Effect of pomegranate solution alone or combined with chlorhexidine against oral multispecies biofilm.

AIM: Natural bioactive products have been tested as alternative antimicrobial agents. This study evaluated the effect of Punica granatum extract (PGE) on oral multispecies biofilms. METHODOLOGY: Lyophilized extracts from pomegranate peel were prepared, and the punicalagin content was assessed by ultra-performance liquid chromatography (UPLC). Oral multispecies biofilms from 2 donors were grown on four collagen-coated hydroxyapatite discs. After incubation for 7 days or 3 weeks, the biofilms were exposed to water (control), 2% CHX, 10% PGE, 20% PGE or 30% PGE for 3 min. The proportions of dead bacteria were assessed by the live/dead staining and confocal microscopy. After the analysis, the best PGE concentration (30%) was combined with CHX. The experimental phases were repeated using water, 2% CHX, 30% PGE and 30% PGE + 2% CHX. Five random areas of the biofilm on each disc were scanned, resulting in 20 scanned areas for each group. RESULTS: Regarding the biofilm volume, no differences were found amongst solutions (p = .111). The PGE solution killed bacteria effectively in 1-week, 2-week and 3-week-old-plaque biofilms, ranging from 37 to 55.3%, depending on the PGE concentration. The 30% PGE (a) (p = .0009) had greater antibiofilm effectiveness than 2% CHX (b), which killed bacteria in the 25.2 to 48.7% range. The 10% and 20% PGE had intermediate values (ab), without significant differences from 30% PGE (p = 1.002). Water (c) had the lowest proportion of dead bacteria (p < .00001) in a range of 5 to 6.7% and lower effectiveness in killing bacteria (p < .05). The PGE alone or mixed with 2% CHX had greater anti-biofilm effectiveness than CHX (p < .05). The old plaque biofilms were more resistant than the 7-day-old plaque (p < .05). CONCLUSIONS: The 30% PGE (alone or combined with CHX) exhibited a greater antibiofilm effect on oral multispecies biofilms grown on hydroxyapatite discs than 2% CHX.

Effect of autoclave sterilization on the cyclic fatigue resistance of thermally treated Nickel-Titanium instruments.

AIM: To compare the cyclic fatigue resistance of HyFlex CM, Twisted Files (TF), K3XF, Race, and K3, and evaluate the effect of autoclave sterilization on the cyclic fatigue resistance of these instruments both before and after the files were cycled. METHODOLOGY: Five types of NiTi instruments with similar size 30, .06 taper were selected: HyFlex CM, TF, K3XF, Race and K3. Files were tested in a simulated canal with a curvature of 60° and a radius of 3 mm. The number of cycles to failure of each instrument was determined to evaluate cyclic fatigue resistance. Each type of instruments was randomly divided into four experimental groups: group 1 (n = 20), unsterilized instruments; group 2 (n = 20), pre-sterilized instruments subjected to 10 cycles of autoclave sterilization; group 3 (n = 20), instruments tested were sterilized at 25%, 50% and 75% of the mean cycles to failure as determined in group 1, and then cycled to failure; group 4 (n = 20), instruments cycled in the same manner as group 3 but without sterilization. The fracture surfaces of instruments were examined by scanning electron microscopy (SEM). RESULTS: HyFlex CM, TF and K3XF had significantly higher cyclic fatigue resistance than Race and K3 in the unsterilized group 1 (P < 0.05). Autoclave sterilization significantly increased the MCF of HyFlex CM and K3XF (P < 0.05) both before and after the files were cycled. SEM examination revealed a typical pattern of cyclic fatigue fracture in all instruments. CONCLUSIONS: HyFlex CM, TF and K3XF instruments composed of new thermal-treated alloy were more resistant to fatigue failure than Race and K3. Autoclaving extended the cyclic fatigue life of HyFlex CM and K3XF.

Comparative analyses of ion release, pH and multispecies biofilm formation between conventional and bioactive gutta-percha.

