Effects of the tea catechin epigallocatechin gallate on Porphyromonas gingivalis biofilms.

AIMS: The aim of this study was to investigate the effects of tea catechin epigallocatechin gallate (EGCg) on established biofilms and biofilm formation by Porphyromonas gingivalis, a major pathogen of periodontal disease. METHODS AND RESULTS: Biofilm cell survival was measured using adenosine triphosphate (ATP) bioluminescence. In the presence of EGCg, the ATP level in cells of established biofilms was significantly decreased compared to the controls (P < 0·0001). Transmission electron microscopy revealed that EGCg damaged the cell membrane and cell wall of P. gingivalis. Confocal laser-scanning microscopy revealed that the proportion of dead cells was higher in biofilms treated with EGCg. Moreover, the effects of subminimal inhibitory concentrations (MICs) of EGCg on P. gingivalis biofilm formation were dose-dependent (P < 0·0001). CONCLUSION: Our results suggest that EGCg destroys established P. gingivalis biofilms and inhibits biofilm formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of chemical control agents against oral biofilms is necessary, because oral biofilms can be only removed using mechanical debridement. This article indicates that EGCg may represent a novel antibiofilm agent that prevents infections involving bacterial biofilms such as periodontitis.

How regular visits and preventive programs affect onset of adult caries.

If it is to be effective, preventive dental treatment must be based on an individual's risk of caries and periodontal disease. However, hitherto, preventive treatment has been generally conducted without proper caries-risk assessment because of lack of knowledge about an individual's caries risk. This study sought to identify high-risk adult patients and examine the effectiveness of preventive programs. Data for 442 patients from a single general practice who met the inclusion criteria were analyzed. Multiple logistic-regression analysis showed that high levels of mutans streptococci (SM) correlated with the onset of primary and secondary caries, with odds ratios of 2.34 (95%CI: 1.15-4.76, p = 0.019) and 2.22 (95%CI: 1.06-4.62, p = 0.034), respectively. Lactobacilli (LB) also correlated with secondary caries, with an odds ratio of 3.34 (95%CI: 1.35-6.85, p = 0.007). When the patients were checked after three years of a preventive program, the correlation with SM and LB disappeared. Survival analyses by Cox regression models and Kaplan-Meier methods showed that secondary caries was difficult to control by conventional preventive programs alone in patients with high levels of cariogenic bacteria. In conclusion, cariogenic bacteria play an important role in the incidence of caries, and additional intensive preventive treatment should be considered for high-risk patients.

Synergistic effects of antibiotics and an N-acyl homoserine lactone analog on Porphyromonas gingivalis biofilms.

AIMS: To investigate the effects of the combined application of an N-acyl homoserine lactone (HSL) analog and antibiotics on biofilms of Porphyromonas gingivalis, a major pathogen of periodontal disease. METHODS AND RESULTS: Antibiotics used were cefuroxime, ofloxacin and minocycline. A flow-cell model was used for biofilm formation. Samples were divided into four groups: control, analog-treated, antibiotic-treated and combined application groups. Biofilm cell survival was determined using adenosine triphosphate (ATP) bioluminescence and confocal laser microscopy (CLSM). In the combined application group, the ATP count in biofilm cells was significantly decreased compared with the antibiotic-treated group (Games-Howell test, P < 0·05). A combination of cefuroxime and the analog was most effective against the P. gingivalis biofilm. CLSM observations revealed that the proportion of dead cells was highest in the combined application group. CONCLUSIONS: The combined application of the N-acyl HSL analog and antibiotics was effective at reducing the viability of P. gingivalis cells in biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined application of the N-acyl HSL analog and antibiotics may be successful for eradicating infections involving bacterial biofilms, such as periodontitis.

Antibiofilm effects of azithromycin and erythromycin on Porphyromonas gingivalis.

