Comparison of the Physicochemical Properties, Antimicrobial Action, and Cytotoxicity of Ambroxol Hydrochloride, N-acetylcysteine, and Calcium Hydroxide Pastes.

OBJECTIVE: To evaluate the solubility, pH, antimicrobial action, and cytotoxicity of ambroxol hydrochloride (AMB), N-acetylcysteine (NAC), and calcium hydroxide (CH) pastes for use as intracanal medications. METHODS: Solubility was determined by micro-CT, based on the paste volume remaining after immersion in water for 7 days. pH was measured by immersing acrylic tubes containing the pastes in ultrapure water and then measuring pH after 3 hours, 3 days, and 7 days. Antimicrobial action against Enterococcus faecalis was assessed based on the percentage of living cells, using the live/dead staining method under confocal microscopy. Cytotoxicity was assessed based on the cell viability of L929 fibroblast-like cells after 6, 24, and 48 hours. Cytotoxicity data were compared using the ANOVA and Tukey tests, and the antimicrobial data were compared using the Kruskal-Wallis and Dunn tests. The significance level used was 5% (α=0.05). RESULTS: The solubility values for all the study groups were significantly different (P<0.05), where the highest values were for NAC, followed by AMB, and then CH. Likewise, the pH levels were all significantly different (P<0.05), where NAC and AMB levels were acidic, and CH levels were alkaline. The antimicrobial action of AMB was significantly higher than that of CH (P<0.05), and that of NAC was also higher than that of CH, albeit not significantly. AMB and NAC were more cytotoxic than CH, and higher dilutions of CH promoted higher cell viability levels than lower dilutions of the same paste (P<0.05). CONCLUSION: The NAC and AMB pastes were more soluble and cytotoxic than the CH paste and had acidic pH levels. The AMB paste displayed the highest antimicrobial action against Enterococcus faecalis biofilm.

Biological and antimicrobial properties of the association Ambroxol and a water-soluble viscous liquid as a vehicle for a tricalcium silicate-based sealer.

This study aimed to investigate the antimicrobial and biological properties of Ambroxol associated with glycerin (GLI), propylene glycol (PG), and polyethylene glycol (PEG) as a possible vehicle for an experimental tricalcium silicate sealer, with the intention of developing a new biomaterial. Mouse undifferentiated dental pulp cells (OD-21) were cultured, and the effects of different association on cell proliferation and inflammatory cytokine production were investigated. Antimicrobial adhesion of Enterococcus faecalis to setting sealers at 2 h was evaluated. Polyethylene tubes containing experimental sealers and empty tubes were implanted into dorsal connective tissues of 12 male 3- to 4-months-old Wistar rats (250-280 g). After 7 and 30 days, the tubes were removed and processed for histological and immunohistochemical analyses. ANOVA followed by Bonferroni correction and ANOVA followed by Tukey test was used for parametric data and Kruskal-Wallis followed by Dunn for nonparametric (p < 0.05). Cell proliferation was dose-dependent, since all association were cytotoxic at higher concentrations; however, Ambroxol-PEG showed significantly higher cytotoxicity than other association (p < 0.05). In addition, irrespective of the association, no cytokine production was observed in vitro. Ambroxol-GLI reduced bacterial viability, whereas Ambroxol-PEG increased (p < 0.05). Histological examination showed no significant difference in the inflammatory response (p > 0.05) and mineralization ability in all association. Additionally, IL-1ß and TNF-α were upregulated on Ambroxol-PEG in relation to Control at 07 days (p < 0.05). Ambroxol-GLI was the best vehicle for experimental tricalcium silicate sealer, as it promoted an increase in antimicrobial activity without altering the inflammatory response or mineralization ability.

Tracing the toxic ions of an endodontic tricalcium silicate-based sealer in local tissues and body organs.