AIM: To analyse the effect of commercial and experimental gutta-percha with the addition of niobium phosphate glass on biofilm formation by oral bacteria from human dental plaque. Additional pH and elemental release of the materials were analysed. METHODOLOGY: The multispecies biofilm was grown anaerobically from plaque bacteria on standardized discs of each material: hydroxyapatite (HA), gutta-percha pellets (OBT) (Obtura pellets, Shoreline, CT, USA), ProTaper gutta-percha (PTP) (ProTaper Universal Gutta-Percha Points, Dentsply Maillefer, Ballaigues, Switzerland), EndoSequence BC gutta-percha (GBC) (Brasseler USA, Savannah, GA, USA), experimental gutta-percha associated with niobium phosphate glass (GNB) and niobium phosphate glass (NPG). Specimens (n = 5 per group and per incubation period) were incubated in brain-heart infusion broth for 3, 14 and 30 days, at 37 °C, and stained using live/dead viability assay. Images were analysed by confocal laser scanning microscopy (CLSM) and the total biovolume (mm3 ), viable bacteria biovolume (mm3 ), and live percentage (%) were quantified. For pH measurement, specimens of each material (n = 3) were immersed in phosphate-buffered saline at 37 °C, and pH was monitored in multiple intervals, up to 30 days. For elemental analysis, additional specimens (n = 3) were immersed in deionized water and elemental release was analysed by ICP-OES (inductively coupled plasma - optical emission spectrometry) at time intervals of 3, 14 and 30 days. Differences between groups were evaluated by the two-way analysis of variation (anova) with Tukey's post hoc test (P < 0.05). RESULTS: The lowest total biovolume at 30 days was found in GNB, GBC and NPG. GNB had the lowest viable bacteria biovolume (mean value) at 30 days (P < 0.05), and the lowest live percentage of bacteria at 3 and 30 days (P < 0.05), whilst NPG had the lowest live percentage at 14 days (P < 0.05). GNB had the highest pH (8.45) after 30 days (P < 0.05), and the greatest Zn and Na release at all time intervals (P < 0.05). Both GBC and GNB had significantly higher Ca release at 14 and 30 days. CONCLUSION: GNB and GBC reduced biofilm formation, GNB had the lowest amount of viable bacteria in biofilms with the highest pH, and high Zn and Na release values after 30 days.

Acidic pH weakens the microhardness and microstructure of three tricalcium silicate materials.

AIM: To investigate the microhardness and microstructural features of three tricalcium silicate materials: mineral trioxide aggregate (MTA), Endosequence Root Repair Material Putty (ERRM Putty) and Endosequence Root Repair Material Paste (ERRM Paste), after exposure to a range of acidic environments in comparison with intermediate restorative material (IRM). METHODOLOGY: Endosequence Root Repair Material Putty (Brasseler, Savannah, GA, USA), ERRM Paste (Brasseler, Savannah, GA, USA), MTA (ProRoot; Dentsply Tulsa Dental, Johnson City, TN, USA) and IRM (Dentsply Caulk, Milford, DE, USA) were set in cylindrical rubber moulds as four groups containing twenty specimens each. Fifteen specimens per each material were randomly distributed into three groups (n = 5) to be exposed to butyric acid buffered at three different pH levels (5.4, 6.4 and 7.4) for 7 days. The remaining five specimens were exposed to distilled water as a control group. The surface microhardness after exposure either to acid or to water was measured after 7-days at 37 °C. The morphology of the internal microstructure was observed using a scanning electron microscope (SEM). Two-way univariate analysis of variance (anova) was applied to evaluate the Vickers microhardness value (VHN). RESULTS: The microhardness values of the materials were significantly higher in the neutral environment of butyric acid at pH 7.4 compared to those in the acidic condition of pH 5.4 for all groups (P < 0.001). MTA, ERRM Putty and ERRM Paste had higher microhardness values than IRM at all pH levels (P < 0.001). Specimens exposed to distilled water displayed significantly higher microhardness values than those values obtained in the presence of butyric acid buffered to all pH levels (P < 0.001). A more porous microstructure was observed following exposure to butyric acid at pH 5.4 than at pH 7.4. Several types of crystalline structures were formed by recrystallization, especially at pH 7.4 in all groups except for IRM. CONCLUSIONS: The microhardness values of ERRM Putty, ERRM Paste and MTA were reduced in an acidic environment, which resulted in these materials having more porous and less crystalline microstructures. MTA seems the most suitable material for application to an area of inflammation where a low pH value may exist.