Antibiotic resistance of biofilm-grown bacteria contributes to chronic infections, such as marginal and periapical periodontitis, which are strongly associated with Porphyromonas gingivalis. Concurrent azithromycin (AZM) administration and mechanical debridement improve the clinical parameters of periodontal tissue in situ. We examined the in vitro efficacy of AZM against P. gingivalis biofilms. The susceptibilities of adherent P. gingivalis strains 381, HW24D1, 6/26, and W83 to AZM, erythromycin (ERY), ampicillin (AMP), ofloxacin (OFX), and gentamicin (GEN) were investigated using a static model. The optical densities of adherent P. gingivalis cells were significantly decreased by using AZM and ERY at sub-MIC levels compared with those of the controls in all the strains tested, except for the effect of ERY on strain W83. AMP and OFX inhibited P. gingivalis adherent cells at levels over their MICs, and GEN showed no inhibition in the static model. The effects of AZM and ERY against biofilm cells were investigated using a flow cell model. The ATP levels of P. gingivalis biofilms were significantly decreased by AZM at concentrations below the sub-MICs; however, ERY was not effective for inhibition of P. gingivalis biofilm cells at their sub-MICs. Furthermore, decreased density of P. gingivalis biofilms was observed three-dimensionally with sub-MIC AZM, using confocal laser scanning microscopy. These findings suggest that AZM is effective against P. gingivalis biofilms at sub-MIC levels and could have future clinical application for oral biofilm infections, such as chronic marginal and periapical periodontitis.

Antibacterial effects of MDPB against anaerobes associated with endodontic infections.

AIM: To investigate the antibacterial effects of 12-methacryloyloxydodecylpyridinium bromide (MDPB), an antibacterial monomer synthesized by combining quaternary ammonium with a methacryloyl group, against three anaerobes associated with endodontic infections using planktonic and biofilm cells. METHODOLOGY: The antibacterial activity of unpolymerized MDPB against Enterococcus faecalis, Fusobacterium nucleatum and Prevotella nigrescens was examined by agar-disc diffusion tests and determination of the minimum inhibitory/bactericidal concentrations (MIC/MBC). Rapid killing effects of MDPB against three bacteria in planktonic form were examined by a cell number counting method, and those against biofilm cells were assessed by a viability staining method. RESULTS: MDPB demonstrated inhibition against all of the bacteria tested by agar-disc diffusion tests. The MIC/MBC values of MDPB for the three anaerobes were much smaller than those of other resin monomers, although greater compared with those of cetylpyridinium chloride or chlorhexidine diacetate for E. faecalis and F. nucleatum. Significant reduction in viable planktonic cells was obtained by contact with 250 microg mL(-1) of MDPB for 20 s (P < 0.05, Fisher's PLSD tests), and 40 s contact with 500 microg mL(-1) or 20 s contact with 1000 microg mL(-1) of MDPB resulted in more than 90% killing. Biofilm cells of all species were completely killed by application of 1000 microg mL(-1) of MDPB for 60 s. CONCLUSION: MDPB was found to have strong antibacterial effects against E. faecalis, F. nucleatum and P. nigrescens, and such effects were rapidly exhibited even against biofilm cells, suggesting the usefulness of application of MDPB to resin-based materials for root canal filling.

Effects of N-acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation.

BACKGROUND AND OBJECTIVE: The gram-negative anaerobic rod Porphyromonas gingivalis in oral biofilms is a primary etiological agent of periodontal disease. Biofilm formation of various gram-negative bacteria is regulated by a quorum-sensing circuit that relies on N-acyl homoserine lactones (HSLs). Some synthetic N-acyl HSL analogues act as quorum-sensing inhibitors and suppress biofilm formation in Pseudomonas aeruginosa. Development of chemical control agents against oral biofilms is necessary, because until now, biofilms have been removed only by mechanical debridement. The present study investigated the effect of N-acyl HSL analogues on P. gingivalis biofilm formation, with the aim of developing new drugs that inhibit oral biofilm formation. MATERIAL AND METHODS: A flow-cell model was used for P. gingivalis biofilm formation. Seventeen synthetic N-acyl HSL analogues were quantitatively assessed by spectrophotometry. The effects of three antagonistic compounds against P. gingivalis biofilm formation were further examined by confocal laser scanning microscopy, and investigated for primary attachment using spectrophotometry and phase contrast microscopy. RESULTS: Ten out of 17 analogues affected P. gingivalis biofilm formation. Three out of 10 analogues significantly decreased biofilm-forming cells (p < 0.05), and these biofilm structures were less well formed three-dimensionally. There were no quantitative or qualitative differences in cell attachment between the control and the three analogue-treated groups. CONCLUSION: Three synthetic N-acyl HSL analogues inhibited biofilm formation in P. gingivalis. We suggest that these analogues influence the development stage of P. gingivalis biofilm formation.

