Effect of ultrasonic activation on the reduction of bacteria and endotoxins in root canals: a randomized clinical trial.

AIM: This randomized clinical trial aimed to compare the effectiveness of ultrasonic activation with that of nonactivated irrigation on the removal of bacteria and endotoxin from root canals. METHODOLOGY: Fifty patients with necrotic pulps and asymptomatic apical periodontitis were randomly allocated into two groups according to the final irrigation protocol after root canal preparation: Group UI - ultrasonic irrigation (n = 25) and Group NI - needle irrigation (n = 25). The root canals were medicated with calcium hydroxide for 14 days. Microbiological sampling was performed before (S1) and after the root canal preparation (S2), after the irrigation protocols (S3) and after the removal of the intracanal medication (S4). Total bacteria counts were determined by qPCR and the endotoxin levels by the limulus amebocyte lysate assay. Intragroup analyses were performed using the Wilcoxon test for related samples, whereas intergroup analyses were performed using the Mann-Whitney U-test (P < 0.05). RESULTS: All S1 samples were positive for bacteria, with median numbers of 1.49 × 106 and 8.55 × 105 bacterial cells for the UI and NI groups, respectively. This number significantly decreased in S2 samples (UI: 1.41 × 104 ; NI: 3.53 × 104 ; both with P < 0.001). After final irrigation protocols, there was a significant decrease in bacterial load from S2 to S3 samples in both groups (UI: 4.29 × 103 ; NI: 1.08 × 104 ; P < 0.01). Intergroup analysis revealed a significant difference between irrigation methods regarding bacterial counts in S3 samples (P < 0.05). In contrast, no significant differences were observed between groups for endotoxin levels (P > 0.05). CONCLUSIONS: Ultrasonic activation was more effective than nonactivated irrigation for reducing the number of bacteria but not the endotoxin levels in root canals of teeth with apical periodontitis.

Counterclockwise or clockwise reciprocating motion for oval root canal preparation: a micro-CT analysis.

AIM: To evaluate oval root canal preparation using one or two instruments in counterclockwise or clockwise reciprocating motion. METHODOLOGY: The radiographic diameter of mandibular human incisors was evaluated, and oval canals were selected (2 ≤ Diameter Ratio ≤ 4). Fifty-seven teeth were assigned to root canal preparation (n = 19): Reciproc 40 (R40) in a counterclockwise reciprocating motion; Mtwo size 40, .06 taper (M 40.06) in a clockwise reciprocating motion or Mtwo size 20, .06 taper and size 40, .06 taper (M 20/40.06) in a clockwise reciprocating motion. Mtwo instruments were coupled to an ENDO DUAL motor, turning 150° clockwise and 30° counterclockwise. Scanning was performed before and after root canal preparation using a SkyScan 1176 micro-computed tomography. Volume, percentage of debris and percentage of uninstrumented surface were analysed in the entire root canal and in each third of the canal. Data were compared using anova and Tukey's tests or Kruskal-Wallis and Dunn tests. RESULTS: The Reciproc and Mtwo systems using different kinematics were associated with a similar increase in root canal volume. Additionally, both system had similar percentage of uninstrumented surface (P > 0.05). Mtwo size 20, .06 taper and size 40, .06 taper was associated with significantly lower debris (P < 0.05) in the middle third (0.56%) when compared to R40 (1.31%) and M size 40, .06 taper (1.54%). CONCLUSIONS: The conventional reciprocation motion for R40 and the clockwise reciprocation motion for Mtwo resulted in similar root canal preparations. Less remaining debris was present in the middle third when two instruments with different diameters were used.

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.

Cytotoxicity, genotoxicity and antibacterial effectiveness of a bioceramic endodontic sealer.

AIM: To compare the characteristics of bioceramic endodontic sealer Endosequence BC sealer with those of AH Plus sealer. METHODOLOGY: Cytotoxicity and genotoxicity were analysed on human gingival fibroblasts submitted to cell culture medium conditioned by sealers using the MTT reduction assay and micronucleus formation test (MNT), respectively. Cells grown on fresh medium served as controls. Cell viabilities were measured at 1, 3, 5 and 7 days. The antibacterial activity was analysed on an Enterococcus faecalis strain (ATCC 29212) using both on agar diffusion test (ADT) and a direct contact test (DCT). The inhibition zones in ADT were measured after 48 h and the colony-forming units counting in the DCT after 1, 24, 72 and 168 h. Data were compared by anova and Tukey's test and MNT by Fisher's exact test (P < 0.05). RESULTS: Cultures submitted to Endosequence BC sealer had a significantly higher number of viable cells (P < 0.01) and less micronucleus formation (P < 0.05) than AH Plus sealer. Endosequence BC sealer exhibited significantly smaller inhibition zones (6.00 ± 0.03 mm) than AH Plus sealer (10.31 ± 0.21 mm) (P < 0.05). Moreover, Endosequence BC sealer had significantly smaller antibacterial activity than AH Plus sealer up to 1 h of direct contact (P < 0.05). On other exposure times, both materials had similar antibacterial effectiveness (P > 0.05). CONCLUSIONS: Bioceramic-based sealer had less cytotoxicity and genotoxicity and similar antibacterial effect against E. faecalis in comparison with AH Plus sealer.

