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Aim: To analyse longitudinally the immune-inflammatory response in teeth of mice that underwent a regenerative protocol with or without the use of ethylenediaminetetraacetic acid (EDTA) to irrigate the root canal system., Methodology: First maxillary molars of mice were devitalized using size 10 and 15 files. Teeth were divided into the following groups: Empty - the canals were left empty; Blood Clot (BC) - the canals were filled with a blood clot; and EDTA + Blood - the canals were irrigated with 0.06 mL of 17% EDTA for 1 min and filled with a blood clot. Access cavities were restored with Coltosol ® . Animals were sacrificed at 7, 14 or 21 days after the operative procedures, and teeth were collected. RNA was extracted, mRNA expression of the cytokines IGF, NGF, IL-1α, IL-10, TGF and VEGF was assessed using real-time PCR, and the anova Kruskal-Wallis test was used., Results: IL-1 mRNA expression was significantly higher in the EDTA + BC group than in the Empty and BC groups at the 7th and 14th days of evaluation (P < 0.05). IL-10 mRNA expression was similar across the three groups at all time periods. TGF-β mRNA expression in the EDTA + BC group was significantly higher on the 7th and 21st days than on the 14th (P < 0.05); at day 21, TGF-β mRNA expression was similar between the BC and EDTA + BC groups but significantly higher than in the Empty group (P < 0.05). IGF mRNA expression was significantly higher in the EDTA + BC group than in the other groups at all time periods. VEGF mRNA expression remained unchanged throughout the experimental period in all groups (P > 0.05). NGF mRNA expression was similar amongst all groups at the 7th and 21st days (P > 0.05). At the 14th day, however, there was a significant increase in NGF mRNA expression in the EDTA + Blood group (P < 0.05) when compared with the expression in the other groups., Conclusion: EDTA promoted increased expression of factors that have the potential to improve the outcome of regenerative endodontic treatment., (© 2019 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the mRNA expression levels of the cytokines interferon-γ, tumour necrosis factor-α, interleukin (IL)-1β, IL-10, IL-6, VEGF, and AGT and the chemokine CCL2/MCP-1 in periapical interstitial fluid associated with root canal infections before and after the reduction of the bacterial load using a cleaning procedure., Methodology: The case group included 11 patients with chronic liver disease, and the control group included 11 healthy patients. Clinical samples were taken from teeth with pulp necrosis. After cleaning and drying the canal, three paper points were introduced into the root canal and passed through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later to characterize those gene expression levels using real-time PCR. The data were subjected to the Shapiro-Wilk and the Wilcoxon tests., Results: In the control group, significantly increased expression of the pro-inflammatory cytokines IFN-γ and TNF-α was observed in teeth with restrained bacterial loads (day 7) (P < 0.05). Similarly, increased TNF-α expression was found on day 7 in the liver group (P < 0.05). No differences were observed in the expression levels of the IL-1β, IL-10 and, IL-6, MCP-1/CCL-2 and VEGF between the first collection (day 0) and second collection (day 7), over time in either group., Conclusion: Chronic liver disease patients exhibited sufficient immunologic ability showing relatively similar expression levels of cytokines, chemokines and angiogenic factors in periapical samples compared with the responses from no-chronic liver disease patients. The outcomes of this study suggest that liver impairment did not compromise the periapical immune response., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the expression of TNF-α, IL-6, IFN-γ, TGF-β, IL-4, IL-10, RANKL, RANK and OPG on mouse calvarial bone treated with MTA, Geristore ® and Emdogain ® ., Methodology: Bone wounds were made on the heads of C57BL/6 mice, breaking the periosteum and the cortical surface of the calvaria. Each repair agent was inserted into sectioned Eppendorf microtubes and placed on the bone wound, and soft tissues were sutured. At 14 and 21 days, animals were sacrificed and the treated region was dissected. The calvaria bone was removed, and RNA was extracted. mRNA expression of the aforementioned cytokines was assessed using real-time PCR. Data were analysed by nonparametric methods, including the Mann-Whitney and Kruskal-Wallis tests (P < 0.05)., Results: Following treatment with Emdogain ® and MTA, mRNA expression of RANKL, RANK and OPG increased significantly (P < 0.05) between days 14 to 21. Geristore ® did not alter the basal expression of these mediators during the same period of evaluation. Whilst treatment with Emdogain ® did cause a significant increase in TNF-α mRNA expression between days 14 and 21 (P < 0.05), treatment with MTA did not alter the basal expression of this cytokine at either experimental time point. However, TNF-α mRNA expression was down-regulated significantly at day 21 (P < 0.05) when Geristore ® was applied. A significant increase in the mRNA expression of IL-6, TGF-β, IL-10, IL-4 and IFN-γ was observed with Emdogain ® and MTA treatment between days 14 to 21, whereas Geristore ® reduced significantly the expression of IL-6, TGF-β and IL-4 (P < 0.05)., Conclusion: The clinical indication of these repair agents depends