatum, Streptococcus oralis, and Veillonella dispar. Uncoated discs were used as controls. Growth of bacteria on the discs was evaluated by culture and by scanning electron microscopy (SEM). Bacterial vitality was examined by fluorescence staining. In the CHX/T-treated group, bacterial accumulation was delayed, and the total number of bacteria was significantly lower than in the controls. In the F-treated group, the total number of bacteria did not differ from the control, although the number of S. oralis was lower. Bacterial vitality in the CHX/T and F groups did not differ from that in the controls. The total number of bacteria on the CHX/T-treated discs immersed in saline was significantly higher than that on the non-immersed discs. Biofilm development was inhibited by the CHX/T varnish but not by the F varnish. The effect of the CHX/T varnish decreased following the immersion of discs in saline.

Ozone gas and photodynamic therapy (PDT) have been claimed to be antimicrobially effective. This study assessed their antimicrobial potential in vitro. Mature six-species oral biofilms were treated as follows (n = 9 per group): (i) a 60-s application of gasiform vacuum-ozone or vacuum alone (on wet or air-dried biofilm samples); (ii) PDT (i.e. methylene blue in combination with or without a diode soft laser, and a soft laser alone); or (iii) antimicrobial solutions: immersion of biofilms for 60 s in 0.2 and 2% chlorhexidine or in 0.5 and 5% hypochlorite solution. Treatment with chlorhexidine or hypochlorite served as a positive control, whereas untreated samples served as negative controls. Colony-forming units on blood agar were counted. Only the 5% hypochlorite solution was able to totally eliminate the microorganisms in the biofilm. The observed reduction of viable counts by vacuum-ozone application and PDT was less than one log(10) step. Under the conditions of the current study, gasiform ozone and PDT had a minimal effect on the viability of microorganisms organized in a cariogenic biofilm.

Glucosyltransferases from Streptococcus mutans are thought to play an important role in bacterial adherence to the tooth surface. The goal of the present study was to determine the effect of the deletion of the gtfC gene, which encodes a glucosyltransferase that catalyses primarily the formation of insoluble glucan (mutan), on colonization of S. mutans in a mixed-species biofilm model of supragingival plaque. A gtfC deletion mutant of S. mutans UA159 grew poorly in biofilms on a polystyrene surface in Todd-Hewitt medium containing sucrose, but biofilm formation in the semi-defined fluid universal medium (FUM) was not affected. The S. mutans gtfC mutant colonized with the same efficiency as the wild-type strain when grown together with five other species in a mixed-species biofilm on hydroxyapatite in a mixture of FUM and saliva with pulses of sucrose and showed the same ability to demineralize enamel in vitro. Colonization of mutant and wild-type strains was also equal in an association experiment in specific-pathogen-free rats. However, the gtfC mutant gave rise to more dentinal fissure lesions and smooth surface caries than the wild-type strain; this could be caused by a change in diffusion properties as a result of to the lack of mutan.

The objective of this study was to quantitatively compare the bacterial population structure in plaque from the gingival margin of two groups of 21 Chinese patients with gingivitis or necrotizing ulcerative gingivitis (NUG). Subjects were recruited in four dental clinics in Eastern China. Samples were quantitatively assessed by immunofluorescence and fluorescent in situ hybridization for taxa known to be associated with periodontal diseases. The analyses showed that the fusiform taxa (Fusobacterium nucleatum/Fusobacterium periodonticum, Leptotrichia buccalis, Tannerella forsythensis, and Capnocytophaga sp.), Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens, Selenomonas sputigena, and treponemes were present in both groups with high prevalence. Porphyromonas gingivalis and Actinomyces gerencseriae were much more prevalent in the NUG group. Quantitatively, most taxa, including P. gingivalis, F. nucleatum and the treponemes, accounted, on average, for < 3% of the total bacterial cell number. Only P. intermedia/P. nigrescens, P. gingivalis, S. sputigena, A. gerencseriae, and the sum of all monitored suspected periodontal pathogens were significantly increased in the NUG group. The present study demonstrates for both groups a highly diverse plaque composition and suggests that, etiologically, the overall concentration and the concerted effects of the entire group of opportunistic pathogens thriving in NUG-associated plaque are of prime importance.

