IgG4-positive plasma cells are more often detected in chronic periapical lesions arising from permanent rather than primary teeth.

AIM: To characterize plasma cell subsets in chronic periapical lesions affecting permanent and primary teeth. METHODOLOGY: Only chronic periapical lesions without root canal treatment were selected. Twenty-one radicular cysts and 7 periapical granulomas affecting permanent teeth and 19 radicular cysts and 4 periapical granulomas affecting primary teeth were assessed for immunoglobulin (Ig) light chain (kappa and lambda), Ig heavy chain (IgG, IgG4, IgA, IgM and IgD) and plasma cell immunohistochemical markers (MUM1/IRF4, EMA and CD138). The data acquired were analysed by Student's t test, Mann-Whitney U, Friedman test followed by Dunn's multiple comparison test and Spearman's rank correlation. RESULTS: All cases were polyclonal (having similar kappa/lambda light chain ratios). IgG was most abundant compared to other Ig heavy chains (all, P < 0.001); like Ig light chains, but unlike IgA, there was greater expression of IgG in the primary compared to the permanent dentition, for both radicular cysts (P < 0.001) and periapical granulomas (P = 0.53). Notably, IgG4 expression was greater in the permanent than the primary dentition, for both radicular cyst (P < 0.05) and periapical granuloma (P = 0.65). IgM and IgD expression was scarce and variable, whereas plasma cell populations were detected efficiently through EMA, CD138 and MUM1/IRF4 markers, the latter being more sensitive in both dentitions. CONCLUSIONS: There were slight variations in the Ig light and heavy chain profiles in chronic periapical lesions when comparing the permanent and primary dentitions. The ability of IgG4+ plasma cell infiltration to modulate inflammatory responses in chronic periapical lesions arising from permanent as opposed to primary teeth should be considered in future studies.

Fibroblast growth factor receptor 2 expression in apical periodontitis in mice.

AIM: To investigate the presence, localization and the possible correlation of the fibroblast growth factor receptor-2 (FGFR2) with inflammatory resorption of cementum, periodontal ligament and alveolar bone during development of apical periodontitis in mice. METHODOLOGY: Apical periodontitis was experimentally induced in mandibular first molars of mice by pulp exposure to the oral environment. Healthy teeth without pulp exposure were used as controls. At 7, 21 and 42 days following pulp exposure, the animals were euthanized and the jaws were prepared for analysis under conventional and fluorescence microscopy, immunohistochemistry (FGFR2), RT-PCR (RNAm levels of RANK, RANKL, OPG, Runx2 and cathepsin K) and enzyme histochemistry (cementoclasts and osteoclasts). Statistical analysis was performed by Kruskal-Wallis tests and Dunn's post hoc tests for multiple comparisons (α = 0.05) using SAS 9.4 software. RESULTS: FGFR2-positive cells were not observed in the tissues surrounding healthy teeth but were observed in teeth with periapical lesions from seven days after root canal contamination. At days 21 and 42 after endodontic infection, the increase in periapical lesion size was accompanied by significantly enhanced expression of FGFR2 (P < 0.0001), significantly increased intensity of inflammatory cells, number of osteoclasts (P < 0.0001) and cementoclasts (P < 0.0001), and significantly enhanced RNAm levels of RANK/RANKL/OPG, Runx2 and cathepsin K compared to day 0 (P < 0.0001). At 21 and 42 days, FGFR2 was also expressed on osteoblasts, fibroblasts and inside enlarged lacunae of cementocytes along with acute and chronic inflammatory cells (macrophages, plasma cells and neutrophils). At all periods and cells, FGFR2 expression was observed in the cell membrane and cytoplasm, but not in the nucleus. CONCLUSION: In mice, FGFR2 was not expressed in tissues surrounding healthy teeth but was expressed in apical periodontitis, specifically in the membrane and cytoplasm of osteoblasts, fibroblasts, lacunae of cementocytes, and acute and chronic inflammatory cells (macrophages, plasma cells and neutrophils). Its expression was correlated with the size of the periapical lesions.

