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ABSTRACT: Chlorhexidine (CHX) gluconate effectively reduces the viability of biofilm-forming bacteria, such as Porphyromonas gingivalis. However, it is impossible to completely remove biofilms. The goal of the present study was to assess the potential pathogenicity of residual P. gingivalis biofilms in vitro after treatment with CHX gluconate. Scanning and transmission electron microscopy and confocal laser imaging revealed that treatment with CHX gluconate disrupted individual biofilm-forming P. gingivalis cells but did not destroy the biofilms. The volumes of the protein and carbohydrate constituents in the residual biofilms were not significantly different from those of the controls. The physical resistance of the residual biofilms to ultrasonication was significantly higher than that of controls. The volume of P. gingivalis adherent to the residual biofilms was higher than that to saliva-coated wells. These findings suggest that although CHX gluconate caused disruption of biofilm-forming cells, the constituents derived from disrupted cells were maintained in the biofilms, which sustained their external structures. Moreover, the residual biofilms could serve as a scaffold for the formation of new biofilms.
European Journal Of Oral Sciences 06/2013; 121(3 Pt 1):162-8. · 1.88 Impact Factor
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ABSTRACT: Eikenella corrodens produces autoinducer-2 (AI-2) in the mid log phase, and AI-2 activity decreases dramatically during the stationary phase. We investigated the mechanism underlying this decrease in AI-2 activity. To analyze the mechanism, we extracted and purified AI-2 from the supernatant of mid-log-phase culture. Simultaneously, the stationary-phase culture supernatant was fractionated by ammonium sulfate precipitation. On incubating purified AI-2 and 4-hydroxy-5-methyl-3(2H)-furanone (MHF) with each fraction, the 30% fraction decreased both AI-2 and MHF activities. The data suggest that AI-2 and MHF were rendered inactive in the same manner. Heat and/or trypsin treatment of the 30% fraction did not completely arrest AI-2 inactivation, suggesting that partially heat-stable proteins are involved in AI-2 inactivation. We observed that an enzyme converted MHF to another form. This suggests that E. corrodens produces an AI-2 inactivating enzyme, and that AI-2 can be degraded or modified by it.
Bioscience Biotechnology and Biochemistry 05/2013; · 1.28 Impact Factor
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ABSTRACT: Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of ΔluxS mutant was greater than that of the wild type (Azakami et al., J. Biosci. Bioeng., 102, 110-117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E. corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E. corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E. corrodens.
Journal of Bioscience and Bioengineering 04/2013; · 1.79 Impact Factor
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ABSTRACT: It is difficult to make a definite diagnosis of a cracked tooth solely based on an inspection within the root canal, especially in case of microcracks. At present, there seems to be no established method to detect dentinal microcracks in roots; therefore, the current detection techniques need to be improved. Vibrothermography (VibroIR) helps to detect microcracks by the friction heat generated from ultrasonic vibration. The purpose of this study was to establish a novel method using VibroIR to detect dentinal microcracks.
The root canals of 20 roots with cracks and control roots were prepared after removing the tooth crowns. A tapered indenter was inserted into the root canal and pressed until a microcrack was created under an optical microscope. Using VibroIR, the detection trials for dentinal microcracks were performed with an ultrasonic vibration power ranging from 0.43 to 1.48 W at an angle of 0°, 30°, 45°, 60°, and 90° between the ultrasonic vibration point and the microcrack line. After the detection test, the microcrack width was measured with an optical microscope.
Frictional heat was detected in the microcracks with thermography at 0.89 to 1.48 W and at an ultrasonic vibration point angle less than 60° from the crack line for 10 seconds. Microcracks with a width of 4 to 35.5 μm were detected with this method.
VibroIR may be an effective method for the diagnosis of root dentinal microcracks.