The aim of the present study was to select bacterial strains with potential properties as oral probiotics, namely for the prevention of dental caries. We examined 23 dairy microorganisms, out of which we identified two Streptococcus thermophilus and two Lactcoccus lactis strains that were able to adhere to saliva-coated hydroxyapatite beads to the same extent as Streptococcus sobrinus OMZ176. Two of them, Strep. thermophilus NCC1561 and Lactoc. lactis ssp. lactis NCC2211, were further successfully incorporated into a biofilm mimicking the dental plaque. Furthermore, they could grow in such a biofilm together with five strains of oral bacterial species, representative of supragingival plaque. In this system, Lactoc. lactis NCC2211 was able to modulate the growth of the oral bacteria, and in particular to diminish the colonization of Streptococcus oralis OMZ607, Veillonella dispar OMZ493, Actinomyces naeslundii OMZ745 and of the cariogenic Strep. sobrinus OMZ176. These findings encourage further research with selected non-pathogenic dairy bacterial strains with the aim to decrease the cariogenic potential of dental plaque.

Proline-rich proteins (PRPs), histatins, and statherin are salivary proteins that exhibit high affinities for hydroxyapatite surfaces. In vitro experiments with parotid submandibular/sublingual or whole saliva have shown these proteins to adsorb selectively to tooth surfaces. This investigation focuses on the histo-morphological identification of PRPs, histatins, and statherin in acquired enamel pellicles. Synthetic hydroxyapatite or bovine enamel were exposed to glandular secretions, and whole saliva and pellicle precursor proteins were identified immunohistologically by electron microscopy. Results obtained by back-scattered scanning electron microscopy showed these proteins to be present in pellicles. Pellicles displayed a distinct structure consisting of a sponge-like meshwork of microglobules. Interconnections between structural elements were identified in submandibular/sublingual and whole saliva pellicles only. Transmission electron microscopy of pellicles formed on bovine enamel surfaces revealed a tendency for preferential localization of precursor proteins within the protein film. Since the data showed the presence of pellicle precursors in pellicles derived both from glandular secretions and from whole saliva, it is likely that PRPs, histatins, and statherin are integral components of acquired enamel pellicles in vivo.

The present study investigated a recently developed automated image analysis technique for its applicability to the enumeration of selected bacteria in supragingival dental plaque. Following initial calibration, the system is capable to count fluorescence-labeled target cells in up to 48 samples without user interference. Test samples contained a characteristic mixture of planktonic bacteria, small almost planar bacterial aggregates, and large, virtually indisruptable clumps with cells from multiple species. Due to their complex composition, these samples provided a challenging validation step for the image analysis system. Automated enumeration of target bacteria was compared with visual counting of the fluorescence-labeled bacteria. Results are shown for six taxa (Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia/Prev. nigrescens, Streptococcus gordonii/Strep. oralis/Strep. sanguis, Strep. sobrinus, and Veillonella dispar/ V. parvula) with characteristic differences in abundance, cell morphology and aggregation behavior. Results revealed good correspondence between the two enumeration techniques (correlation coefficients ranging from 0.77 to 0.92) provided that the portion of target bacteria exceeded 0.05% of the total bacterial cell number. This work demonstrates the applicability and usefulness of fully automated immunofluorescence to analyze such complex ecosystems as supragingival dental plaque.

This study validates an in situ model for ecological studies of dental plaque exposed to various antimicrobial agents with different modes of action on plaque bacteria. Eleven subjects wore two acrylic appliances, each containing two bovine enamel discs, during two 1-wk test periods. Using a split-mouth crossover design, the appliances were dipped twice daily for 1 min into water (control; treatment A), fluoride (26.3 mM NaF; B), zinc acetate (20.0 mM; C), or fluoride plus zinc acetate (D). Four of the subjects used also chlorhexidine diacetate (2.2 mM; E) and chlorhexidine plus fluoride (F). At the end of each period, plaque was collected from the discs, after which the microbiota were analyzed by culture, automated quantitative immunofluorescence, and a viability fluorescence stain. As compared to control, treatments B, C, and D resulted in a significant reduction of individual taxa as detected by immunofluorescence, whereas similar bacterial viability and total bacterial numbers were observed. In contrast, chlorhexidine significantly reduced bacterial viability, total cell numbers, and the abundance of most of the enumerated taxa. We conclude that this in situ model is well suited to study effects of antimicrobial agents on dental plaque ecology. Combined with viability testing, immunofluorescence is obviously superior to culture in detecting taxa-specific shifts caused by antimicrobial agents.