Micropush-out dentine bond strength of a new gutta-percha and niobium phosphate glass composite.

AIM: To characterize an experimental gutta-percha and niobium phosphate glass composite (GNB) applied with a thermoplastic technique to the root canals without sealer in a moist environment and to evaluate its micropush-out bond strength to root canal wall dentine. METHODOLOGY: The root canals of sixty human mandibular pre-molars were prepared using rotary NiTi instruments and irrigation with sodium hypochlorite and EDTA. The teeth were then randomly divided into three groups according to the root filling material used: AH plus sealer and gutta-percha (AH), EndoSequence BC gutta-percha without sealer (GBC), and GNB without sealer. The root canals were filled with a single cone using warm vertical condensation. Push-out bond strengths associated with the filling materials in slices from middle root thirds was determined 30 days after root filling. The failure mode was analyzed with SEM. Analysis using EDX and SEM-EDS was carried out to verify the composition and distribution of the particles of the tested materials. Data were statistically analyzed by one-way anova and Tukey's test (P < 0.05). RESULTS: AH and GNB groups had bond strengths of 2.83 ± 0.64 MPa and 2.68 ± 0.84 MPa, respectively, with no significant difference between them (P > 0.05). The GBC group had the lowest mean bond strength (1.34 ± 0.42 MPa), which was significantly different compared with the other groups (P < 0.05). Cohesive failures prevailed in the AH group, whereas failures were mixed in the GBC and GNB groups. The SEM-EDS analysis on the surface and in the bulk of GBC revealed only a superficial coating of bioceramic particles. Glass particles were detected both on the surface and in the bulk of GNB. CONCLUSIONS: The experimental root filling composite (GNB) had an ability to adhere to root canal wall dentine equal to the current gold standard root filling with gutta-percha and sealer (AH Plus).

Micro-computed tomography evaluation of the removal of calcium hydroxide medicament from C-shaped root canals of mandibular second molars.

AIM: To use micro-computed tomography (µ-CT) to evaluate the amount of calcium hydroxide [Ca(OH)2 ] remaining in the C-shaped root canals of mandibular second molars after attempting to remove it with passive ultrasonic and sonic irrigation. METHODOLOGY: Thirty mandibular second molars, 15 in C1 and 15 in C2 configurations as first identified by µ-CT, were divided into three groups (five C1 and five C2 in each group) for the three irrigation methods. All teeth were prepared to ProTaper Universal F2 and filled with Ca(OH)2 paste. The Ca(OH)2 was removed with F2 files and irrigation without agitation or with agitation using either EndoActivator or ultrasonics. µ-CT was used to measure the initial amount of Ca(OH)2 present. After removal of Ca(OH)2, µ-CT imaging was used to assess the percentage of volume of residual Ca(OH)2 in the canal. Data were analysed using one-way anova test. RESULTS: There was no significant difference in the mean volume of the root canal systems after instrumentation amongst the three groups. The three irrigation techniques left 2-17% of Ca(OH)2 in the root canals after removal. The mean volume of the remaining Ca(OH)2 was higher in the group without agitation than in the groups with sonic or ultrasonic agitation (P < 0.05). In the apical third, 68% of the canal space remained occupied by Ca(OH)2 when no agitation was used, whereas 28% and 31% remained filled by Ca(OH)2 in the EndoActivator and ultrasonic groups, respectively. There was no significant difference in the amount of residual Ca(OH)2 between the EndoActivator and ultrasonic groups. The proportion of remaining Ca(OH)2 in the apical canals was higher than in the middle and coronal canals in all groups (P < 0.05). CONCLUSIONS: A considerable proportion of the apical canal space remained filled with Ca(OH)2 in the C-shaped root canals after instrumentation and conventional needle irrigation. Although combining rotary instrumentation and irrigation with sonic or ultrasonic agitation reduced the amount of residual Ca(OH)2 in the C-shaped root canals, the large amount of calcium hydroxide in the critical apical area remains a concern. Alternative strategies should be considered in medication of the apical canal in C-shaped teeth.

