Effect of ethylenediaminetetraacetic acid irrigation on immune-inflammatory response in teeth submitted to regenerative endodontic therapy.

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-ß 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-ß 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.

Immunological profile of teeth with inflammatory periapical disease from chronic liver disease patients.

AIM: To evaluate the mRNA expression levels of the cytokines interferon-γ, tumour necrosis factor-α, interleukin (IL)-1ß, 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-1ß, 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.

Immunological profile of periapical endodontic infections from HIV- and HIV+ patients.

AIM: To evaluate CD4(+) CD28(+) and CD8(+) T-cell genes and the gene expression of IFN-γ, TNF-α, IL-1-ß, 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-ß and CCL5. However, in HIV+ patients an increase in cytokines IFN-γ, IL-1-ß, 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.

Microbiologic profile of endodontic infections from HIV- and HIV+ patients using multiple-displacement amplification and checkerboard DNA-DNA hybridization.

OBJECTIVE: To compare the microbiota of endodontic infections in necrotic pulp from HIV-negative and HIV-positive subjects. MATERIALS AND METHODS: Root canal samples from necrotic pulp were collected from 40 HIV- and 20 HIV+ subjects. Pulps were amplified using multiple displacement amplification (MDA). Then, checkerboard DNA-DNA hybridization was employed to assess the levels of 107 microbial taxa. The percentage of DNA probe count and the percentage of teeth colonized by each test species were investigated. Significant differences between groups regarding proportions of taxa and prevalence of the test species were sought using the Mann-Whitney test and the Chi-square analysis, respectively. RESULTS: The most prevalent taxa detected were Dialister pneumosintes, Stenotrophomonas maltophilia, Streptococcus sobrinus, Corynebacterium diphteriae, and Helicobacter pylori among HIV- subjects and D. pneumosintes, Prevotella tannerae, Porphyromonas gingivalis, Parvimonas micra, Prevotella nigrescens, and Corynebacterium diphtheriae among HIV+ individuals. D. pneumosintes, C. diphtheria, and C. albicans were the most abundant species in the HIV- group, whereas the predominant taxa in HIV+ samples were P. tannerae, D. pneumosintes and Olsenella uli. P. tannerae, O. uli, Veilonella dispar, Bacteroides fragilis, and Actinomyces meyeri were significantly more abundant in HIV+ samples. CONCLUSIONS: There were significant differences in the prevalence and proportions of specific microbial taxa between HIV- and HIV+ individuals. The root canal microbiota may represent a reservoir of important oral and medical pathogens, mainly in HIV+ individuals.

Use of multiple-displacement amplification and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections.

Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. Sixty-six samples were collected from teeth with endodontic infections. Nonamplified and amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, percentages of DNA probe counts, and percentages of teeth colonized for each species in amplified and nonamplified samples were computed. Significance of differences for each species between amplified and nonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. The amount of DNA in the samples ranged from 6.80 (+/- 5.2) ng before to 6.26 (+/- 1.73) mug after MDA. Seventy of the 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels of >10(4) in both amplified and nonamplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89 and 100% of samples. The mean number of species at counts of >10(4) in amplified samples was 51.2 +/- 2.2 and in nonamplified samples was 14.5 +/- 1.7. The endodontic microbiota was far more complex than previously shown, although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris, and A. baumannii.