The purpose of this study was the clinical and microbiological re-examination of dental hygienists, who, 30 months before, had shown remarkably high supragingival levels of periodontitis-associated micro-organisms. Interdental plaque was collected from the same molar sites and investigated by the same immunofluorescence assay with taxa-specific monoclonal antibodies as at the initial examination. On average, the 15 re-examined subjects showed slightly increased plaque levels but unchanged bleeding on probing scores (0.3-1.4). Pocket formation was restricted to a single subject. Prevotella intermedia/P. nigrescens and Peptostreptococcus micros were present in every plaque sample. Prevalences of Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Campylobacter rectus were again between 20-40%, but some fluctuation within subjects was noted. The data confirm supragingival plaque as a natural habitat for periodontitis-associated bacteria in periodontially healthy persons, and indicate that colonization with A. actinomycetemcomitans, B. forsythus or C. rectus is mostly stable in spite of better than average personal plaque control.

The effectiveness of 5 different brands of dentifrices on caries and plaque were tested in rats in 2 experiments. 10 litters of rats each were infected with Streptococcus sobrinus OMZ-176 and Actinomyces viscosus Ny1. The rats were offered the diet 2000a containing either 40% of sucrose (study I) or 10% of sucrose (study II). The following products were tested topically: (1) H2O, (negative control); (2) chlorhexidine diacetate, (positive control); (3) Parodontax NF; (4) Meridol; (5) Colgate Total; (6) Mentadent C; (7) Dent Xpress. Comparisons with the water control in study I showed that plaque extent was decreased by chlorhexidine diacetate, Dent Xpress and by Mentadent C only. All fluoride-containing dentifrices tested reduced caries. In study II, comparisons with the water control showed that plaque extent was reduced by chlorhexidine diacetate, Parodontax, Meridol, Dent Xpress and by Mentadent C. All dentifrices reduced caries, however, the incidence was low. These 2 studies confirm that fluorides incorporated in dentifrices decrease dental caries in the rat. Differences in the effectiveness of caries inhibition between the 5 dentifrices could not be explained either by the form of fluoride utilized or by the antimicrobials added. Rather, the performance of a given dentifrice depended upon the concerted action of the particular ingredients. It appears that the effect of antiplaque agents should be evaluated in the rat model using a diet containing sucrose at a low level.

The present study details the structural and ultrastructural features of healing events between the regenerated periodontal ligament and the root surface after experimental periodontal disease. Experiments were performed on dogs, and the concept of guided tissue regeneration was tested using resorbable polyurethane membranes. Light microscopy, scanning- and transmission electron microscopy were employed to determine if healing events involve regeneration (ie, process by which the architecture and the function are completely renewed) or periodontal repair (ie, healing of a wound by tissue that does not fully restore the architecture of a part). Regeneration occurred in parts of the roots only if some original cementum remained on the root surface after root planing. Repair was observed if peripheral dentin was removed by root planing as this layer was not reestablished. In areas remote from the base of the defect, new collagen fibrils, synthesized by fibroblasts and oriented perpendicular to the root surface, were spliced with severed ends of Sharpey's fiber bundles of original cementum. If circumpulpal dentin was exposed, intermingling between new fibrils with dentinal matrix fibrils occurred. In areas near the base of the defect, the first event was the formation of a cementoid by a cementoblast monolayer and subsequent formation of intrinsic fibrils oriented parallel to the root surface. Afterwards, the cementoblast monolayer disintegrated and extrinsic fiber bundles became anchored in the new cellular mixed fiber cementum. In these areas, linkage between new cementum and pre-existing tissues always occurred by interfacial intermingling of the fibrils, regardless of whether new attachment occurred at circumpulpal dentin or original cementum.