Dental Caries, Developmental Defects of Enamel and Enamel Microhardness Associated with Genetic Polymorphisms in the RANK/RANKL/OPG System.

Purpose: Recent studies have suggested that disruptions in the RANKL/RANK/OPG system might be involved in enamel conditions. The aim of this study was to test whether genetic polymorphisms in RANK, RANKL and OPG are associated with dental caries, developmental defects of enamel (DDE) and enamel microhardness. Study design: Saliva samples were collected from two subsets for the purpose of genomic DNA extraction. In the first subset, composed of 248 children, dental caries and DDE were evaluated during their clinical examination. In the second subset, composed of 72 children, enamel samples from the buccal surface of primary teeth were used for enamel microhardness analysis. Genetic polymorphisms in RANK, RANKL and OPG were genotyped by real-time polymerase chain reactions in all samples from both populations. The chi-square test was used for dental caries and DDE analysis while, one-way ANOVA with Tukey's post-test was used for microhardness analysis. Hardy-Weinberg equilibrium was also calculated. The established alpha was 5%. Results: Caries experience analysis demonstrated a statistically-significant difference for OPG allele distribution in primary dentition (p=0.033). The studied polymorphisms in RANK, RANKL and OPG were not associated with DDE or enamel microhardness (p>0.05). Conclusion: The genetic polymorphism rs2073618 in OPG is associated with dental caries experience in primary dentition.

Alendronate inhibits osteocyte apoptosis and inflammation via IL-6, inhibiting bone resorption in periapical lesions of ovariectomized rats.

AIM: To evaluate the effect of alendronate (ALN) on the development of periapical lesions induced in ovariectomized rats. METHODOLOGY: Twenty-five rats were divided into three groups: sham (control), ovariectomy (OVX) and OVX + ALN. One day after OVX, animals from the OVX + ALN group received the medication via gavage. After 9 weeks, the first molars of all animals were submitted to periapical lesion induction. After 21 days, the animals were euthanized. Femurs were analysed for bone mineral density. The blocks of bone tissue containing the mandibular first molars were submitted to histotechnical processing and staining with haematoxylin and eosin (HE) for periapical lesion analysis under conventional microscopy. At the same time, the morphometric analysis of the periapical lesion area was performed in the fluorescence mode, as well as the histoenzimology for the quantification of osteoclasts and 4'-6-diamidino-2-phenylindole staining for the quantification of apoptotic osteocytes. In addition, the first maxillary molars were used for analysis of the gene expression of proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) and osteoclastogenesis markers (RANKL/OPG). The results were submitted to ANOVA and Kruskal-Wallis tests and Tukey and Dunn post-tests (significance level of 5%). RESULTS: Ovariectomy reduced bone mineral density of the femur, and treatment with ALN was able to prevent bone loss (P < 0.001). Regarding the microscopic analysis of the periapical region, the sham and OVX + ALN groups had moderately increased periodontal ligament and inflammatory infiltrate, while the OVX group had these parameters increased intensely. The periapical lesions of the OVX group were significantly larger in area in comparison to the other groups (P < 0.001). The OVX group had the largest amount of apoptotic osteocytes, and ALN was able to prevent the apoptosis of these cells, in addition to significantly reducing IL-6 expression (P < 0.05). OVX and ALN had no effect on RANKL/OPG expression and did not influence the number of osteoclasts around the periapical lesion (P > 0.05). CONCLUSION: The hypoestrogenic condition induced by OVX aggravated bone resorption, inducing the death of osteocytes and provoking larger periapical lesions. ALN treatment inhibited osteocyte apoptosis and inflammation via IL-6, inhibiting bone resorption in periapical lesions of ovariectomized rats.

Immunophenotypic quantification of M1 and M2 macrophage polarization in radicular cysts of primary and permanent teeth.