on the root resorption diagnosis. Whilst MTA and Emdogain ® induce a pro- and anti-inflammatory response early and late, respectively, Geristore ® was not associated with an inflammatory reaction when compared with both repair agents., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate CD4(+) CD28(+) and CD8(+) T-cell genes and the gene expression of IFN-γ, TNF-α, IL-1-β, IL-17A, IL-10, CCL-2/MCP-1, CCL-4, CCL-5 (RANTES), CXCR4, CCR5 and RANKL from cells in the periapical interstitial fluid from root canal infections in healthy patients (HIV-) and HIV-positive individuals (HIV+)., Methodology: Subjects included 20 HIV- and 23 HIV+ patients referred to the School of Dentistry at the Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brazil). Almost all HIV+ patients were undergoing highly active antiretroviral therapy (HAART). Clinical samples were taken from teeth with pulp necrosis, and no patients had acute periapical symptoms at the time of the appointments. After cleaning and drying, 3 paper points were introduced into the root canal, passing passively through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions using real-time PCR., Results: Significantly higher levels of CD4(+) CD28(+) and CD8(+) T cells in teeth with restrained bacterial loads (second collection) compared with the first collection were observed in both HIV- and HIV+ samples. In HIV- patients, an increase in IL-10 and CXCR4 expression was demonstrated as well as a decrease in pro-inflammatory cytokines such as RANKL, IFN-γ, IL1-β and CCL5. However, in HIV+ patients an increase in cytokines IFN-γ, IL-1-β, TNF-α and IL-17A, and chemokines CCL-2, CXCR4 and CCR5 were observed. The chemokine CCL-5 was not detected in HIV+ individuals., Conclusions: These findings suggest that after reducing the root canal bacterial load in HIV- individuals an anti-inflammatory response is generated whilst in HIV+ patients a pro-inflammatory response is sustained in the periapical area., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To test the effects of a mineral trioxide aggregate-based sealer (MTA Fillapex(®)) and MTA (MTA-Ângelus(®)) on viability and on the production of cytokines, reactive oxygen species (ROS) and nitrogen species (NO) by M1 and M2 inflammatory macrophages., Methodology: M1 (from C57BL/6 mice) and M2 (from BALB/c mice) peritoneal inflammatory macrophages were obtained and cultured in vitro in the presence of original and diluted extracts of MTA and MTA Fillapex (FLPX). The cell viability, ROS release and the release of tumour necrosis factor-a, interleukin (IL)-12, IL-10 and NO in response to stimulation with interferon-γ and Fusobacterium nucleatum or Peptostreptococcus anaerobius were evaluated. The data were analysed using the Mann-Whitney test and Student's t-test., Results: Fillapex was cytotoxic at the highest concentrations (1:1;1:2) and decreased the viability (P < 0.05) of both macrophage types (<20%). MTA did not interfere with cellular viability. FLPX inhibited the release of ROS and decreased NO release in F. nucleatum and P. anaerobius -stimulated M1 and M2 macrophages (≤25 μ mol L(-1)). F. nucleatum-stimulated M2 macrophage cultures released lower levels of TNF-α when FLPX was added (≤1 ng mL(-1)). M2 macrophages released higher (>5 ng mL(-1)) levels of IL-10 than M1 macrophages. Only M1 macrophage cultures produced IL-12p70., Conclusions: Fillapex impaired effector immune responses during inflammation (M1 macrophages), as well as during healing (M2 macrophages) responses., (© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To examine cytokine expression profiles during periapical lesion development in response to synergetic human pathogens in a gnotobiotic mouse model., Methodology: Human strains of Fusobacterium nucleatum and Peptostreptococcus prevotii were inoculated into the root canals of germ-free mice in either mono- or bi-association. Animals were killed 7 and 14 days after infection, and periapical tissues were collected. mRNA expression of the cytokines IFN-γ, TNF-α, Receptor activator of nuclear factor kappa-B ligand (RANKL), IL-10, IL-4 and transforming growth factor β (TGF-β) was assessed using real-time PCR. Levene's test was used to assess the equality of variance of the data, whereas a t-test for independent samples was used to evaluate the significance of the differences between groups (P < 0.05)., Results: The mRNA expression of IFN-γ and TNF-α was up-regulated by F. nucleatum during the acute (day 7) and chronic phase (day 14) of periapical lesion development. However, in bi-infection the expression of IFN-γ and TNF-α were effectively absent at both time-points. RANKL mRNA expression was down-regulated during dual infection at the chronic phase. As IL-4 expression was similar at both time-points, IL-4 does not appear to be involved in the periapical response to these bacterial strains. IL-10 was up-regulated during the chronic phase by mono-infection with either F. nucleatum or P. prevotii. Dual infection increased TGF-β mRNA expression on day 7, which paralleled the decrease in IFN-γ and TNF-α mRNA levels at the same time-point. F. nucleatum increased TGF-β mRNA expression during the chronic phase., Conclusion: Cytokine profiles depend on the nature of the bacterial challenge. Both TGF-β and IL-10 appeared to be regulating the proinflammatory cytokine responses at both time-points of the periapical immune response., (© 2012 International Endodontic Journal.)