Tissue samples from 2 humans suffering from severe periodontitis were investigated by transmission electron microscopy. Confirming earlier observations in gnotobiotic rats, bacteria were found in various regions of gingival tissues. We observed bacteria invading the pocket epithelium, the underlying connective tissue, and microorganisms were also present deep in the connective tissue. The bacterial invasion of the periodontium was accompanied by different stages of tissue degradation. Various morphologically distinct types of Gram-negative and Gram-positive bacteria were evident. These microorganisms were found in the apical part of the periodontium. More coronally, a heavy infiltrate consisting predominantly of plasma cells was present. It is suggested that bacteria cannot invade regions of connective tissue protected by a massive cellular infiltrate. It seems that they rather circumvent this strong defense by penetrating more apically through the pocket epithelium while producing as camouflage leukochemophobic compounds which paralyze the chemotactically regulated mobile defense. It seems to us that bacterial invasion is a consistent feature of advanced periodontitis, leading to focal necrosis or microabscesses, and may well explain the cyclic nature of this disease.

Plaque accumulation and characteristics were determined gravimetrically, planimetrically, visually, and microbiologically in 24 human subjects who consumed a carbohydrate-free diet for 4 days. In addition, the effects of xylitol-, sorbitol- and sucrose-containing candies upon plaque formed during consumption of the diet were ascertained. Experimental design was such that all 24 subjects traversed each 4-day test period; a 9-day interval between test periods was observed. All plaque measurement methods demonstrated that the subjects in all groups, including a control group which received no candies, accumulated significant amounts of dental plaque. No significant differences in plaque amount could be detected among the xylitol, sorbitol and control groups. However, subjects receiving sucrose-containing candies clearly exhibited larger amounts of plaque. The bacteriological investigations corroborated the clinical observations. The number of microbial colonies from plaque samples taken from the sucrose test group was clearly higher than in the other three groups. In addition, the number of bacteria in plaque samples from the sorbitol group was higher than in the xylitol group. A statistically significant increase in the proportion of anaerobic sorbitol- and xylitol- fermenting bacteria was detected in the sorbitol and xylitol test groups during the 4-day test periods.

The prevalence of Bacteroides forsythus and Bacteroides gingivalis in subgingival plaque of patients on short recall was analyzed in relation to the probing depth of the test sites. The subjects had excellent oral hygiene and therefore were unlikely to suffer from active periodontal destruction. Sixty-four subgingival plaque samples, taken from gingival or periodontal pockets with probing depths ranging from 1 to 8 mm, were quantitatively assessed for the presence of the two species using species-specific monoclonal antibodies in conjunction with a very sensitive indirect immunofluorescence technique. Both organisms were encountered in probes from sites as shallow as 2 mm, but the percentage of positive samples clearly rose in relation to the probing depth of the test sites. Overall, B. forsythus was found to colonize lesions earlier, that is at smaller probing depths, than B. gingivalis. Interestingly, whenever a sample was found to be positive for B. gingivalis it was also positive for B. forsythus. The numbers of B. forsythus and B. gingivalis and the total bacterial cell number found in the pockets were significantly correlated to the probing depth. However, with advancing probing depth the increase of the total cell numbers of the two Bacteroides species was considerably more pronounced than the increase of the total subgingival plaque cell number. The recall interval neither affected the frequency of sites positive for B. forsythus or B. gingivalis nor influenced significantly the proportions of the two species in subgingival plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

The histopathology of human root caries was examined in extracted teeth by different optical methods. The present part of the study details the sequential stages of initial caries in both cementum and peripheral dentin. Significant differences are shown between the mechanisms operating on the various dental hard tissues during development of caries. Histologically distinguishable reaction patterns depended on both the degree of the cariogenic challenge and the respective structural features of cementum and peripheral dentin. Earliest lesions, histologically visible as small clefts traversing cementum and extending into peripheral dentin, were clinically not detectable. Cementum was stepwise destroyed until peripheral dentin became exposed. The exposed peripheral dentin was sclerosed and tubule-free. Demineralization of dentin preceded the degradation of the organic matrix. Frequently, a hypermineralized layer occurred in both cementum and exposed dentin. Its localization was not identical with the anatomical surface. Initial penetration of bacteria into peripheral dentin occurred along small clefts. Breakdown of the organic matrix was the final step in the destructive phase of the carious process.