Journal of endodontics 01/2013; 39(1):88-91. · 2.95 Impact Factor
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ABSTRACT: Biofilm-forming cells are distinct from well characterized planktonic cells and aggregate in the extracellular matrix, the so-called extracellular polymeric substances (EPS). The sinR gene of Bacillus subtilis encodes a transcriptional regulator that is known to be involved in the biosynthesis of EPS in biofilms. Porphyromonas gingivalis inhabits the subgingival and extraradicular biofilm of humans and is one of the primary pathogens that cause progressive marginal and refractory apical periodontitis. Furthermore, P. gingivalis possesses PGN_0088, which encodes a putative ortholog of B. subtilis sinR. Here, we investigated the role of PGN_0088 (sinR) on biofilm formation. P. gingivalis strains formed biofilms on saliva-coated glass surfaces in phosphate buffered saline. Quantitative analysis indicated that the biofilm of the sinR null mutant consisted of dense exopolysaccharide. Microscopic observations showed that the increased levels of exopolysaccharide produced by the sinR mutant changed the morphology of the EPS to a mesh-liked structure. Furthermore, physical analyses suggested that the enrichment of exopolysaccharide in the EPS enhanced the resistance of the biofilm to hydrodynamic shear force. The results presented here demonstrate sinR plays important roles in the ability of P. gingivalis strain ATCC 33277 to act as a negative mediator of exopolysaccharide accumulation and is indirectly associated with the structure of the EPS and the force of its adhesion to surfaces.
PLoS ONE 01/2013; 8(2):e56017. · 4.09 Impact Factor
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ABSTRACT: Tertiary dentin is deposited inside teeth after various stimuli and serves as a major defensive wall to preserve pulp cells. However, the molecular mechanisms of the activation of quiescent odontoblasts, immature pulp cells and tertiary dentin formation are still unclear. Therefore, we performed a comprehensive gene expression analysis of pulp cells after cavity preparation of 9-week-old rat molars to clarify the critical molecules in tertiary dentinogenesis. As a result, mRNA expression of various molecules was up- or down-regulated. Notably, several members of the matrix metalloprotease family and their endogenous inhibitors were up-regulated after cavity preparation. In situ hybridization showed that tissue inhibitor of metalloprotease 1 (Timp1) was widely and continuously distributed in the pulp beneath the cavity in vivo. We also observed accumulation of β-catenin in the pulp cells beneath the cavity by fluorescence immunohistochemistry. Furthermore, Timp1 transcription was repressed by a dominant-negative TCF4 in immature undifferentiated mesenchymal cells, but not altered in mature odontoblast-like cells. These results indicate that cavity preparation may activate the Wnt/β-catenin pathway, and the Wnt/β-catenin pathway and Timp1 may be correlatively involved in pulp repair. Timp1 might play crucial roles in reactivation of immature pulp cells for tertiary dentinogenesis.
Journal of biochemistry 10/2012; · 1.95 Impact Factor
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ABSTRACT: Pretreatment of dentin using colloidal platinum nanoparticles (CPtN) can enhance the bond strength of dentin adhesives. However, the combination of CPtN, which is negatively charged, with cationic monomer-containing adhesive may reduce the antibacterial activity of the original material. Thus, the purpose of this study was to assess the effect of CPtN on the bactericidal activity of two cationic antibacterial monomers, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB). The rapid killing effects of the two monomers against planktonic or attached Streptococcus mutans in the presence or absence of CPtN were examined by viable cell counts. The measurement of minimum inhibitory and bactericidal concentrations demonstrated that CPtN up to 2.5 mM has no antibacterial activity. In the absence of CPtN, rapid killing of both planktonic and attached Streptococcus mutans were achieved by the two cationic monomers. Combination with 0.1 mM CPtN did not reduce the bactericidal effects of the two monomers, indicating that CPtN may be used as a pretreatment with antibacterial adhesives.
Dental Materials Journal 02/2012; 31(1):150-6. · 1.14 Impact Factor
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ABSTRACT: We recently succeeded in purifying a novel multipotential progenitor or stem cell population from bone marrow stromal cells (BMSCs). This population exhibited a very high frequency of colony forming units-osteoblast (CFU-O; 100 times higher than in BMSCs) and high expression levels of osteoblast differentiation markers. Furthermore, large masses of mineralized tissue were observed in in vivo transplants with this new population, designated highly purified osteoprogenitors (HipOPs). We now report the detailed presence and localization of HipOPs and recipient cells in transplants, and demonstrate that there is a strong relationship between the mineralized tissue volume formed and the transplanted number of HipOPs.
International Journal of Molecular Sciences 01/2012; 13(8):10229-35. · 2.60 Impact Factor
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ABSTRACT: We had previously discovered that the flexural and tensile strengths of human dentin were 2-2.4 times greater after being heated to 140°C, and deduced that the generation of higher-density structures and therefore dehydration probably promoted the increased strength. Our test hypotheses were that intertubular dentin, which constitutes a major part of organic components, was selectively affected by heating, and such changes could happen without critical damages to the basic structure of dentin type I collagen.