Ca(OH)2 application modes: in vitro alkalinity and clinical effect on bacteria.

AIM: To compare five calcium hydroxide (CH) products, CH-gutta-percha Plus points(®) (CHGP) and conventional CH paste, for their ability to maintain alkalinity and to assess the clinical antimicrobial effect of CHGP. METHODOLOGY: Calcium hydroxide products were tested in the presence of standardized dentine powder or by titrating them with 1 mol HCl, expressed in mL (±SD). In a clinical trial, 21 single-rooted teeth with primary apical periodontitis were medicated with CHGP or with conventional CH paste. Bacterial samples were taken before and after chemo-mechanical preparation, after dressing and after leaving canals empty but sealed. To compare groups, anova with Tukey's test was used in the laboratory study and Fisher's exact test in the clinical study. Significance level was set at 5%. RESULTS: Pure CH with water (8.5 ± 0.1) and Calasept (9.3 ± 0.1) maintained the highest alkalinity, followed by the gel-like products DS CaOH gel (7.3 ± 0.3) and Ultracal XS (6.8 ± 0.2) and then Biokalkki (6.3 ± 0.3) and Calxyl blue (5.1 ± 0.2). All CH paste products had higher values compared with CHGP (1.6 ± 0.1) (P < 0.05). Saturated solutions of the products were all neutralized to pH 8.6 within 24 h by dentine powder addition. Clinically, culture-negative results were obtained in 5/10 canals in the CHGP group and 7/11 with conventional CH (P > 0.05). CONCLUSIONS: Aqueous CH mixtures kept high pH better than viscous gel products or CHGP. Dentine powder had equal buffering effect on each product tested. CHGP and traditional CH paste both had an antimicrobial effect in the clinical setting, but there was no significant difference between the groups.

Fatigue and nanomechanical properties of K3XF nickel-titanium instruments.

AIM: To examine the fatigue behaviour of heat-treated NiTi instruments when immersed in aqueous media and to determine the effect of cyclic fatigue on the hardness and elastic modulus of NiTi instruments using a nanoindentation technique. METHODOLOGY: K3XF and K3 NiTi instruments, both in sizes 25, 0.04 taper and 40, 0.04 taper, were subjected to rotational bending at a curvature of 42° either in air or under deionized water, and the number of revolutions to fracture (Nf ) was recorded. The fracture surface of all fragments was examined with a scanning electron microscope. The hardness and elastic modulus of the fracture surface of instruments sized 25, 0.04 taper were then measured using a nanoindentation test. RESULTS: The K3XF instruments had a fatigue resistance superior to K3 instruments under dry and aqueous environments (P < 0.05). The fatigue life of K3 instruments was similar under both conditions, whereas the Nf of K3XF was greater under water than in air, especially at the size 40, 0.04 taper (P < 0.05). The values for the fraction of the area occupied by the dimple region were significantly smaller in K3XF instruments than in K3 instruments, especially under water (P < 0.05). There was no difference in hardness on K3XF instruments between new files and instruments subjected to the fatigue process. The hardness of instruments subjected to the fatigue process was significantly lower in K3XF than in K3 instruments (P < 0.05). CONCLUSION: The fatigue life of K3XF instruments under water is longer than it is for K3XF instruments in air. There was no work-hardening effect on K3XF instruments subjected to the fatigue process.

Hydrogen sulphide increases hepatic differentiation of human tooth pulp stem cells compared with human bone marrow stem cells.

