Effects of several storage media on viability and proliferation capacity of periodontal ligament cells.

AIM: The purpose of this study was to investigate the effect of different storage media on viability and proliferation capacity of periodontal ligament cells. METHODS: Plates with periodontal ligament fibroblasts (PDLF) cells were incubated in skimmed and whole milk, recently prepared Hank's balanced salt solution (HBSS), Save-A-Tooth system's, coconut water, propylene glycol with 20% propolis, egg white, tap water (negative control) at 5 °C and 20 °C, for 24 h. In one of the plates of each temperature, cell viability was determined by MTT assay. In the remaining plates, the wells were filled and incubated with Minimum Essential Medium (MEM) at 37 °C for 24, 48, 72, 96, and 120 h. The proliferation capacity of PDFL cells was also evaluated by MTT assay. Data were statistically analyzed by Kruskal-Wallis, Scheffé and Mann-Whitney tests (α = 5%). RESULTS: At 5 °C, milk maintained more viable cells than other storage media immediately after exposure (0 h) and allowed greater proliferation capacity. At 20 °C, milk and HBSS had similar and allowed similar proliferation ability at 24 and 48 h. From 72 h onwards, capacity to maintain cell viability the proliferation rate of cells incubated in HBSS was superior than milk. At both temperature and experiments, Save-A-Tooth system was similar to tap water. CONCLUSION: Milk and HBSS were more effective in maintaining cellular viability and proliferation capacity than any other storage media. At 5 °C, the most viable alternative was milk. At 20 °C, HBSS had better results.

Effects of the addition of nanoparticulate calcium carbonate on setting time, dimensional change, compressive strength, solubility and pH of MTA.

AIM: To evaluate nanoparticulate calcium carbonate (NPCC) using transmission electron microscopy and the effects of NPCC addition to MTA in regard to the setting time, dimensional change, compressive strength, solubility and pH. METHODOLOGY: The experimental groups were G1 (MTA), G2 (MTA with 5% NPCC) and G3 (MTA with 10% NPCC). The tests followed ISO and ADA standards. The specimens in the dimensional change and compressive strength tests were measured immediately after setting, after 24 h and after 30 days. In the solubility test, rings filled with cement were weighed after setting and after 30 days. The pH was measured after 24 h and 30 days. The data were analysed with the ANOVA, Tukey's and Kruskal-Wallis tests (α = 5%). RESULTS: The setting time was reduced (P < 0.05) in samples from G2 and G3 compared to G1. After 24 h, the dimensional change was similar amongst the groups, and after 30 days, G2 was associated with less alteration than G1 and G3. There was a difference in the compressive strength (P < 0.001) after 24 h and 30 days (G1 > G2 > G3). The solubility test revealed a difference amongst the groups when the specimens were hydrated: G2 > G1 > G3 and dehydrated: G3 > G2 > G1. The pH of the groups was similar at 24 h with higher values in each group after 30 days (P < 0.05), and G2 and G3 had similar mean pH values but both were higher than G1. CONCLUSIONS: Nanoparticulate calcium carbonate had a cubic morphology with few impurities. The addition of nanoparticulate calcium carbonate to MTA accelerated the setting time, decreased compressive strength and, after 30 days, resulted in lower dimensional change (G2), higher solubility and a higher pH.

Subcutaneous connective tissue reactions to iRoot SP, mineral trioxide aggregate (MTA) Fillapex, DiaRoot BioAggregate and MTA.

AIM: To evaluate connective tissue reactions to iRoot SP (Innovative Bioceramics, Vancouver, BC, Canada), mineral trioxide aggregate (MTA) Fillapex (FLPX) (Angelus Soluções Odontológicas, Londrina, Brazil), DiaRoot Bioaggregate (DiaDent Group International, Burnaby, BC, Canada) and white MTA (Angelus, Londrina, Brazil) in Wistar rats. METHODOLOGY: A total of 128 dentine tubes filled with the materials and 32 empty tubes (control) were implanted into 32 rats. After 7, 15, 30 and 90 days (n = 8 per period), the animals were euthanized, and the tissues were processed for histological evaluation using haematoxylin-eosin (H&E) and Von Kossa (VK) staining. Observations were made for cellular inflammatory components and the presence of multinucleated giant cells (MNGC), macrophages and tissue necrosis. Data were analysed by Fisher's exact and Kruskal­Wallis tests (P < 0.05). RESULTS: In all experimental periods, MTA FLPX and iRoot SP scored higher than the other groups for the variable macrophages (P < 0.05). After 30- and 90-day experimental periods, MTA FLPX scored higher than the other groups for the variable MNGC (P < 0.05). After 90 days, the only group that exhibited samples with severe inflammatory response was MTA FLPX. VK positivity was observed in areas of necrosis in all groups, except in the control group. CONCLUSIONS: The materials were considered biologically acceptable except MTA FLPX, which remained toxic to subcutaneous tissue even after 90 days.