AIM: To quantify M1 and M2 macrophages in radicular cysts of permanent (n = 14 cases) and primary teeth (n = 15 cases). METHODOLOGY: All patients who attended the School of Dentistry Ribeirão Preto, University of São Paulo with primary teeth or permanent molars that were scheduled for extraction and fulfilled the inclusion criteria: absence of pain; presence/absence of fistulae; extensive coronal destruction due to caries lesions without possibility of restoration; pulp necrosis; radiographically visible apical periodontitis; and no previous treatment, were selected. The radicular cysts were removed and subsequently submitted to histopathologic analysis in order to classify the type of inflammatory infiltrate. In addition, CD68 (M1+, M2+) and CD163 (M1-, M2+) markers were quantified through an immunohistochemistry analysis. The data acquired were submitted to a Mann-Whitney test, with a 5% significance level. RESULTS: The patients had a mean age of 38.6 years and 5.9 years for cysts associated with permanent and primary teeth, respectively. In the histopathological analysis, no significant difference (P = 0.87) was found between radicular cysts in primary and permanent teeth regarding the intensity of the chronic inflammatory infiltrate. A significantly greater prevalence of M2 macrophages (P < 0.05) was observed in the lesions of both permanent and primary teeth, even though both M1 and M2 macrophages were detected. No significant difference (P > 0.05) was found for M1 and M2 macrophages associated with the cysts of primary and permanent teeth. CONCLUSION: M1 and M2 macrophages were present in radicular cysts associated with primary and permanent teeth, with a greater quantity of M2 cells. The immunophenotypic quantification of M1 and M2 macrophage polarization in radicular cysts associated with primary and permanent teeth were similar.

Effect of intercellular adhesion molecule 1 deficiency on the development of apical periodontitis.

AIM: To evaluate the specific role of ICAM-1 in host responses against endodontic infection. METHODS: Apical periodontitis was experimentally induced in the mandibular first molars of ICAM-1 knockout and wild-type (WT) mice by pulp exposure to the oral environment. At 7, 21 and 42 days following pulp infection, the animals were euthanized and the jaws were prepared for analysis under conventional and fluorescence microscopy (histopathologic and morphometric analysis), immunohistochemistry (polymorphonuclear leucocytes), enzyme histochemistry (osteoclasts and cementoclasts) and RT-PCR (IL-1 α, TNF-α, INF-γ, IL-10, RANK, RANKL and OPG). A generalized linear model with GLIMMIX procedure with Satterthwaite approximation method of degrees of freedom, Tukey-Kramer, pseudo-ranking nonparametric, Bonferroni-Holm multiple testing adjustment, analysis of variance (ANOVA) and the Tukey's multiple comparisons tests were used to evaluate the statistical differences between the groups using SAS 9.4 and the GraphPad Prism 5.0 software (α = 0.05). RESULTS: Compared to WT mice, ICAM-1 knockout mice had significantly greater bone resorption (P < 0.05), reduced recruitment of neutrophils to periapical inflammatory tissues (P < 0.05) and an increased number of fibroblasts (P < 0.05) at all experimental periods. The osteoclast number was significantly higher in ICAM-1 KO than that of WT animals at all times (P < 0.05), while there was no significant difference between the groups regarding cementoclasts. At day 21, the level of IL-1α, RANK, RANKL and IL-10 had increased significantly in tissues from ICAM-1 KO versus WT mice (P < 0.05), while no significant difference was observed in TNF-α and OPG levels (P > 0.05). Tissue levels of INF-γ were significantly lower in ICAM-1 KO than those in WT mice (P < 0.05). CONCLUSION: ICAM-1 deficiency impaired the host response against endodontic infection, resulting in increased tissue destruction.

Histopathological, histoenzymological, immunohistochemical and immunofluorescence analysis of tissue response to sealing materials after furcation perforation.