Micro-mechanical changes of human dentin by heating at 140°C were investigated by nano-indentation. Chemical changes in dentin collagen after heating were also investigated by X-ray diffraction study, a microscopic Fourier transform infrared (micro-FTIR) and a laser Raman spectroscopic analyses, and a cross-linking analysis by high-performance liquid chromatography.
The results of nano-indentation showed that the micro-hardness of intertubular dentin increased after heating at 140°C to 1.8 times more than unheated dentin; on the other hand, peritubular dentin was unchanged. Results of X-ray diffraction showed that the lateral packing of collagen molecules shrank from 13.6±0.3 to 10.6±0.1Å after heating, but the shrinkage reversed to the original after rehydration for seven days. After heating, no substantial chemical changes in the collagen molecules were detected in tests by micro-FTIR or Raman analyses, or by cross-linking analysis.
These results suggest that intertubular dentin, which contains most of the type I collagen, was selectively affected by heating at 140°C without critical damage to its collagen.
Dental materials: official publication of the Academy of Dental Materials 12/2011; 28(4):385-91. · 2.88 Impact Factor
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ABSTRACT: This study evaluated the cytotoxicity of self-etching primers/adhesives by direct contact and dentin barrier tests. The three two-step self-etching systems Clearfil SE Bond (CSE), Clearfil Protect Bond (CPB), Prime&Bond NT/NRC (PB) and one-step self-etching systems Reactmer Bond (RB), Clearfil Tri-S Bond (CTS), and Adper Prompt L-Pop (AP) were examined. In direct contact tests, L929 cells were cultured in the presence of diluted solutions (50, 20, 10, and 1%) of primer/conditioner of adhesive systems. For dentin barrier tests, each system was applied onto 0.5 or 1.5 mm thick human dentin assembled in a simple pulp chamber device and incubated for 24 h at 37°C to make the diffusive components contact the L929 cells placed at the bottom of the chamber. The cytotoxic effects were assessed by MTT assay. Cell culture without application of any primers/adhesives served as the control for both tests. One-way ANOVA and Tukey HSD tests were used for statistical analyses. The direct contact tests demonstrated that CSE and CPB were less toxic than the other materials at all dilutions. In the dentin barrier tests, toxic effects of materials were reduced with an increase in thickness of intervening dentin. CSE and CPB showed less cytotoxicity than the other adhesives (p<0.05) when applied to 0.5 mm-thick dentin, and CSE was the least toxic in the 1.5 mm-dentin group (p<0.05). Dentin thickness positively affected biocompatibility of the tested bonding systems. Two-step self-etching systems with HEMA-based primers were more biocompatible than other self-etching adhesives.
Dental Materials Journal 11/2011; · 1.14 Impact Factor
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ABSTRACT: The aim of this study was to investigate the cross-sectional association of dental restorations with salivary cariogenic pathogens among the elderly to establish effective parameters of caries risk for this population.
Stimulated whole saliva was collected from 289 community-dwelling older adults (66.2 ± 3.9 years old) who had 20 or more teeth. Salivary levels of three cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus and lactobacilli) were estimated using quantitative polymerase chain reaction (real-time PCR) method.
The mean number of residual teeth was 26.4, and restored teeth with crowns, inlays and composite resin were 7.35, 3.88 and 0.68, respectively. The number of crowns correlated positively with salivary S. mutans, S. sobrinus and lactobacilli. Multiple linear regression analyses showed that the number of restored teeth with crowns was independently associated with salivary S. mutans, S. sobrinus and lactobacilli after controlling for age, gender, number of residual teeth and salivary flow rate. Salivary flow rate was independently associated with salivary S. mutans and lactobacilli.
The number of crowns had an association with salivary levels of cariogenic bacteria, suggesting that this parameter may be a caries risk indicator for the elderly population.
Gerodontology 10/2011; 29(2):e845-50. · 1.03 Impact Factor
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ABSTRACT: Chronological gene expression patterns of biofilm-forming cells are important to understand bioactivity and pathogenicity of biofilms. For Porphyromonas gingivalis ATCC 33277 biofilm formation, the number of genes differentially regulated by more than 1.5-fold was highest during the growth stage (312/2,090 genes), and some pathogen-associated genes were time-dependently controlled.