An explanation of the mineralization mechanism in osteoblasts induced by calcium hydroxide.

Calcium hydroxide (Ca(OH)(2)) has been broadly used in endodontics, including apexification to obtain apical closure by mineralization. However, the detailed mechanism of mineralization induced by Ca(OH)(2) is still unclear. This study focuses on the function of calcium and hydroxyl ions which dissociate from Ca(OH)(2) during the mineralization process. Though primary osteoblasts cultured in the medium without or with 0.025mgml(-1) Ca(OH)(2) did not show mineralization, they did exhibit mineralization when they were cultured with a higher concentration of Ca(OH)(2) (0.25mgml(-1)). Mineralization induced in the presence of 0.25mgml(-1) Ca(OH)(2) was greater at pH 7.4 than at pH 8.5. The high mineralization activity observed under neutral conditions was caused by the prolonged activation of p38 and JNK. Hydroxyl ions did not have any effect on the mineralization. The results demonstrate that calcium ions dissociated from Ca(OH)(2) are critical for inducing the mineralization of osteoblasts.

Effects of rehydration on dentin strengthened by heating or UV irradiation.

Type I collagen, the major organic component of human dentin, plays an important role in regulating the mechanical strength of dentin. Collagen in dentin can be strengthened by heating. We hypothesized that UV irradiation could produce similar strengthening effects and might maintain the strength of dentin after rehydration. Beam-shaped dentin specimens from the crowns of human third molars were subjected to flexural testing. Flexural strengths were two and three times greater than those in the control group after 5 minutes' UV irradiation and heating to 140 degrees C, respectively. After 30 days of rehydration, the heated specimens reverted to their original strength, whereas the UV specimens were 69% stronger than the original. Raman spectra of dental collagen were unchanged after heating, whereas several peaks, including a C-C bond in a proline ring, were amplified by UV irradiation. It is concluded that dentin strengthened by UV irradiation retains strength after rehydration because of chemical changes in collagen.

Heat treatment strengthens human dentin.

The flexural strength of Type I collagen, the major organic component of human dentin, increases with heat. We hypothesized that human dentin can be strengthened by heating, which may help prevent fracture of non-vital teeth after restoration. Beam-shaped dentin specimens were obtained from the crowns of human third molars. The dentinal tubular orientations were arranged to run parallel or perpendicular to loading surfaces. The flexural and microtensile strengths of dentin in the parallel specimens were 2- to 2.4-fold greater after being heated between 110 degrees C and 140 degrees C for 1 hr. The stress intensity factors at fracture also increased after specimens were heated. The x-ray diffraction analyses suggested that shrinking of the lateral packing of the collagen triple-helices from 14 A to 11 A was the probable cause of the strengthening of heated dentin. We conclude that heat treatment strengthens human dentin.

Antibacterial effect of chlorhexidine- containing glass ionomer cement in vivo: a pilot study.

This in vivo pilot study was carried out to test the antibacterial effect of glass ionomer containing chlorhexidine (test group) in comparison to conventional glass ionomer (control group). Fifty 6- to 11-year-old children with one occlusal lesion in a molar were randomly allocated to test and control groups in a parallel-group design. The cavity walls and one half of the floor were cleaned and restored with one of the materials without dentine conditioning. The restorations were removed after 7 days. Dentine samples were taken from the cleaned (affected dentine) and noncleaned area (infected dentine) at baseline and at day 7. Samples were anaerobically and aerobically cultivated for mutans streptococci, lactobacilli and total viable bacterial count (TVC) following common laboratory procedures. ANCOVA was used to test for treatment effects. Seven days after treatment, a significant decrease in anaerobic and aerobic bacterial counts (p = 0.0001) was shown. Lower numbers of anaerobic lactobacilli (p = 0.02), TVC (p = 0.008) and aerobic lactobacilli and TVC (p = 0.03), but not of mutans streptococci, were indicated in the test group compared to the control group. A significant reduction in aerobic lactobacilli from infected dentine treated with the glass ionomer containing chlorhexidine (p = 0.05) was observed whereas in affected dentine, anaerobic mutans streptococci, lactobacilli and TVC and aerobic TVC and mutans streptococci were significantly lower in the test group 7 days after treatment (p = 0.01). We conclude that the present pilot study revealed lower microorganism counts in chlorhexidine-containing glass ionomers than in conventional glass ionomers for both affected and infected dentine over a 7-day period.