AIM: To evaluate in vivo tissue responses after sealing furcation perforations in dog's teeth with either Biodentine™, mineral trioxide aggregate (MTA) or gutta-percha, by means of histopathological, histoenzymological, immunohistochemical and immunofluorescence analysis. METHODOLOGY: After root canal treatment, perforations were created in the central region of the pulp chamber floor using a round diamond bur and filled with one or other of the materials. The animals were euthanized after 120 days, and the teeth (n = 30) were processed for histopathological analysis of new mineralized tissue formation and collagen fibre reinsertion, immunohistochemical analysis of osteopontin (OPN) and alkaline phosphatase (ALP) and immunofluorescence analysis for bone morphogenetic protein (BMP-2), cementum attachment protein (CAP), bone sialoprotein (BSP), osteocalcin (OCN) and cementum protein1 (CEMP1). Histoenzymology was performed for TRAP activity and osteoclast count. Data were analysed statistically (α = 0.05) using chi-square and Kruskal-Wallis tests. RESULTS: Gutta-percha did not induce mineralized tissue formation. MTA and BiodentineTM formed mineralized tissue in 88% and 92% of specimens, respectively, with no significant difference (P > 0.05). Gutta-percha was associated with scattered collagen fibres parallel to the perforations. Groups treated with MTA or BiodentineTM had partial fibre reinsertion perpendicular to the newly formed mineralized tissue. All materials induced OPN and ALP expression, weakest for gutta-percha and strongest for MTA (P < 0.05). Only MTA induced BMP-2, BSP, OCN, CAP and CEMP1 expression. Osteoclast counts were similar in all groups (P = 0.97). CONCLUSIONS: Mineral trioxide aggregate and BiodentineTM were biocompatible, with formation of mineralized tissue and partial reinsertion of collagen fibres. In addition, the participation of several molecules by which calcium silicate-based materials induce the formation of mineralized tissue were noted, with expression of ALP and OPN mineralization markers, without interference in the number of osteoclasts. Only MTA stimulated the expression of proteins associated with the formation of a cementum-like mineralized tissue.

Estrogen receptor gene is associated with dental fluorosis in Brazilian children.

OBJECTIVES: The aim of this study was investigate the association between genetic polymorphisms in ESR1, ESR2, and ESRRB and dental fluorosis (DF) in a well-characterized sample of children from Curitiba, Brazil. MATERIAL AND METHODS: From a representative sample of 538 children, 12-year-old were evaluated. DF was assessed in erupted permanent teeth by the Dean's index modified. Fourteen polymorphisms were selected in intronic and intergenic regions of ESR1, ESR2, and ESRRB and genotyped in genomic DNA source from saliva using TaqMan chemistry and end-point analysis. Allele and genotype distributions between DF and DF free groups were analyzed using the Epi Info 7.2. Chi-square or Fisher's exact tests at a level of significance of 5% and odds ratios calculations with 95% confidence intervals were used to determine the statistical associations. RESULTS: Among 538 children, 147 were DF and 391 were DF free. Genotype distribution for the polymorphism rs12154178 in ESR1 was different between the two groups (p = 0.037; OR = 0.91; CI = 0.67-1.22). The dominant model analysis (AA+AC vs. CC) demonstrated that CC is a protective factor for DF (p = 0.038; OR = 0.51, 0.27-0.97 95% CI). We did not find differences in frequency distributions in the other evaluated polymorphisms. CONCLUSION: This study provides evidence that ESR1 is associated with DF. CLINICAL RELEVANCE: Dental fluorosis is an important condition that affects the mineralized tissues of the teeth. In severe cases, the treatment takes time and is extremely costly. This research provides evidences that there are genetic factors involved in dental fluorosis and will help professionals to plan more precise strategies to reduce dental fluorosis occurrence.

One-session root canal treatment with antimicrobial photodynamic therapy (aPDT): an in vivo study.