Applied and environmental microbiology 07/2011; 77(18):6733-6. · 3.69 Impact Factor
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ABSTRACT: This study investigated the relationship between caries assessment using a laser fluorescence device (DIAGNOdent), and bacterial invasion in arrested carious dentin detected by polymerase chain reaction (PCR). The ten extracted human molars used in this study had black or dark brown, hard occlusal carious lesions, and were found to be only weakly stained or unstained with a caries detector dye of 1% acid red in propylene glycol. In those extracted human molars, dentin was removed in the direction of the pulp chamber at 150-μm intervals. During each removal (104 sections in total), the dentin surface was assessed with DIAGNOdent, and a dentinal tissue sample was taken with a round bur. Bacterial DNA of each tissue sample was examined using PCR and primers based on the nucleotide sequence of a conserved region of bacterial 16S rDNA. Rates of bacterial detection increased as the DIAGNOdent values increased. When the DIAGNOdent values were <10, the rate of bacterial detection was 0%. The area under the receiver operating characteristic curve of the DIAGNOdent values was 0.87. These results indicate that the DIAGNOdent values of arrested dentinal carious lesion were closely related to the rates of bacterial detection.
Lasers in Medical Science 07/2011; 26(4):439-44. · 2.00 Impact Factor
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ABSTRACT: Being able to predict an individual's risks of dental caries would offer a potentially huge natural step forward toward better oral heath. As things stand, preventive treatment against caries is mostly carried out without risk assessment because there is no proven way to analyse an individual's risk factors. The purpose of this study was to try to identify those patients with high and low risk of caries by using Classification and Regression Trees (CART).
In this historical cohort study, data from 442 patients in a general practice who met the inclusion criteria were analysed. CART was applied to the data to seek a model for predicting caries by using the following parameters according to each patient: age, number of carious teeth, numbers of cariogenic bacteria, the secretion rate and buffer capacity of saliva, and compliance with a prevention programme. The risks of caries were presented by odds ratios. Multiple logistic regression analysis was performed to confirm the results obtained by CART.
CART identified high and low risk patients for primary caries with relative odds ratios of 0.41 (95%CI: 0.22-0.77, p = 0.0055) and 2.88 (95%CI: 1.49-5.59, p = 0.0018) according the numbers of cariogenic bacteria. High and low risk patients for secondary caries were also identified with the odds ratios of 0.07 (95%CI: 0.01-0.55, p = 0.00109) and 7.00 (95%CI: 3.50-13.98, p < 0.0001) according the numbers of bacteria and existing caries.
Cariogenic bacteria play a leading role in the incidence of caries. CART proved effective in identifying an individual patient's risk of caries.
Journal of dentistry 06/2011; 39(6):457-63. · 2.00 Impact Factor
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ABSTRACT: The periodontopathogenic bacterium Eikenella corrodens has an N-acetyl-D-galactosamine (GalNAc)-specific lectin, that contributes significantly to the pathogenicity of the bacterium. Recently, we reported that plasmid-mediated genomic recombination enhances the activity of this lectin. In this study, we investigated the effects of genomic recombination on certain virulence factors. Introduction of the recombinase gene resulted in hemolysis and significantly increased bacterial adhesion to epithelial cells. It was suggested that the enhanced adhesion was attributable to increased lectin activity due to genomic recombination, because it was inhibited by the addition of GalNAc. In contrast, invasion of the epithelial cells was remarkably reduced by genomic recombination. Although we assumed that this decrease in invasion resulted from a loss of type-IV pili, the phase variant did not show any decrease in invasion activity. This suggests that type-IV pili do not contribute to the invasive ability of E. corrodens. Our results suggest that genomic recombination enhances the pathogenicity of E. corrodens.
Bioscience Biotechnology and Biochemistry 05/2011; 75(4):748-51. · 1.28 Impact Factor
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ABSTRACT: The antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) is a strong bactericide when unpolymerized and has the potential to be utilized in various resinous biomaterials. To analyze the antibacterial characteristics of this monomer in detail, the ability of high concentrations of unpolymerized MDPB to kill Streptococcus mutans in planktonic or biofilm forms within a short time-period of contact, and the inhibitory effects of low concentrations of MDPB on the metabolic function of S. mutans, were examined. High concentrations of MDPB showed effective killing of planktonic and biofilm S. mutans cells within 60 s, and complete killing was obtained by contact with 1,000 μg ml(-1) of MDPB for 60 s. At a concentration of 4-8 μg ml(-1) , MDPB demonstrated growth inhibition, inducing elongation of the lag phase and of the doubling time, when the bacterial number was low. Inhibition of the production of acid from S. mutans by 8 μg ml(-1) of MDPB may have been caused by the inhibition of lactate dehydrogenase activity. At high concentrations, MDPB is lethal to both planktonic and biofilm forms of S. mutans in a short time-period, and at low concentrations, MDPB inhibits metabolic enzymatic activity.