Influence of the exposure of MTA with and without calcium chloride to phosphate-buffered saline on the push-out bond strength to dentine.

AIM: To analyse the influence of exposure of mineral trioxide aggregate (MTA) with and without calcium chloride (CaCl2 ) to phosphate-buffered saline (PBS) on the push-out bond strength, over different experimental periods. METHODOLOGY: One hundred and twenty dentine discs with standardized cavities were filled with MTA with and without 10% CaCl2 . The specimens were randomly divided as follows (n = 30): (G1) MTA in contact with a moistened cotton pellet, (G2) MTA immersed in PBS, (G3) MTA + CaCl2 in contact with a moistened cotton pellet and (G4) MTA + CaCl2 immersed in PBS. The samples were stored for 3, 28 and 60 days. The bond strengths were measured with the Instron Testing machine. Data were analysed using the three-way anova and Tukey test (P < 0.05). RESULTS: In general, the samples of MTA with and without CaCl2 , exposed to PBS, had higher bond strength values in all study periods (P < 0.05). Analysis of the influence of addition of CaCl2 to MTA (G1 × G3) evidenced significant differences in bond strength in the different periods (P < 0.05). CONCLUSIONS: The exposure of MTA to PBS positively influenced the push-out bond strength, whereas the addition of CaCl2 had a negative influence.

Endodontic re-instrumentation enhances hydroxyl ion diffusion through radicular dentine.

AIM: To evaluate the diffusion of hydroxyl ions from calcium hydroxide paste (CH) before root canal filling and after retreatment. METHODOLOGY: After preparation of 60 root canals, the cementum layer was removed, and the canals and root surfaces were treated for smear layer removal. The apical third of roots was covered with adhesive. The canals were filled with CH, and the teeth were placed in individual vials containing 10 mL of distilled water, which had its pH measured after 7, 14, 21 and 28 days (pH1). The root canals were then divided into five groups and filled with Resilon/Real Seal (G1) or gutta-percha and Endofill (G2), Sealapex (G3), AH Plus (G4) or MTA Fillapex (G5) sealers. After storage for 7 days, the root canals were retreated. The CH was again inserted into the canals, and the teeth were placed in new vials containing 10 mL of distilled water. The pH was again measured at 7, 14, 21 and 28 days (pH2). The initial and final pH readings (pH1 and pH2) were compared by anova, anova2 and Tukey's tests (P < 0.05). RESULTS: The pH1 and pH2 measurements increased with time. The measurements obtained after retreatment were significantly higher than those obtained before root canal filling. CONCLUSIONS: Hydroxyl ions are able to diffuse through dentinal tubules. Regardless of the filling material, it was possible to re-establish the permeability of dentine to ionic diffusion after retreatment. Time had a positive influence on ionic diffusion.

Viability of human periodontal ligament fibroblasts in milk, Hank's balanced salt solution and coconut water as storage media.

AIM: To evaluate the effectiveness of various storage media at 5 °C for maintaining the viability of human periodontal ligament fibroblasts (PDLF). METHODOLOGY: Plates with PDLF were soaked in recently prepared Hank's balanced salt solution (HBSS), skimmed milk, whole milk, Save-A-Tooth(®) system's HBSS (Save), natural coconut water, industrialized coconut water or tap water (negative control) at 5 °C for 3, 6, 24, 48, 72, 96 and 120 h. Minimum essential medium (MEM) at 37 °C served as the positive control. PDL cell viability was determined by MTT assay. Data were statistically analysed by Kruskal-Wallis test complemented by the Scheffé test (α=5%). RESULTS: The greatest number of viable cells was observed for MEM. Skimmed and whole milk, followed by natural coconut water and HBSS, were the most effective media in maintaining cell viability (P<0.05). From 24 to 120 h, Save, industrialized coconut water and tap water were the worst storage media. CONCLUSIONS: Skimmed and whole milk had the greatest capacity to maintain PDLF viability when compared with natural coconut water, HBSS, Save, industrialized coconut water and tap water.

Ex vivo evaluation of the ability of the ROOT ZX II to locate the apical foramen and to control the apical extent of rotary canal instrumentation.