AIM: To evaluate the response of the apical and periapical tissues of dog teeth with apical periodontitis after one-session root canal treatment with and without antimicrobial photodynamic therapy (aPDT) compared with the use of an intracanal dressing. METHODOLOGY: Sixty root canals with an induced periapical lesion were instrumented and assigned to three groups: I, two-session root canal treatment using antibacterial dressing with calcium hydroxide-based paste; II, one-session root canal treatment using aPDT; and III, one-session root canal treatment in which the root canals were filled immediately after biomechanical preparation. The animals were euthanized after a 90-day experimental period. The maxillas and mandibles with teeth were submitted to histotechnical processing and haematoxylin-eosin staining. Descriptive microscopic analysis of the apical and periapical region characteristics was performed, as well as morphometric assessment of the periapical lesion areas in fluorescence microscopy. Quantitative data were analysed statistically by the nonparametric Kruskal-Wallis test and Dunn's post-test (α = 0.05). RESULTS: Group I was characterized by progressive repair, with the presence of fibres, cells and blood vessels. Group II had periodontal ligaments with the presence of collagen fibres and residual inflammatory cells. Group III had a dense inflammatory infiltrate with extensive oedematous areas and fibrillar dissociation, suggesting a persistent inflammatory and resorptive condition. Regarding periapical lesion size, group I had significantly smaller lesions (P < 0.05) than groups II and III, which did not differ significantly from each other. CONCLUSION: Two-session root canal treatment using a calcium hydroxide-based dressing was associated with significantly smaller periapical lesions at 90 days and characterized by progressive repair.

Absence of interleukin 22 affects the oral microbiota and the progression of induced periapical lesions in murine teeth.

AIM: To evaluate the absence of IL-22 on the progression of periapical lesions in wild-type (WT) and IL-22 knockout (IL-22 KO) mice. METHODOLOGY: The evaluation of the oral microbial profile of mice was performed by Checkerboard DNA-DNA hybridization from saliva samples. Periapical lesions were induced in manbibular first molars by pulpal exposure and evaluated after 7, 21 and 42 days (n = 15). Haematoxylin-eosin-stained sections were analysed under conventional and fluorescence microscopy to evaluate the tissue features and size of periapical lesions and tartrate-resistant acid phosphatase histoenzymology (TRAP), Brown & Brenn staining and immunohistochemistry. The scores of the number of bacterial cells present in the oral cavity were analysed by the Mann-Whitney test, and the results and comparisons for periapical lesion size and number of osteoclasts were subjected to one-way anova and Bonferroni's post-test (α = 0.05). RESULTS: Significant differences were observed for bacterial load between the groups of animals for 6 bacterial species (P < 0.05), with five species found in higher levels in the WT group, and one in the IL-22 KO group. WT mice had significantly larger periapical lesions (P < 0.05) between 7 and 42 days and between 21 and 42 days, with an increase in the mean size and number of osteoclasts. IL-22 KO mice had an increase in periapical lesion size and number of osteoclasts between 7 and 21 days (P < 0.05). No differences were found between bacteria localization in the root canal system between the experimental groups. Small variations related to the location of immunostaining were found between the groups. CONCLUSION: This study revealed differences in the composition of oral microbiota between mice that may be taken into account in the susceptibility to infections and development of periapical lesions. The absence of IL-22 in mice resulted in smaller periapical lesions with fewer osteoclasts at the final experimental period, suggesting the participation of IL-22 in the host immune and inflammatory response to a periradicular infection.

MyD88 knockout mice develop initial enlarged periapical lesions with increased numbers of neutrophils.

AIM: To characterize the formation and progression of experimentally induced periapical lesions in teeth of MyD88 knockout (MyD88 KO) mice compared with wild-type (WT) mice. METHODOLOGY: Periapical lesions were induced in the mandibular first molars of 30 WT and 30 MyD88 KO mice. After 7, 21 and 42 days, the animals were euthanized and the mandibles were subjected to histotechnical processing. Histological sections were stained with haematoxylin and eosin (HE), TRAP histoenzymology, Brown and Brenn staining and immunohistochemistry (RANK, RANKL, OPG). Data were subjected to statistical analysis by the nonparametric Mann-Whitney and Kruskal-Wallis tests and the Dunn post-test, using the SPSS software, version 17.0 (α = 0.05). RESULTS: Regarding the periapical lesion size, the MyD88 KO group had significantly higher values than the WT group in the periods of 7 (P = 0.001) and 21 days (P = 0.05). A larger number of neutrophils in the MyD88 KO group were observed (P = 0.01 at 7 days, P = 0.004 at 21 days and P < 0.001 at 42 days). Regarding the number of osteoclasts, no statistically significant difference was observed between the groups at any of the experimental periods (P = 0.884 at 7 days, P = 0.506 at 21 days and P = 0.211 at 42 days). CONCLUSIONS: In the absence of MyD88, the animals had larger periapical lesions, with a severe inflammatory infiltrate and a significantly larger number of neutrophils.