A micro-computed tomography evaluation of long-oval canal preparation using reciprocating or rotary systems.

AIM: To evaluate, using micro-computed tomography, the preparation of long-oval root canals using a single reciprocating system versus a multiple-file rotary system. METHODOLOGY: Distal canals of thirty mandibular molars were selected and randomly assigned to one of two instrument groups (n = 15): Reciproc 40 (VDW, Munich, Germany) or BioRaCe system (FKG Dentaire, La Chaux-de-Fonds, Switzerland). The teeth were scanned before and after preparation of the canal by a SkyScan 1172 micro-computed tomography scanner at 11-µm resolution. Morphometric variations were measured by volume increases and by the remaining untreated canal surface area in the entire canal and as well as in each third of the canal. Data were compared using the Mann-Whitney test. RESULTS: The Reciproc system left significantly more areas untouched (P < 0.001) in the cervical and middle thirds (18.14% and 21.82%) as compared to BioRaCe (8.14% and 11.35%). The Reciproc system had the greatest increase in volume of both the entire canal and the apical third (P < 0.5). CONCLUSIONS: Neither technique was able to completely prepare the outline of long-oval canals. The Reciproc system removed more tooth structure. The BioRaCe left fewer untouched dentine walls in the more coronal thirds of the canal, whilst Reciproc left fewer in the apical third.

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).

Ex vivo evaluation of the effects of several root canal preparation techniques and irrigation regimens on a mixed microbial infection.

AIM: To assess the ex vivo effectiveness of the alternated use of 1% NaOCl and 15% citric acid in association with two instrumentation techniques for the disinfection of root canals infected with Enterococcus faecalis and Candida albicans. METHODOLOGY: Eighty human mandibular premolars with straight, oval root canals standardized to 15 mm in length were infected with a mixed culture of E. faecalis and C. albicans for 28 days. Five other teeth were used as controls and were neither contaminated nor instrumented. Specimens were divided into two groups (n = 40), according to whether the canal preparation technique used manual (K-type) or rotary (Protaper Universal) instruments. These groups were further divided into four subgroups (n = 10) according to the irrigation solution used: saline, 1% NaOCl, 1% NaOCl with alternated use of 15% citric acid and 5.25% NaOCl. Root canals were prepared with a crown-down technique until a size 50 K-file or with rotary preparation until an F5 instrument. Microbiological sampling was performed before (S1) and after (S2) the chemomechanical preparation, using sterile paper points. The specimens were split, and 0.02 g of dentine chips was collected from the root thirds to verify the presence of microorganisms in root canal walls. RESULTS: Saline and 1% NaOCl were less effective in reducing microorganisms compared with 1% NaOCl with alternated use of 15% citric acid or 5.25% NaOCl alone (P < 0.05). Both manual and rotary preparations significantly reduced microorganisms regardless of the irrigation solution used (P < 0.05). However, there was no significant difference between the canal preparation techniques (P > 0.05). CONCLUSIONS: Irrigation with 5.25% NaOCl and 1% NaOCl alternated with 15% citric acid reduced microorganisms in infected root canals significantly more than saline and 1% NaOCl.

Determination of pulp vitality in vivo with pulse oximetry.

AIM: To evaluate the use of pulse oximetry as a test for pulp vitality, by comparing in the same patient, the levels of oxygen saturation of the index finger and of the maxillary central incisor and canine teeth without clinically detectable pulp inflammation. METHODOLOGY: Seventeen male and female patients aged between 26 and 38 years participated and a total of 32 maxillary central incisor and 32 canine teeth were analysed. Selection criteria required the teeth to have healthy crowns, or with restorations no more than 2 mm in diameter and no clinical and radiographical signs or symptoms of pulp or periapical inflammatory changes. The negative control group consisted of 10 root filled teeth. Measurements were first taken from the index finger of patients. Their teeth were then subjected to a thermal test with refrigerant gas and then to a vitality test with pulse oximetry. Data were analysed by Pearson's and paired t-tests. RESULTS: There were no significant statistical correlations between blood oxygen levels in the index finger and in the teeth of the patient (P > 0.05). There was a statistically significant difference in the oxygen levels between the two tooth groups studied and the index finger (P

Cytotoxicity analysis of EDTA and citric acid applied on murine resident macrophages culture.