BACKGROUND: This study aimed to track the toxic ions released by MTA Fillapex, BioRoot RCS, and an experimental tricalcium silicate-based sealer (CEO) into local and distant tissues as well as to investigate their potential adverse effects. In addition, the chemical constituents of the sealers were also evaluated. The main components of the dry powders, pastes, and mixed sealers were characterized. MATERIAL AND METHODS: Dry powder and sealer discs were each set for 72 h and their main components were characterized by energy dispersive X-ray spectroscopy. Polyethylene tubes filled with sealers were used to measure silicon and calcium ions. Polyethylene tubes filled with sealers or empty tubes were implanted into the dorsal connective tissue of Wistar rats. On days 7, 15, 30, and 45, the animals were euthanized and their brains, livers, kidneys, and subcutaneous tissues were removed and processed to determine the concentrations of chromium, cobalt, copper, lead, iron, magnesium and nickel using an inductively coupled plasma optical emission spectrometer. RESULTS: The main compounds in all sealers were carbon, oxygen, silicon, and calcium. MTA Fillapex release more Si while highest levels of Si were found in presence of BioRoot. The release of Si and Ca ions promoted by MTA Fillapex raise by time. No traces of cobalt, chromium, or magnesium were detected in any tissue. Irrespective of the sealer, no traces of copper and lead were found in the subcutaneous tissue; however, they were observed in the organs. The highest concentration of iron was identified in the liver. All sealers exhibited similar nickel traces in the brain, kidney, and liver except for MTA Fillapex, which demonstrated levels higher than CEO in the subcutaneous tissue on day 7. Tracing nickel ions over time revealed that lowest concentrations were found in subcutaneous tissue. CONCLUSION: Taken together, our data demonstrate that CEOs have chemical compositions similar to those of other commercial sealers. Furthermore, none of them exhibited a threat to systemic health. Moreover, the minimal amounts of iron and nickel detected were not related to the sealers.

Biological assessment of a new ready-to-use hydraulic sealer.

OBJECTIVES: This study compared the cytotoxicity, biocompatibility, and tenascin immunolabeling of a new ready-to-use hydraulic sealer (Bio-C Sealer) with MTA-Fillapex and white MTA-Angelus. MATERIALS AND METHODS: L929 fibroblasts were cultivated and exposed to undiluted and diluted material extracts. Polyethylene tubes with or without (the control) the materials were implanted into the dorsa of rats. At 7 days and 30 days, the rats were euthanized, and the specimens were prepared for analysis; inflammation and immunolabeling were measured, and statistical analysis was performed (p < 0.05). RESULTS: MTA-Fillapex exhibited greater cytotoxicity than the other materials at all time points (p < 0.05). The undiluted Bio-C Sealer exhibited greater cytocompatibility at 6 and 48 hours than white MTA-Angelus, with higher cell viability than in the control (p < 0.05). White MTA-Angelus displayed higher cell viability than the control at 24 hours, and the one-half dilution displayed similar results at both 6 and 48 hours (p < 0.05). At 7 days and 30 days, the groups exhibited moderate inflammation with thick fibrous capsules and mild inflammation with thin fibrous capsules, respectively (p > 0.05). At 7 days, moderate to strong immunolabeling was observed (p > 0.05). After 30 days, the control and MTA-Fillapex groups exhibited strong immunolabeling, the white MTA-Angelus group exhibited moderate immunolabeling (p > 0.05), and the Bio-C Sealer group exhibited low-to-moderate immunolabeling, differing significantly from the control (p < 0.05). CONCLUSIONS: Bio-C Sealer and white MTA-Angelus exhibited greater cytocompatibility than MTA-Fillapex; all materials displayed adequate biocompatibility and induced tenascin immunolabeling.

Hyperglycemic condition influence on mineral trioxide aggregate biocompatibility and biomineralization.