The treatment of a maxillary lateral incisor with unusual morphology with long-term follow-up.

AIM: To report the treatment of an unusual combination of one dens evaginatus and two dens invaginatus in a single tooth and its healing outcome after 10 years. SUMMARY: The long-term outcome of a maxillary lateral incisor with dens evaginatus combined with two Oehlers type II dens invaginatus and a large periradicular lesion in an 11-year-old female treated endodontically and restoratively is described. The endodontic treatment included intracanal medication with calcium hydroxide and canal filling using a thermoplastic root canal filling technique. The crown was restored with conventional composite resin. During periodic clinical and radiographic follow-up, the patient remained symptom free, and the periradicular region was completely healed, meeting both aesthetic and functional expectations after 10 years. KEY LEARNING POINTS: The co-occurrence of dens evaginatus and two dens invaginatus in the same tooth is an unusual finding that compromises aesthetics and predisposes the patient to dental pulp infection. The complex morphology observed in this case represented both endodontic and restorative challenges.

Pulpal and periapical response after restoration of deep cavities in dogs' teeth with Filtek Silorane and Filtek Supreme XT systems.

OBJECTIVE: This study evaluated, histopathologically, the pulpal and periapical response to a silorane-based resin (Filtek Silorane) and a methacrylate-based nanoparticle resin (Filtek Supreme XT) in deep cavities in dogs, having zinc oxide and eugenol-based cement (ZOE) as a control. METHODS: The tooth/bone blocks were collected after 10 and 90 days and processed for microscopic analysis of the dentin, pulp, and periapical tissues using a score system. Data were analyzed statistically by Kruskal-Wallis and Dunn post-test (α=0.05). RESULTS: At 10 days, the pulp, connective tissue, and periodontal ligament showed normal characteristics. No resorption areas were observed. Both resins caused significantly less (p<0.05) periapical and pulpal inflammatory response than ZOE. At 90 days, for all materials, the connective pulp tissue was healthy and dense, with a normal blood vessel system. The apical and periapical region had normal structure and thickness. CONCLUSIONS: The use of the Filtek Silorane and the Filtek Supreme XT resins caused no adverse pulpal and periapical reactions after restoration of deep dentin cavities in vivo.

Preliminary study of the inflammatory response to subcutaneous implantation of three root canal sealers.

AIM: To evaluate the kinetics of the inflammatory tissue response to three root canal sealers using a physicochemical method for quantification of the enhanced vascular permeability and histopathological analysis. METHODOLOGY: Twenty-eight male Wistar rats randomly assigned to four groups according to the evaluation periods (1, 3, 7 and 14 days) were used to assess the vascular permeability and histopathological reaction to RoekoSeal, AH Plus and Sealapex (new formulation) sealers, using saline and Chloropercha as negative and positive controls, respectively. Seven rats were sacrificed per period. The biocompatibility of the sealers was evaluated spectrophotometrically and histopathologically. RESULTS: At day 14, Sealapex produced significantly more inflammatory exudate than AH Plus and RoekoSeal (P < 0.05); however, there was no significant difference between AH Plus and RoekoSeal (P > 0.05). Sealapex (new formulation) was the most irritating sealer, producing severe inflammation with the presence of multinucleated giant cells. RoekoSeal was the most biocompatible sealer, producing the least amount of inflammatory exudate. CONCLUSIONS: RoekoSeal root canal sealer was biocompatible when implanted in connective tissue.

Accuracy of the iPex multi-frequency electronic apex locator in primary molars: an ex vivo study.