AIM: To evaluate the capacity of the ROOT ZX II to locate the apical foramen and to control the apical extent of rotary instrumentation. METHODOLOGY: Sixty-five extracted human single rooted teeth were selected and measured directly using a size 15 K-Flexofile introduced in the canal until the tip was visible at the major foramen (direct length, DL). The teeth were then measured electronically (EL1) with the ROOT ZX II when used passively, that is without rotation. To test the auto reverse function, the root canals were instrumented with nickel titanium rotary instruments. Instrumentation was carried out apically until rotation was reversed by the automatic apical reverse (AAR) function at different levels (2, 1 and 0.5). The instrumented length at each level was measured and registered as AAR2, AAR1 and AAR0.5, respectively. After instrumentation, a second passive electronic measurement was conducted and noted as electronic length 2 (EL2). All measurements were expressed in millimetres with accuracy set to 0.5 mm. Percentages of acceptable measurements for each electronic reading were calculated and compared using the proportions test. The Wilcoxon's signed rank test was used to compare the differences between DL/EL1 and DL/EL2, and to compare EL2 with the different AAR measurements. The critical value of statistical significance was 5%. RESULTS: EL1 and EL2 measurements were coincident to DL in 56 (86%) and 54 (83%) of the cases, respectively. The proportions test showed no statistically significant difference between these percentages (P > 0.05). The Wilcoxon's signed rank test did not show any differences (P > 0.05) when comparing the mean difference between DL with EL1 (0.03) and DL with EL2 (0.10). Statistically significant differences were observed when comparing EL2 with AAR2 and with AAR1. CONCLUSIONS: The ROOT ZX II reliably located the major apical foramen, but was not an accurate method for controlling the apical extent of rotary instrumentation. Rotary instrumentation with the automatic apical reverse feature was always closer to the foramen than expected.

The effect of mineral trioxide aggregate on the apexification and periapical healing of teeth with incomplete root formation.

AIM: To evaluate the influence of mineral trioxide aggregate (MTA) on apexification and periapical healing of teeth in dogs with incomplete root formation and previously contaminated canals and to verify the necessity of employing calcium hydroxide paste before using MTA. METHODOLOGY: Twenty premolars from two 6-month old dogs were used. After access to the root canals and complete removal of the pulp, the canal systems remained exposed to the oral environment for 2 weeks. Canal preparation was then carried out using Hedström files, under irrigation with 1% sodium hypochlorite, 1 mm short of the radiographic apex. After drying, the canals of two premolars in each dog were left empty (control group). The other eight teeth in each animal were divided into two experimental groups. The apical thirds of the canals of group 1 were filled with MTA. In the teeth of group 2, the canals were dressed with a calcium hydroxide-propylene glycol paste. After 1 week, the paste was removed and the apical third was filled with MTA. All teeth were restored with reinforced zinc oxide cement (IRM) and amalgam. The animals were killed 5 months later, and blocks of the teeth and surrounding tissues were submitted to histological processing. The sections were studied to evaluate seven parameters: formation of an apical calcified tissue barrier, level of barrier formation, inflammatory reaction, bone and root resorption, MTA extrusion, and microorganisms. Results of experimental groups were analysed by Wilcoxon's nonparametric tests and by the test of proportions. The critical value of statistical significance was 5%. RESULTS: Significant differences (P < 0.05) were found in relation to the position of barrier formation and MTA extrusion. The barrier was formed in the interior of the canal in 69.2% of roots from MTA group only. In group 2, it was formed beyond the limits of the canal walls in 75% of the roots. MTA extrusion occurred mainly in roots from group 2. There was similarity between the groups for the other parameters. CONCLUSIONS: Mineral trioxide aggregate used after root canal preparation favoured the occurrence of the apexification and periapical healing. The initial use of calcium hydroxide paste was not necessary for apexification to occur, and has shown to be strongly related to the extrusion of MTA and formation of barriers beyond the limits of the root canal walls.

Ex vivo evaluation of the capacity of the Tri Auto ZX to locate the apical foramen during root canal retreatment.