Soluble products from Eikenella corrodens induce cell proliferation and expression of interleukin-8 and adhesion molecules in endothelial cells via mitogen-activated protein kinase pathways.

The periodontal vasculature is profoundly affected during the progression of periodontitis, and several specific bacteria are believed to be involved in this inflammatory disease. Eikenella corrodens is one of the common bacteria detected in periodontitis diseased lesions; however, the function of this organism in periodontitis is not well understood. In this study, we investigated the E. corrodens-induced endothelial cell alteration and inflammation process that leads to leukocyte infiltration in inflamed regions. Soluble products from E. corrodens (EcSP) induced the gene expression and protein production of vascular endothelial growth factor in oral epithelial cells and human umbilical vein endothelial cells (HUVEC). Direct stimulation by EcSP also activated endothelial cell proliferation. Moreover, EcSP induced ERK1/2 (p44/42) and p38 mitogen-activated protein kinase (MAPK) phosphorylation within 10-30 min in HUVEC, as demonstrated by Western blot analysis and up-regulated intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin and interleukin-8 (IL-8) production demonstrated by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. The specific p38 MAPK inhibitor SB203580 reduced the expression of ICAM-1, VCAM-1 and IL-8, whereas the blockade of p44/42 by MAPK kinase (MEK1) inhibitor, PD98059, inhibited only IL-8 expression. Our results indicate that E. corrodens can trigger a cascade of events that induce inflammatory responses in periodontal tissue via the MAPK cascade and may promote chronic periodontitis without bacteria-cell contact.

Fatigue behavior of resin composites in aqueous environments.

OBJECTIVES: Interaction between mechanical and chemical fatigue of resin composites was investigated. METHODS: Fatigue fracture resistance of composites after (1) water immersion and under (2) dry and (3) aqueous conditions was analyzed using a fatigue crack propagation test. The characteristics of fatigue crack propagation were expressed as the correlation between the fatigue crack growth rate (da/dN) and stress intensity factor range (DeltaK) and by the fatigue crack growth threshold (DeltaKth). Following the fatigue test, a fractographic examination was performed by scanning electron microscopy. RESULTS: Contrary to common perception, fatigue crack propagation was retarded under aqueous conditions but accelerated after water immersion. In addition, fatigue cracks detoured around inorganic macro-fillers, but penetrated organic fillers. SIGNIFICANCE: It was concluded that the inverse effects of water on fatigue resistance of composites are dependent on the experimental conditions. Moreover, inorganic macro-fillers were shown to be functional in retarding fatigue cracks in composites.

Prevalence of root caries in a selected population of older adults in Japan.

In this study, the prevalence of root caries in a sample of Japanese elders was investigated along with various aetiological factors. Two hundred and eighty-seven independently living elderly people over the age of 60 years were examined for the prevalence of active, inactive, and filled root lesions as well as the number of teeth. General health, oral status and brushing habits were surveyed, and salivary flow was determined in terms of flow rate and subjective feeling of dry mouth during eating. About 39% of the subjects had one or more decayed roots and 53.3% had at least one decayed or filled lesion. Root caries was seen most frequently on canine teeth followed by the first premolar. The number of decayed or filled lesions increased with increasing number of residual teeth (Kruskal-Wallis test, P = 0.033), however, there was no association between the proportion of teeth with root decay and the number of teeth. For subjects with 20 or more teeth, the number of decayed roots is likely to be lower in subjects who reported brushing frequently (P = 0.058), and higher in subjects with low salivary flow (P = 0.059) or subjective feeling of dry mouth (P = 0.052) (Chi-squared). The presence of decayed lesions is likely to be related to dry mouth (P = 0.057, odds ratio 3.13) and brushing frequency (P = 0.088, odds ratio 1.76) (Multiple logistic regression analysis). The results indicate a high frequency of root surface caries in this Japanese community-dwelling older population. Oral hygiene status and low salivary flow or perceived dry mouth were potential risk factors for disease.

