Effect of chronic unpredictable stress on the progression of experimental apical periodontitis in rats.

AIM: To establish an experimental model combining chronic stress and apical periodontitis by assessing the development of periapical lesions in rats in three different time points. METHODOLOGY: Forty-eight male Wistar rats were randomly assigned into two equal groups: Apical periodontitis (AP) and AP + Stress (AP + S). The animals of the AP group were not exposed to stressful conditions whereas the AP + S group were exposed to a variety of stressors on a daily basis until the end of the experiment. After three weeks of chronic unpredictable stress, apical periodontitis was induced in both groups by exposing the pulpal tissue of the mandibular first molar to the oral environment. Each group was further subdivided into three subgroups according to the euthanasia period: 14, 21 and 28 days after pulp exposure. The animals were weighed, and the blood was collected for corticosterone serum dosage by radioimmunoassay. The mandibles were removed and submitted to histopathological and microtomography analyses to assess the inflammatory response and the progression of periapical lesions. Comparisons between the AP and AP + S groups were performed using Student's t-test and Mann-Whitney U-test for parametric and nonparametric data, respectively. The one-way anova test followed by Tukey's test (parametric data) and Kruskal-Wallis followed by Dunn's test (nonparametric data) were used for comparisons between the three time points within the same group (P < 0.05). RESULTS: The AP + S group had a significantly lower average percentage of weight gain at 14 days and 21 days after AP induction (P < 0.05). Significantly higher levels of corticosterone were found in the AP + S group at 21 days (P < 0.05). The AP + S group had a significantly greater intensity and extension of inflammatory infiltrate with larger areas of bone loss compared to the AP groups at 21 days (P < 0.05). The volume of the periapical lesions in the AP + S group was significantly larger than that of the AP group 21 days following pulp exposure (P < 0.05). CONCLUSIONS: The chronic unpredictable stress model applied for 6 weeks exacerbated the inflammatory response and increased bone loss associated with AP, especially 21 days after its induction. This model appears to be suitable for investigating the bidirectional relationship between apical periodontitis and chronic stress.

Clinical influence of calcium hydroxide and N-acetylcysteine on the levels of resolvins E1 and D2 in apical periodontitis.

AIM: To investigate the presence of resolvins E1 (RvE1) and D2 (RvD2) in teeth with primary endodontic infections and apical periodontitis, and to assess the influence of calcium hydroxide medication [Ca(OH)2 ], in association with 2% chlorhexidine gel (2% CHX gel), and N-acetylcysteine (NAC) on the levels of RvE1 and RvD2 in periapical tissues. METHODOLOGY: Thirty-six single-rooted teeth with primary endodontic infections and apical periodontitis were selected and randomly divided into three groups according to the medication: [Ca(OH)2 ] + saline solution (SSL) [Ca(OH)2  + SSL group] (n = 12), Ca(OH)2  + 2% chlorhexidine gel [Ca(OH)2  + 2% CHX gel group] (n = 12) and NAC [NAC group] (n = 12). Samples were collected from the periapical interstitial fluid at two different sampling times: before (S1) and after 14 days of intracanal medications (S2). Resolvins were measured using the enzyme-linked immunosorbent assay. Data were analysed using paired t-test, Wilcoxon test and Kruskal-Wallis test, followed by Dunn's post hoc test; all statistical tests were performed at a significance level of 5%. RESULTS: RvE1 and RvD2 were detected in 100% of the samples (36/36) at S1 and S2. Ca(OH)2 medication did not increase the levels of RvE1 or RvD2 (both P > 0.05); however, NAC significantly increased the levels of RvE1 and RvD2 after 14 days of treatment (P < 0.05). CONCLUSIONS: RvE1 and RvD2 were detected in periapical tissues from teeth with root canal infections. Moreover, calcium hydroxide medication did not increase the levels of resolvins in apical periodontitis. In contrast, the use of NAC intracanal medication significantly increased the levels of RvE1 and RvD2 after 14 days of treatment.

Comparison of cytotoxicity, genotoxicity and immunological inflammatory biomarker activity of several endodontic sealers against immortalized human pulp cells.

AIM: To establish an SV40 T-Ag-transfected cell line of human pulp-derived cells in order to compare the cytotoxicity, genotoxicity and to investigate the activities of immunological biomarkers of several endodontic sealers. METHODOLOGY: Primary human pulp cells and transfected cells were cultured. Cell morphology and proliferation were analysed, and the expression of cell-specific gene transcripts and proteins was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining phenotypic characteristics from the primarily cells tested. The SV40 T-Ag-transfected cells were cultured and stimulated by sealers (Apexit Plus, Real Seal, AH Plus, and EndoREZ) to evaluate the cytotoxicity and genotoxicity by MTT and MTN assays, respectively. Immunological inflammatory biomarkers (IL6, IL8 and TNF-α) were determined by ELISA assay. The differences between median values were statistically analysed using Kruskal-Wallis and Dunn's tests at 5% significance level. RESULTS: The cytotoxicity assay revealed that multimethacrylate (Real Seal) was the most cytotoxic sealer (P < 0.05) and exhibited the highest inflammatory potential against the SV40 T-Ag-transfected cells (P < 0.05). All root canal sealers tested were able to stimulate the immortalized pulp cells to produce IL-6, IL-8 and TNF-α, with differences in relation to the control group (P < 0.05). Higher levels of IL-6, IL-8 and TNF-α were found in cell supernatant after stimulation with multimethacrylate (Real Seal) compared to all other sealers tested (P < 0.05). No differences were found comparing epoxy resin-based sealer (AHPlus), single-methacrylate sealer (EndoREZ) and calcium hydroxide-based sealer (Apexit Plus), regardless of the cytokine investigated (all P > 0.05). CONCLUSIONS: A SV40 T-Ag-transfected cell line of human pulp-derived cells was established. The methacrylate resin-based sealer (Real Seal) exhibited the greatest cytoxicity and inflammatory potential against immortalized pulp cells compared to an epoxy resin-based sealer (AH Plus), a methacrylate-based sealer (EndoRez) and a calcium hydroxide-based sealer (Apexit).