AIM: To determine the differences in stem cell properties, in hepatic differentiation and in the effects of hydrogen sulphide (H2 S) on hepatic differentiation between human bone marrow stem cells (hBMC) and stem cells from human exfoliated primary tooth pulp (SHED). METHODOLOGY: CD117(+) cells were magnetically separated and subjected to hepatic differentiation. CD117(+) cell lineages were characterized for transcription factors indicative of stem cells by qRT-PCR. For the last 9 days of the differentiation, the test cells were exposed to 0.1 ng mL(-1) H2 S. Immunocytochemistry and flow cytometry of albumin, alpha-fetoprotein and carbamoyl phosphate synthetase were carried out after differentiation. Urea concentration and glycogen synthesis were also determined. RESULTS: Genes expressed in SHED were also expressed in BMC. No difference in expression level of hepatic markers was shown by immunofluorescence. SHED showed more positive cells than hBMC (P < 0.01). H2 S increased the number of positive cells in both cultures (P < 0.01). Urea concentration and glycogen synthesis increased significantly after H2 S exposure (P < 0.001 and P < 0.05, respectively). Real-time PCR data were analysed by RT(2) profiler RT-PCR Array Data Analysis version 3.5 (Qiagen), and ELISA data were analysed by Bonferroni's multiple comparison using Windows spss version 16 (SPSS Inc, Chicago, IL, USA). Bonferroni's multiple comparison test was also carried out after angle transformation for the percentage data of flow cytometer using Windows spss(®) version 16 (SPSS Inc). Statistical significance was accepted at P < 0.05. CONCLUSIONS: Stem cells from human exfoliated primary tooth pulp and BMC have similar properties. The level of hepatic differentiation in SHED compared with BMC was the same or higher. H2 S increased the level of hepatic differentiation.

Ex vivo killing of Enterococcus faecalis and mixed plaque bacteria in planktonic and biofilm culture by modified photoactivated disinfection.

AIM: To compare the efficacy of conventional and modified photoactivated disinfection (PAD) against Enterococcus faecalis and mixed plaque bacteria in suspension and biofilms. METHODOLOGY: Enterococcus faecalis (four strains) and mixed plaque bacteria from three adult volunteers were suspended in water, added to methylene blue (MB, 15 µmol L⁻¹), MB mixed with 0.5% hydrogen peroxide and 0.05% chlorhexidine (CHX), MB mixed with 0.5% hydrogen peroxide and 0.05% EDTA or MB mixed with 0.05% EDTA and 0.05% CHX and exposed to laser irradiation from 10 s to 5 min. After exposure, samples were taken, serially diluted and grown aerobically and anaerobically on Tryptic Soy Agar plates or on blood agar plates for 24 and 72 h, respectively. For biofilm experiments, E. faecalis and mixed plaque biofilms were grown on sterile hydroxyapatite (HA) discs coated overnight with bovine dermal collagen type I for 3 weeks. After exposure to MB or MB and low concentration of EDTA with either hydrogen peroxide or CHX, the percentage of killed bacteria by PAD was evaluated using viability staining and confocal laser scanning microscope. For statistical analysis, one-way analysis of variance was performed. RESULTS: Conventional PAD killed from 90.76% to 100% E. faecalis for 3 min, but failed to kill all plaque bacteria even after 5 min of laser irradiation. In modified PAD, up to 100% of suspended E. faecalis and mixed plaque bacteria were killed after 1 min and 30 s of irradiation. Up to twenty times more biofilm bacteria were killed by modified PAD than by conventional PAD with 15 µmol L⁻¹ MB (P < 0.001) and up to eight times more than 2% CHX (P < 0.001) and 1% sodium hypochlorite (P < 0.001). CONCLUSION: Modified PAD was superior to conventional PAD against planktonic and biofilm bacteria.

HyFlex nickel-titanium rotary instruments after clinical use: metallurgical properties.

AIM: To analyse the type and location of defects in HyFlex CM instruments after clinical use in a graduate endodontic programme and to examine the impact of clinical use on their metallurgical properties. METHODOLOGY: A total of 468 HyFlex CM instruments discarded from a graduate endodontic programme were collected after use in three teeth. The incidence and type of instrument defects were analysed. The lateral surfaces of the defect instruments were examined by scanning electron microscopy. New and clinically used instruments were examined by differential scanning calorimetry (DSC) and x-ray diffraction (XRD). Vickers hardness was measured with a 200-g load near the flutes for new and clinically used axially sectioned instruments. Data were analysed using one-way anova or Tukey's multiple comparison test. RESULTS: Of the 468 HyFlex instruments collected, no fractures were observed and 16 (3.4%) revealed deformation. Of all the unwound instruments, size 20, .04 taper unwound the most often (n = 5) followed by size 25, .08 taper (n = 4). The trend of DSC plots of new instruments and clinically used (with and without defects) instruments groups were very similar. The DSC analyses showed that HyFlex instruments had an austenite transformation completion or austenite-finish (Af ) temperature exceeding 37 °C. The Af temperatures of HyFlex instruments (with or without defects) after multiple clinical use were much lower than in new instruments (P < 0.05). The enthalpy values for the transformation from martensitic to austenitic on deformed instruments were smaller than in the new instruments at the tip region (P < 0.05). XRD results showed that NiTi instruments had austenite and martensite structure on both new and used HyFlex instruments at room temperature. No significant difference in microhardness was detected amongst new and used instruments (with and without defects). CONCLUSIONS: The risk of HyFlex instruments fracture in the canal is very low when instruments are discarded after three cases of clinical use. New HyFlex instruments were a mixture of martensite and austenite structure at body temperature. Multiple clinical use caused significant changes in the microstructural properties of HyFlex instruments. Smaller instruments should be considered as single-use.