AIM: To evaluate ex vivo the accuracy of the iPex multi-frequency electronic apex locator (NSK Ltd, Tokyo, Japan) for working length determination in primary molar teeth. METHODOLOGY: One calibrated examiner determined the working length in 20 primary molar teeth (total of 33 root canals). Working length was measured both visually, with the placement of a K-file 1 mm short of the apical foramen or the most coronal limit of root resorption, and electronically using the electronic apex locator iPex, according to the manufacturers' instructions. Data were analysed statistically using the intraclass correlation (ICC) test. RESULTS: Comparison of the actual and the electronic measurements revealed high correlation (ICC = 0.99) between the methods, regardless of the presence or absence of physiological root resorption. CONCLUSIONS: In this laboratory study, the iPex accurately identified the apical foramen or the apical opening location for working length measurement in primary molar teeth.

Nitric oxide detection in cell culture exposed to LPS after Er:YAG laser irradiation.

AIM: To evaluate in vitro the effect of calcium hydroxide [Ca(OH)(2)] and Er:YAG laser on bacterial endotoxin [also known as lipopolysaccharide (LPS)] as determined by nitric oxide (NO) detection in J774 murine macrophage cell line culture. METHODOLOGY: Samples of LPS solution (50 microg mL(-1)), Ca(OH)(2) suspension (25 mg mL(-1)) and LPS suspension with Ca(OH)(2) were prepared. The studied groups were: I - LPS (control); II - LPS + Ca(OH)(2); III - LPS + Er:YAG laser (15 Hz 140 mJ); IV - LPS + Er:YAG laser (15 Hz 200 mJ); V - LPS + Er:YAG laser (15 Hz 250 mJ), VI - Pyrogen-free water; VII - Ca(OH)(2). Murine macrophage J774 cells were plated and 10 microL of the samples were added to each well. The supernatants were collected for NO detection by the Griess reaction. Data were analysed statistically by one-way anova and Tukey's test at 5% significance level. RESULTS: The mean and SE (in micromol L(-1)) values of NO release were: I - 10.48 +/- 0.58, II - 6.41 +/- 0.90, III - 10.2 +/- 0.60, IV - 8.35 +/- 0.40, V - 10.40 +/- 0.53, VI - 3.75 +/- 0.70, VII - 6.44 +/- 0.60; and the values for the same experiment repeated after 1 week were: I - 21.20 +/- 1.50, II - 9.10 +/- 0.60, III - 19.50 +/- 1.00, IV - 18.50 +/- 0.60, V - 21.30 +/- 0.90, VI - 2.00+/- 0.20, VII - 6.80 +/- 1.70. There was no significant difference (P > 0.05) between the control and the laser-treated groups (III, IV and V), or comparing groups II, VI and VII to each other (P > 0.05). Group I had significantly higher NO release than group II (P < 0.05). Groups II and VI had similar NO release (P > 0.05). CONCLUSIONS: Calcium hydroxide inactivated the bacterial endotoxin (LPS) whereas none of the Er:YAG laser parameter settings had the same effectiveness.

Biofilm on the apical region of roots in primary teeth with vital and necrotic pulps with or without radiographically evident apical pathosis.

AIM: To evaluate, by scanning electron microscopy (SEM), the presence of biofilms on the external surfaces of the apical third of roots of human primary teeth with vital or necrotic pulps with and without radiographically evident periradicular pathosis. METHODOLOGY: Eighteen teeth were selected: group I - normal pulp (n = 5), group II - pulp necrosis without radiographic evidence of periapical pathosis (n = 7) and group III - pulp necrosis with well-defined radiographic periapical pathosis (n = 6). After extraction, the teeth were washed with saline and immersed in 0.03 g mL(-1) trypsin solution for 20 min. The teeth were then washed in sodium cacodilate buffer and stored in receptacles containing modified Karnovsky solution. The teeth were sectioned, dehydrated in an ethanol series, critical-point dried with CO(2), sputter coated with gold and the external root surface in the apical third examined by SEM. RESULTS: In the teeth of groups I and II, the apical root surfaces were covered by collagen fibres, with no evidence of bacteria (100%). In the teeth of group III, the root apices had no collagen fibres but revealed resorptive areas containing microorganisms (cocci, bacilli, filaments and spirochetes) in all cases (100%). CONCLUSION: Microorganisms organized as biofilms on the external root surface (extraradicular infection) were detected in primary teeth with pulp necrosis and radiographically visible periapical pathosis.