Influence of explorer tip diameter in identifying restoration margin discrepancies.

OBJECTIVES: Evaluations of the marginal adaptation of restorations in clinical trials rely on subjective assessments by evaluators. The purpose of the present study was to assess the use of dental explorers with different tip diameters to detect simulated marginal discrepancies. METHODS: Using three devices simulating vertical steps, horizontal gaps, and the combination of vertical and horizontal discrepancies and explorers with five different tip diameters, 10 experienced dental faculty members were asked to identify discrepancies at the boundary between Alfa (replacement unnecessarily) and Bravo (replacement questionable) ratings, according to modified United States Public Health Service criteria. RESULTS: A significant correlation was found between the tip diameters of the explorers and the Alfa/Bravo boundaries for the horizontal gaps, but not for the vertical steps. CONCLUSION: It was concluded that the tip diameter of dental explorers had no significant effect on the detection of vertical steps, but had a significant effect on the detection of horizontal gaps. The diagnosis of restoration marginal discrepancies and the rating of marginal adaptation in clinical trials may subsequently be found to be best achieved using techniques other than the probing of restoration margins.

Ammonium hexafluorosilicate increased acid resistance of bovine enamel and dentine.

Although diamine silver fluoride (AgF: (NH3)2AgF) stains teeth black, it is known as a very effective agent to prevent the dental caries progress. In order to find another fluoride that has a similar anticariogenic effect without changing tooth color, we prepared ammonium hexafluorosilicate (SiF: (NH4)2SiF6), in which the silver of AgF is replaced with silicon. In this study, the anticariogenic effect of SiF was evaluated using bovine teeth. Fluoride solutions, SiF, AgF, acidulated phosphate fluoride (APF), and sodium fluoride (NaF), were applied to bovine enamel and dentine blocks, and the depth of demineralization was measured after exposure to a demineralizing solution for 24 h. Also, fluoride was applied to a simulated dentine caries specimen to evaluate the caries progress-preventing ability. For the dentine specimens, mineral loss (Delta Z) was also measured with microradiography. We found that SiF treated enamel showed better acid resistance than specimens treated with NaF or APF. AgF treated enamel also showed similar acid resistance, but was stained black. SiF and AgF treated caries-affected dentine showed reduced demineralization when exposed to a demineralization solution for 24 h. Mineral loss (Delta Z) was reduced to 85% and 75%, respectively. Although the acid resistance of the SiF treated teeth was inferior to that of the AgF treated teeth, we consider that SiF has good potential as anticariogenic agent, since it increased acid resistance without changing tooth color.

Inhibition of root caries progression by an antibacterial adhesive.

A dentin primer containing the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) has been shown to penetrate and kill the bacteria in artificially demineralized dentin. We hypothesized that an experimental adhesive system, which incorporates the MDPB-containing primer, would be effective in inhibiting the progression of root caries in vitro. Artificial caries lesions were prepared by either an acid-gel or a Streptococcus mutans culture technique on the roots of extracted human teeth. The progression of these lesions after the application of the experimental or proprietary adhesive system was examined. Further demineralization was completely prevented by the experimental adhesive system, while lesions managed with the proprietary materials showed limited ability to inhibit further demineralization. We conclude that the experimental adhesive system can inhibit the progression of root-surface caries in vitro, through a combination of its antimicrobial activity and sealing of the demineralized dentin.

Localization of Porphyromonas gingivalis-carrying fimbriae in situ in human periodontal pockets.