Antibacterial and smear layer removal ability of a novel irrigant, QMiX.

AIM: To assess in a laboratory experimental model the efficacy of a novel root canal irrigant, QMiX, against Enterococcus faecalis and mixed plaque bacteria in planktonic phase and biofilms. In addition, its ability to remove smear layer was examined. METHODOLOGY: Enterococcus faecalis and mixed plaque bacteria were exposed to QMiX, 2% chlorhexidine (CHX), MTAD and 1% sodium hypochlorite (NaOCl) for 5 s, 30 s and 3 min. Following exposure, samples were taken, serially diluted and grown aerobically and anaerobically on tryptic soy agar (TSA) plates or on blood agar plates for 24 and 72 h, respectively, to measure killing of bacteria. E. faecalis and plaque biofilms were grown for 3 weeks on collagen-coated hydroxyapatite or dentine discs and exposed for 1 and 3 min to QMiX, 2% CHX, MTAD, 1% and 2% NaOCl. The amount of killed bacteria in biofilms was analysed by confocal laser scanning microscopy using viability staining. Dentine blocks were exposed to QMiX and 17% EDTA for 5 min. The effectiveness of smear layer removal by the solution was evaluated using scanning electron microscopy. For statistical analysis, one-way analysis of variance and comparison of two proportions were used. RESULTS: QMiX and 1% NaOCl killed all planktonic E. faecalis and plaque bacteria in 5 s, while 2% CHX and MTAD were unable to kill all plaque bacteria in 30 s, and some E. faecalis cells survived even 3 min of exposure. QMiX and 2% NaOCl killed up to 12 times more biofilm bacteria than 1% NaOCl (P < 0.01), 2% CHX (P < 0.05; P < 0.001) and MTAD (P < 0.05; P < 0.001). QMiX removed smear layer equally well as EDTA (P = 0.18 × 10(-5)). CONCLUSION: QMiX and NaOCl were superior to CHX and MTAD under laboratory conditions in killing E. faecalis and plaque bacteria in planktonic and biofilm culture. Ability to remove smear layer by QMiX was comparable to EDTA.

In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions.

AIM: To investigate the antibacterial effect of Tetraclean, MTAD and five experimental irrigants using both direct exposure test with planktonic cultures and mixed-species in vitro biofilm model. METHODOLOGY: Tetraclean, MTAD and five experimental solutions that were modifications of existing formulae including MTAD + 0.01% cetrimide (CTR), MTAD + 0.1% CTR, MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD), MTAC-2 (Tween 80 replaced by 0.1% CTR) and MTAD-D (MTAD without the Tween 80 and no CTR added) were used as disinfectants in the experiments. In the direct exposure test, a suspension of Enterococcus faecalis was mixed with each of the solutions. After 0.5, 1, 3 and 10 min, an inactivator was added and the number of surviving bacteria was calculated. A mixed-species biofilm from subgingival plaque bacteria was grown in brain heart infusion broth in anaerobic conditions on synthetic hydroxyapatite discs. Two-week-old biofilms were exposed to the solutions for 0.5, 1 and 3 min. The samples were observed by confocal laser scanning microscopy after bacterial viability staining. The scans were quantitatively analysed, and the volume of killed cells of all cells was calculated for each medicament. RESULTS: Tetraclean and MTAC-2 (0.1% CTR) killed planktonic E. faecalis in <30 s. Complete killing of bacteria required 1 min by MTAC-1, 3 min by MTAD + 0.1% CTR and 10 min by MTAD, MTAD-D and MTAD + 0.01% CTR. In the biofilm test, there were significant differences in microbial killing between the different solutions and times of exposure (P < 0.005). MTAC-2 showed the best performance, killing 71% of the biofilm bacteria in 3 min, followed by MTAC-1 and Tetraclean. MTAD and the three MTAD modifications demonstrated the lowest antibacterial activity. CONCLUSION: Tetraclean was more effective than MTAD against E. faecalis in planktonic culture and in mixed-species in vitro biofilm. CTR improved the antimicrobial properties of the solutions, whereas Tween 80 seemed to have a neutral or negative impact on their antimicrobial effectiveness.