Ex vivo evaluation of the accuracy of two electronic apex locators during root canal length determination in primary teeth.

AIM: To evaluate ex vivo the accuracy of two electronic apex locators during root canal length determination in primary incisor and molar teeth with different stages of physiological root resorption. METHODOLOGY: One calibrated examiner determined the root canal length in 17 primary incisors and 16 primary molars (total of 57 root canals) with different stages of root resorption based on the actual canal length and using two electronic apex locators. Root canal length was measured both visually, with the placement of a K-file 1 mm short of the apical foramen or the apical resorption bevel, and electronically using two electronic apex locators (Root ZX II--J. Morita Corp. and Mini Apex Locator--SybronEndo) according to the manufacturers' instructions. Data were analysed statistically using the intraclass correlation (ICC) test. RESULTS: Comparison of the actual root canal length and the electronic root canal length measurements revealed high correlation (ICC = 0.99), regardless of the tooth type (single-rooted and multi-rooted teeth) or the presence/absence of physiological root resorption. CONCLUSIONS: Root ZX II and Mini Apex Locator proved useful and accurate for apex foramen location during root canal length measurement in primary incisors and molars.

Microbial contamination of toothbrushes and their decontamination.

PURPOSE: The objective was to determine the level of contamination of toothbrushes by mutans streptococci using microbiological identification, to access the bacterial contamination using scanning electron microscopy (SEM) and to evaluate the efficacy of two toothbrush disinfectants. METHODS: Nineteen children used their toothbrushes once a day, for five consecutive days. The toothbrushes were then immersed into disinfectant solutions for 20 h: Group I--0.12% chlorhexidine gluconate; Group II--1% sodium hypochlorite; Group III--sterile tap water. They were then placed into test tubes containing CaSa B, for 3 to 4 days at 37 degrees C. The number of MS cfu was counted and the toothbrushes were submitted to SEM analysis. RESULTS: There was no bacterial growth in Groups I and II; Group III showed MS growth (range, 21 to 120 cfu). Scanning electron microscopy showed biofilm formation on toothbrush bristles. CONCLUSION: Immersion in 0.12% chlorhexidine gluconate and 1% sodium hypochlorite are efficient methods for toothbrush disinfection.

Connective tissue responses to calcium hydroxide-based root canal medicaments.

AIM: The objective of the present study was to evaluate the tissue inflammatory response induced by calcium hydroxide pastes, with or without paramonochlorophenol and camphor. METHODOLOGY: Isogenic BALB/c mice were inoculated into the subcutaneous tissue with either 0.1 mL of a suspension of Calen, Calen with camphorated paramonochlorophenol, Calen with paramonochlorophenol, Calasept paste or phosphate-buffered saline (control). After 6, 12 and 24 h and 2, 3, 5, 7 and 15 days, three animals in each group were sacrificed and the excised lesions processed for histopathological evaluation of the inflammatory response. Events monitored and graded included the assessment of vascular congestion, oedema, haemorrhage, inflammatory infiltrate, necrosis and tissue repair. RESULTS: The pastes induced an inflammatory response at every observation period, although the intensity, duration and extension of inflammation varied. Calen paste always produced an initial short-term inflammatory response whilst the other pastes produced extended reactions. All pastes allowed repair to take place by the end of the experimental period, although the speed of this process varied between the materials. Calen presented the best biocompatibility; the phenolic compound caused greater tissue response, which was even more severe in the absence of camphor. Calasept paste was damaging and the repair process slower. CONCLUSIONS: All calcium hydroxide formulations caused an inflammatory response. The severity and longevity of the responses varied between pastes as a result of the various antiseptic agents. Although irritating, repair was apparent with all formulations.