Aims: To evaluate the microbiota of endodontic infections in deciduous teeth by Checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA)., Methodology: Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interradicular bone resorption were collected and 32 were analysed, with three individuals contributing two samples; these were MDA-amplified and analysed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species and the mean proportion of each bacterial taxon present across all samples., Results: The mean amount of DNA in the samples prior to amplification was 5.2 (±4.7) ng and 6.1 (±2.3) μg after MDA. The mean number of species detected per sample was 19 (±4) (range: 3-66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples., Conclusion: Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp. were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens amongst the most prominent species detected., (© 2010 International Endodontic Journal.)

Aim: To assess the influence of co-culture with mineral trioxide aggregate (MTA) on phagocytosis and the production of reactive oxygen intermediates (ROI) and nitrogen (NO) species and the arginase activity by M1 and M2 peritoneal macrophages., Methodology: Cellular viability, adherence and phagocytosis of Saccharomyces boulardii were assayed in the presence of MTA. Macrophages were stimulated with zymosan for ROI assays and with Fusobacterium nucleatum and Peptostreptococcus anaerobius and IFN-gamma for NO production and arginase activity, when in contact with capillaries containing MTA. Data were analysed by T, anova, Kruskall-Wallis and Mann-Whitney tests., Results: M2 macrophages displayed greater cellular viability in polypropylene tubes, greater ability to ingest yeast and smaller production of ROI and higher arginase activity when compared with M1 macrophages. Both macrophages, M1 and M2, presented similar cell adherence and NO production. The addition of bacterial preparations to macrophages interfered with NO and arginase productions. MTA did not interfere with any of the parameters measured., Conclusions: Phagocytosis and the ability of the two macrophage subtypes to eliminate microbes were not affected by MTA.

Aim: The objective of the present study was to assess the efficacy of 2.5% sodium hypochlorite and 2.2% glutaraldehyde ('Cidex') as sterilizing agents for gutta-percha cones. The efficacy of storage of gutta-percha cones in the presence or absence of paraformaldehyde was also evaluated., Methodology: Gutta-percha cones artificially contaminated with a suspension of Bacillus stearothermophilus (ATCC/7953) were treated with either 2.2% glutaraldehyde for 10, 15, 30 and 60 min and 10 and 12 h, or 2.5% sodium hypochlorite for 5, 10 and 15 min. The cones were then incubated in thioglycollate medium for the determination of microbial growth. In parallel, additional sterile gutta-percha cones were stored in sealed containers with or without paraformaldehyde tablets for 30 days. The containers were opened 30 min a day and exposed to the environment of a functioning dental clinic. Twelve cones were removed weekly from the containers to determine whether contamination had occurred., Results: The results showed that 2.5% sodium hypochlorite was effective after 5, 10 and 15 min, whereas 10 and 12 h contact with 2.2% glutaraldehyde was necessary to obtain sterilization. There was no contamination of the gutta-percha cones when stored with or without paraformaldehyde., Conclusions: Sodium hypochlorite (2.5%) and 2.2% glutaraldehyde ('Cidex') proved to be effective as sterilizing agents for gutta-percha cones, with sodium hypochlorite requiring shorter periods of use. No difference was observed between the two methods of cone storage.