Functions of S-layers.

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Development of a Cavity Disinfectant Containing Antibacterial Monomer MDPB.

An experimental cavity disinfectant (ACC) that is intended to be used for various direct and indirect restorations was prepared by adding an antibacterial monomer 12-methacryloyloxydodecylpyridinum bromide (MDPB) at 5% into 80% ethanol. The antibacterial effectiveness of ACC and its influences on the bonding abilities of resin cements were investigated. To examine the antibacterial activity of unpolymerized MDPB, the minimum inhibitory and bactericidal concentrations (MIC and MBC) were determined for Streptococcus mutans, Lactobacillus casei, Actinomyces naeslundii, Parvimonas micra, Enterococcus faecalis, Fusobacterium nucleatum, and Porphyromonas gingivalis Antibacterial activities of ACC and the commercial cavity disinfectant containing 2% chlorhexidine and ethanol (CPS) were evaluated by agar disk diffusion tests through 7 bacterial species and by MIC and MBC measurement for S. mutans The effects of ACC and CPS to kill bacteria in dentinal tubules were compared with an S. mutans-infected dentin model. Shear bond strength tests were used to examine the influences of ACC on the dentin-bonding abilities of a self-adhesive resin cement and a dual-cure resin cement used with a primer. Unpolymerized MDPB showed strong antibacterial activity against 7 oral bacteria. ACC produced inhibition zones against all bacterial species similar to CPS. For ACC and CPS, the MIC value for S. mutans was identical, and the MBC was similar with only a 1-step dilution difference (1:2). Treatment of infected dentin with ACC resulted in significantly greater bactericidal effects than CPS (P < 0.05, analysis of variance and Tukey's honest significant difference test). ACC showed no negative influences on the bonding abilities to dentin for both resin cements, while CPS reduced the bond strength of the self-adhesive resin cement (P < 0.05). This study clarified that the experimental cavity disinfectant containing 5% MDPB is more effective in vitro than the commercially available chlorhexidine solution to eradicate bacteria in dentin, without causing any adverse influences on the bonding abilities of resinous luting cements.

Epithelial Microvesicles Promote an Inflammatory Phenotype in Fibroblasts.

Microvesicles (MVs) are extracellular vesicles secreted by various cell types that are involved in intercellular communication. We hypothesized that in human periodontal disease, the pocket epithelium releases MVs, which then modulate gene expression in the underlying fibroblasts to control periodontal inflammation. MVs were isolated from culture medium of gingival epithelial cells (GECs) treated with oral bacterial biofilm extract or left untreated. Biofilm treatment significantly increased MV release from the GECs. Mass spectrometry of GEC-MVs identified a total of 2,173 proteins, of which about 80% were detected in MVs from both control and biofilm-treated GECs. Among 80 signature genes of human gingival fibroblasts, 20 were significantly regulated (P < 0.05) by MVs from control and biofilm-treated GECs in a similar manner. Matrix metalloproteinase 1 and 3 and interleukin 6 and 8 showed the strongest regulation at the mRNA and protein levels. Several cellular signaling pathways were activated by GEC-MVs in human gingival fibroblasts, including Smad and mitogen-activated protein kinase-associated pathways ERK1/2, JNK, and p38. However, ERK1/2 signaling dominated in the MV-induced gene expression changes. The results demonstrate that GEC-MVs have a strong regulatory effect on the expression of fibroblast genes associated with inflammation and matrix degradation and that bacterial biofilm stimulates the generation of GEC-MVs. This suggests that bacterial biofilms can contribute to the initiation and progression of periodontal disease by promoting a tissue-destructive phenotype in gingival fibroblasts via the enhanced secretion of epithelial MVs.