AIM: To evaluate ex vivo the capacity of the Tri Auto ZX to locate the apical foramen during root canal retreatment. METHODOLOGY: The root canals of 62 maxillary and mandibular canines were prepared to a length 1 mm short of the apical foramen, to an apical size 35 using 1% sodium hypochlorite as an irrigant. Once prepared, the length of each tooth was measured directly using a size 15 K-Flexofile introduced in the canal until the tip was visible at the apical foramen. After the file was removed, its length was recorded to a precision of 0.01 mm using a calliper. These direct lengths (DL) became the 'gold standard' for comparison with the electronic lengths (EL) derived from the Tri Auto ZX. After direct measurement, the tooth was measured electronically (EL1) and the canals were filled using lateral condensation of gutta-percha cones and sealer. Seven days later the root filling was removed using solvent, Gates-Glidden burs, and K-files, and new electronic lengths determined (EL2). The electronic lengths (EL1 and EL2) were compared with the DL, and the differences were analysed statistically using the proportions test and Student's t-test. RESULTS: At a tolerance limit of +/-0.5 mm, EL1 coincided with the DL in 76% of cases. Lengths obtained in the presence of remnant of filling material (EL2) coincided in 81% of cases. The proportions test used to compare these percentages showed no statistically significant difference between EL1 and EL2 (P > 0.05). The Student's t-test revealed a statistically significant difference (P < 0.05) between the means of the differences between DL/EL1 (-0.36 mm) and DL/EL2 (-0.04 mm). CONCLUSION: The Tri Auto ZX was accurate to +/-0.5 mm in more than 80% of teeth following removal of root fillings.

The effect of the renewal of calcium hydroxide paste on the apexification and periapical healing of teeth with incomplete root formation.

AIM: To evaluate the influence of renewing calcium hydroxide paste on apexification and periapical healing of teeth in dogs with incomplete root formation and previously contaminated canals. METHODOLOGY: Forty premolars from four 6-month-old dogs were used. After access to the root canals and complete removal of the pulp, the canal systems remained exposed to the oral environment for 2 weeks. Canal preparation was then carried out using Hedströem files, under irrigation with 1% sodium hypochlorite, 1 mm short of the radiographic apex. After drying, the canals of one premolar in each dog were left empty (group 4-control), and those of the other nine teeth in each animal were filled with a calcium hydroxide-propylene glycol paste. All teeth were restored with reinforced zinc oxide cement (IRM) or IRM and amalgam (group 4). The paste was renewed and the teeth restored again 1 week later. Then, the nine teeth in each animal were divided into three experimental groups: group 1 - paste not changed; group 2 - paste renewed every 4 weeks for 5 months; and group 3 - paste renewed after 3 months had elapsed. The teeth were restored with IRM and amalgam (groups 1 and 3) or IRM (group 2). The animals were killed 5 months later, and blocks of the teeth and surrounding tissues were submitted to histological processing. The sections were studied to evaluate six parameters: apical calcified tissue barrier, inflammatory reaction, bone and root resorption, paste extrusion and microorganisms. Results of experimental groups were analysed by Kruskal-Wallis nonparametric tests and by the test of proportions. The critical value of statistical significance was 5%. RESULTS: Significant differences (P < 0.05) were found in relation to the presence of bone resorption and paste in the periradicular area, the formation of a calcified tissue barrier at the apex, and the intensity of the apical inflammatory reaction. Bone resorption was more evident in group 1 (medicament not changed), and the presence of paste in the periodontal tissues was more common in groups 2 and 3. Renewal of the paste reduced the intensity of the inflammatory reaction (groups 2 and 3), but the formation of apical calcified tissue was more noticeable in the teeth where the paste had not been renewed. CONCLUSIONS: Replacement of calcium hydroxide paste was not necessary for apexification to occur, however, it did reduce significantly the intensity of the inflammatory process. Monthly renewal of calcium hydroxide paste reduced significantly the occurrence of apexification.

The effect of application time of EDTA and NaOCl on intracanal smear layer removal: an SEM analysis.

AIM: To verify, under the scanning electron microscope (SEM), the influence of irrigation time with ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite (NaOCl) on intracanal smear layer removal. METHODOLOGY: Twenty-one extracted human permanent teeth with single straight root canals were included. The root canals of the teeth were instrumented and, at the end of preparation, were irrigated with 3 mL of 15% EDTA, followed by 3 mL of 1% NaOCl for 1 min (group 1), for 3 min (group 2), and for 5 min (group 3). The canals of teeth in group 4 (control) did not receive the final irrigation. The teeth were sectioned longitudinally and prepared for an SEM. The dentinal wall of cervical, middle and apical thirds was graded according to the amount of debris and smear layer remaining on the walls. The results were analysed using the Kruskal-Wallis and Conover-Inman tests. RESULTS: In all the canals of experimental groups irrigation with EDTA and NaOCl completely removed the smear layer from the cervical and middle thirds. In the apical third, the dentine surface were partially covered, particularly in the teeth of group 1, where there was significantly more smear layer when compared with the other thirds in the same group (P<0.007). However, the Kruskal-Wallis test showed overall that there were no significant differences between groups 1, 2 and 3 (P>0.05). CONCLUSION: In this limited laboratory study, canal irrigation with EDTA and NaOCl for 1, 3 and 5 min were equally effective in removing the smear layer from the canal walls of straight roots.