Fimbriae, which are involved in adherence, constitute an important pathogenic factor of Porphyromonas gingivalis. In vivo, however, the distribution of P. gingivalis-carrying fimbriae is unknown. The localization of P. gingivalis-carrying fimbriae was examined in situ. From 19 patients with severe periodontitis and P. gingivalis, we obtained 20 teeth with periodontal tissue attached, with and without immunolocalized fimbriae. Eleven teeth were subjected to light microscopy, 9 to electron microscopy. In 6 of the 11 samples examined, we detected positive reactions with an anti-P. gingivalis-fimbriae serum, located in the cementum-attached plaque area in the deep pocket zones. In the so-called 'plaque-free zones', P. gingivalis-carrying fimbriae were immunocytochemically observed to reside in contact with the dental cuticle in 6 of the 9 samples examined. These findings suggest that P. gingivalis-carrying fimbriae are strongly related to adherence to the root surface at the bottoms of human periodontal pockets.

Effects of cyclosporin-A-induced immunosuppression on periapical lesions in rats.

Cyclosporin A (CsA) might induce immune response alterations in periapical lesions and modify bone remodeling. This study determined the changes that occur in the periapical lesions of rats during CsA administration and after CsA withdrawal. After the induction of periapical lesions, the animals were treated with CsA (0-20 mg/kg/day) for 4 wks. Lesion volumes were measured by computed tomography. Histological observations and immunohistochemical evaluations were performed with anti-CD3 and anti-CD25 antibodies. CsA administration reduced lesion volumes, and the lesions significantly expanded after CsA withdrawal. CsA inhibited the proliferation and activation of T-cells at lesion sites. The effects of CsA on T-cells were dose-dependent up to 10 mg/kg/day, after which no significant difference was evident. These results suggest that CsA inhibits periapical destruction by interfering with T-cell function in periapical lesions.

Analysis of longitudinal marginal deterioration of ceramic inlays.

This study quantitatively and morphologically analyzed and clarified the longitudinal marginal changes of ceramic inlays and determined the mechanism for those changes. Epoxy replicas of 15 Class II ceramic inlays in permanent premolars prepared at baseline, 6, 12, 24, 48, 72 and 96 months after placement were selected. A CCD optical laser scanner was employed to measure quantitative changes in the occlusal surfaces of restored teeth. Longitudinal cross-sections of marginal areas of a ceramic inlay were computed, and two profiles of the same location obtained at different periods were superimposed using software. The area enclosed by the two profiles obtained at different periods was defined as the quantitative marginal change, and both the area and maximum depth in the area enclosed were calculated with picture analysis software. The marginal deterioration pattern was analyzed by drawing a longitudinal curve of quantitative change for each restoration. Morphological observation was carried out by scanning electron microscopy at magnifications from 20x to 75x. Quantitative measurement and morphological observation identified a sequential three-stage pattern of marginal deterioration; initial rapid progress of wear of resin composite cement in the first stage, followed by a second stage without any remarkable visible change, then rapid progression of microfractures of ceramics and/or enamel in the third stage. Boundaries between the first and second stage were found in the six and 21-month period, and those between the second and third stage at 72 months. It was concluded that longitudinal marginal deterioration of fired ceramic inlays progressed in a sequential three-stage pattern.

Influence of resin monomers on growth of oral streptococci.

Ethyleneglycol dimethacrylate monomers have been previously reported to stimulate the growth of certain caries-associated bacteria on the basis of turbidity measurements. To elucidate the detail of this effect, we examined the influence of resin monomers on the growth of Streptococcus sobrinus and Streptococcus sanguis by determination of bacterial numbers (colony-forming units), morphological observation, and chemical analysis. Although the absorbance values in the stationary phase of bacterial suspension were increased in the presence of ethyleneglycol monomers, no significant differences were observed for bacterial numbers throughout the incubation period. Scanning electron microscopy observation revealed the formation of sparse vesicular material surrounding bacterial cells when incubated with ethyleneglycol monomers, and these products were proved to be resin polymers. The results demonstrate that the apparent biomass increase during incubation with ethyleneglycol monomers is due not to promotion of bacterial multiplication, but to the polymerization of resin monomers to form vesicular structures attached to cells.