Epidemiology and pathogenesis of Bacillus cereus infections.

Bacillus cereus is a causative agent in both gastrointestinal and in nongastrointestinal infections. Enterotoxins, emetic toxin (cereulide), hemolysins, and phoshpolipase C as well as many enzymes such as beta-lactamases, proteases and collagenases are known as potential virulence factors of B. cereus. A special surface structure of B. cereus cells, the S-layer, has a significant role in the adhesion to host cells, in phagocytosis and in increased radiation resistance. Interest in B. cereus has been growing lately because it seems that B. cereus-related diseases, in particular food poisonings, are growing in number.

Radiation sensitivity of Bacillus cereus with and without a crystalline surface protein layer.

The radiation sensitivity of four strains of Bacillus cereus was investigated with attention to bacterial surface structure. All four strains were sensitive to radiation with gamma rays (D(10)=0.4 kGy). No crystalline surface protein layer could be detected on the cell surface. When cultured on solid media, an S-layer covered the cells of the two strains, and they were 2.6 times as resistant to radiation as the two reference strains without an S-layer. In SDS-PAGE, a major 97-kDa band from the resistant strains from plate cultures was replaced by a ca. 85-kDa protein band in samples from broth cultures. Electron microscopy, SDS-PAGE, Western blot and fluorescent antibody staining indicated that the higher resistance to radiation of the clinical strains from plate cultures was associated with the presence of the S-layer on the cell surface.

Black-pigmented gram-negative anaerobes in endodontic infections.

Necrotic dental root canal infections are polymicrobial infections dominated by anaerobic bacteria. The number of different species in one canal is usually low, approx. 4-7 species. The species isolated most frequently belong to the genera Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Eubacterium and Streptococcus. The frequency of isolation of black-pigmented Gram-negative anaerobes in endodontic infections varies from 25% to > 50%. Pr. intermedia is the most commonly found pigmented species, followed by Pr. denticola and two Porphyromonas species, P. gingivalis and P. endodontalis. Several studies have shown that P. gingivalis and P. endodontalis are closely related to the presence of acute symptoms in endodontic infections, whereas other black-pigmented Gram-negative anaerobes are not. However, several other species may also be involved in acute infections. Moreover, Porphyromonas species have occasionally been isolated from cases with no symptoms. Although Porphyromonas spp. are clearly related to symptoms at the beginning of therapy, they are not important for the prognosis of the treatment.

In vitro infection and disinfection of dentinal tubules.

An in vitro model for dentinal tubule infection of root canals was developed. Cylindrical dentin specimens, 4 mm high with a diameter of 6 mm and a canal 2.3 mm wide, were prepared from freshly extracted bovine incisors. The cementum was removed from all dentin blocks. The tubules were opened by four-minute treatments with 17% EDTA and 5.25% NaOCl before being infected with Enterococcus faecalis ATCC 29212 in yeast extract-glucose broth. Bacteria rapidly invaded the tubules. After three weeks of incubation, a heavy infection was found 400 micron from the canal lumen, and the front of the infection reached 1000 micron in some blocks. Camphorated paramonochlorophenol (CMCP) and a calcium hydroxide compound, Calasept, were tested for their disinfecting efficacy toward E. faecalis-infected dentin. Liquid CMCP rapidly and completely disinfected the dentinal tubules, whereas CMCP in gaseous form disinfected tubules less rapidly. Calasept failed to eliminate, even superficially, E. faecalis in the tubules. The method used in bacteriological sampling allowed for sequential removal of 100-micron-thick zones of dentin from the central canal toward the periphery. Control specimens were uniformly infected and yielded growth in bur samples up to some 500 microns from the surface. The model proved quite sensitive and seems suitable for in vitro testing of root canal medicaments.