AIM: To assess the ex vivo cytotoxicity of EDTA and citric acid solutions on macrophages. METHODOLOGY: The cytotoxicity of 17% EDTA and 15% citric acid was evaluated on murine macrophage cultures using MTT-Tetrazolium method [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide]. A total of 5 x 10(5) cells were plated in medium culture with 17% EDTA or 15% citric acid. Fresh medium was used as a control. Toxicity values were analysed statistically by anova and Tukey's test (P<0.05) at short (0, 6, 12, 24 h) and medium periods (1, 3, 5, 7 days), using ELISA absorbance. RESULTS: On the short term, both EDTA (0.253 nm) and citric acid (0.260 nm) exhibited cytotoxic effects on macrophage cultures (P<0.05). On the medium term, statistical differences were observed (P<0.05) between the groups. EDTA (0.158 nm) and citric acid (0.219 nm) were cytotoxic when compared with the control group; EDTA-reduced macrophage viability significantly more than citric acid (P<0.05). CONCLUSIONS: Both EDTA and citric acid had effects on macrophages cells ex vivo, but citric acid was less toxic in periods from 1 to 7 days of use.

In vitro evaluation of the cytotoxic effects of acid solutions used as canal irrigants.

Solutions of EDTA and citric acid have been used as canal irrigants. These substances must be compatible with apical periodontal tissue. The aim of this study was to evaluate comparatively the cytotoxicity of a 17% EDTA solution and that of three solutions with different concentrations of citric acid (10, 15, and 25%) on cultured fibroblasts. The solutions were diluted to 0.1% and 0.5% in culture medium and then applied to NIH 3T3 cells. After 0, 6, 12, and 24 h (short-term assay; viability) and 1, 3, 5, and 7 days (long-term assay; survival), the cells were counted. The data were compared by ANOVA. In the short-term experiments, all solutions presented a percentage of cell viability similar to that of control cells, except for the 17% EDTA solution diluted to 0.5%. After the long-term assay, all groups presented a continuous and progressive cell growth except for the 17% EDTA solution and for the 25% citric acid solution at a 0.5% dilution. The citric acid solution did not impair cell growth and viability, proving to be noncytotoxic in vitro.

Effect of nitric oxide synthase inhibition on high affinity Ca(2+)-ATPase during hypoxia in cerebral cortical neuronal nuclei of newborn piglets.

Previous studies have shown that during hypoxia, neuronal nuclear high affinity Ca(2+)-ATPase activity is increased in the cerebral cortex of newborn piglets. The present study tests the hypothesis that pretreatment with N-nitro-L-arginine (NNLA) will prevent the hypoxia-induced increase in high affinity Ca(2+)-ATPase activity in cortical neuronal nuclear membrane of newborn piglets. We also tested the hypothesis that nitration is a mechanism of elevation of the high affinity Ca(2+)-ATPase activity during hypoxia. Studies were performed in five normoxic, five hypoxic, and six NNLA-pretreated (40 mg/kg) hypoxic newborn piglets. Cerebral cortical neuronal nuclei were isolated and the high affinity Ca(2+)-ATPase activity was determined. Further, normoxic samples were aliquoted into two sub-groups for in vitro nitration with 0.5 mM peroxynitrite and subsequent determination of the high affinity Ca(2+)-ATPase activity. The activity increased from 309+/-40 nmol Pi/mg protein/h in the normoxic group to 520+/-108 nmol Pi/mg protein/h in the hypoxic group (P<0.05). In the NNLA-pretreated group, the activity was 442+/-53 nmol Pi/mg protein/h (P<0.05), which is 25% lower than in the hypoxic group. In the nitrated group the enzyme activity increased to 554+/-59 nmol Pi/mg protein/h (P<0. 05). Thus peroxynitrite-induced nitration in vitro increased the high affinity Ca(2+)-ATPase activity and NNLA administration in vivo partially prevented the hypoxia-induced increase in neuronal nuclear high affinity Ca(2+)-ATPase activity. We conclude that the hypoxia-induced increase in nuclear membrane high affinity Ca(2+)-ATPase activity is NO-mediated and that nitration of the enzyme is a mechanism of its modification.