European Journal Of Oral Sciences 04/2011; 119(2):175-81. · 1.88 Impact Factor
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ABSTRACT: The Gram-negative anaerobic bacterium Porphyromonas gingivalis is a major pathogen in periodontal disease, one of the biofilm-caused infectious diseases. The bacterium possesses potential virulence factors, including fimbriae, proteinases, hemagglutinin, lipopolysaccharide (LPS), and outer membrane vesicles, and some of these factors are associated with biofilm formation; however, the precise mechanism of biofilm formation is still unknown. Colonial pigmentation of the bacterium on blood agar plates is related to its virulence. In this study, we isolated a nonpigmented mutant that had an insertion mutation within the new gene PGN_1251 (gtfB) by screening a transposon insertion library. The gene shares homology with genes encoding glycosyltransferase 1 of several bacteria. The gtfB mutant was defective in biosynthesis of both LPSs containing O side chain polysaccharide (O-LPS) and anionic polysaccharide (A-LPS). The defect in the gene resulted in a complete loss of surface-associated gingipain proteinases, strong autoaggregation, and a marked increase in biofilm formation, suggesting that polysaccharide portions of LPSs influence attachment of gingipain proteinases to the cell surface, autoaggregation, and biofilm formation of P. gingivalis.
Infection and immunity 09/2010; 78(9):3801-12. · 4.21 Impact Factor
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ABSTRACT: This study investigated the proliferation and differentiation potential of pluripotent mesenchymal cells on three resin-based restoratives using a typical pluripotent mesenchymal precursor cell line, C2C12. C2C12 cells were cultured for 3-21 days on cured specimens of a Bis-GMA/TEGDMA-based composite resin (APX; Clearfil AP-X), a 4-META/MMA-based resin cement (SB; Superbond C&B) or a HEMA-containing resin modified glass-ionomer (LC; Fuji Ionomer Type II LC). To examine the influences on differentiation potential, alkaline phosphatase (ALP) activity of the cells cultured on each material was determined. On APX and SB, cells adhered and proliferated well, and no significant influences on ALP activity were observed. In contrast, poor cell proliferation and significant suppression of ALP activity were observed for cells cultured on LC, similar to those cultured on a zinc oxide EBA cement used as a control material. Bis-GMA/TEGDMA-based composite resin and 4-META/MMA-based resin exhibited better biocompatibility for C2C12 cells than HEMA-containing resin modified glass-ionomer, suggesting a potential advantage of the former two resins to show smaller influences on regeneration of periapical or periodontal tissue.
Dental Materials Journal 05/2010; 29(3):341-6. · 1.14 Impact Factor
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ABSTRACT: To evaluate the durability of a resin coating system after toothbrushing abrasion resistance and surface hardness of the coating resin.
Rectangular blocks of Top Coat (TC) or the bonding resin of the two-step self-etching adhesive Clearfil SE Bond (SEB) were prepared by irradiation with halogen light units. After immersion in distilled water at 37 degrees C until stabilized water absorption, the specimens were subjected to toothbrushing abrasion tests in which a toothbrush was moved on the specimen at 60 strokes/minute with a 400 gf vertical load with toothpaste slurry. The specimens were weighed after every 10,000 strokes until 200,000 strokes. Hardness of the specimen was measured with a Knoop hardness tester, and the degree of cure was determined using ATR-FTIR spectroscopy. Statistical differences between materials were analyzed using a Student's t-test.
TC demonstrated significantly less weight loss than SEB after 40,000 strokes (P<0.05), showing 0.87 +/- 0.64 mg loss after 200,000 strokes. The Knoop hardness number of TC was significantly greater than that of SEB (P<0.05), and TC presented a greater degree of cure.
American journal of dentistry 04/2010; 23(2):70-4. · 0.76 Impact Factor