This study aimed to investigate in vitro and in vivo the influence of hyperglycemic condition on biocompatibility and biomineralization of gray mineral trioxide aggregate (GMTA) and white mineral trioxide aggregate (WMTA). For the in vitro study, fibroblast-like cells L929 were cultured under high or normal glucose concentration to investigate the effects of both MTA's on cell proliferation and inflammatory cytokines production IL-1ß, IL-6, and TNF-α. For the in vivo study, polyethylene tubes containing MTA materials and empty tubes were implanted into dorsal connective tissues of Wistar rats previously assigned normal and hyperglycemic. After 7 and 30 days, the tubes with surrounding tissues were removed and subjected to histological, fluorescence and immunohistochemical analyzes of IL-1ß, IL-6, and TNF-α. In vitro study showed that, under high glucose condition, GMTA reduced cell proliferation and IL-6 production compared with WMTA. Moreover, in vivo study revealed that hyperglycemic condition did not modify the inflammatory response and cytokines production in the tissue close to both materials. Independently of hyperglycemic status, mineralized areas were observed with both materials, but the fluorescence intensity of WMTA was diminished on 14 days in hyperglycemic animals. It is possible to conclude that GMTA was able to inhibit the proliferation rate and IL-6 production under high glucose concentration in vitro. Furthermore, cytokines production and inflammatory response were not upregulated in hyperglycemic animals; however, a decrease in the calcium deposition was observed in presence of WMTA, suggesting a delay in the mineralization process.

Cytotoxicity and biocompatibility of a new bioceramic endodontic sealer containing calcium hydroxide.

This study evaluated the cytotoxicity and biocompatibility of a new bioceramic endodontic sealer (i.e., Sealer Plus BC) in comparison with those of MTA Fillapex and AH Plus. L929 fibroblasts were cultured and Alamar Blue was used to evaluate cell viability of diluted extracts (1:50, 1:100, and 1:200) from each sealer at 24 h. Polyethylene tubes that were filled with material or empty (as a control) were implanted in the subcutaneous tissue of rats. The rats were killed after 7 and 30 d (n = 8), and the tubes were removed for histological analysis. Parametric data was analyzed using a one-way ANOVA test, and nonparametric data was analyzed via the Kruskal-Wallis test followed by the Dunn test (p < 0.05). A reduction in cell viability was observed in the extracts that were more diluted for Sealer Plus BC when compared to that of Control and AH Plus (p < 0.05). However, the 1:50 dilution of the Sealer Plus BC was similar to that of the Control (p > 0.05). Conversely, more diluted extracts of MTA Fillapex (1:200) and AH Plus (1:100 and 1:200) were similar to the Control (p > 0.05). Histological analysis performed at 7 d did not indicate any significant difference between tissue response for all materials, and the fibrous capsule was thick (p > 0.05). At 30 d, Sealer Plus BC was similar to the Control (p > 0.05) and MTA Fillapex and AH Plus exhibited greater inflammation than the Control (p < 0.05). The fibrous capsule was thin for the Control and for most specimens of Sealer Plus BC and AH Plus. Thus, Sealer Plus BC is biocompatible when compared to MTA Fillapex and AH Plus, and it is less cytotoxic when less-diluted extracts are used.

Th1/Th2/Th17/Treg Balance in Apical Periodontitis of Normoglycemic and Diabetic Rats.

INTRODUCTION: The aim of this study was to evaluate the inflammatory profile of T helper (Th) cells in normoglycemic (N) and diabetic rats with apical periodontitis (AP). METHODS: Twenty male Wistar rats were divided in 2 groups: N rats and rats with diabetes mellitus (DM). DM was induced using streptozotocin, and AP was induced by dental pulp exposure of the first mandibular molar to the oral environment. After 30 days, the mandibles were removed and processed for histologic analysis, bacterial analysis, and immunochemical assays for interleukin (IL)-6, tumor necrosis factor alpha, IL-17, IL-23, interferon gamma, and IL-10. The Mann-Whitney U test and Student t test were used for statistical analysis (P < .05). RESULTS: The DM group showed more intense inflammatory infiltrate with larger sizes of bone reabsorption and a greater presence of bacteria than the N group (P < .05). Proinflammatory cytokine levels in the DM group were also greater than those in the N group (P < .05). However, interferon gamma was more intense in the N group than in the DM group (P < .05). CONCLUSIONS: The inflammatory profile of AP in DM is different from that in the N group, suggesting that Th1 is a secondary strain and the Th17 strain is predominant in DM.

Evaluation of the Cytotoxicity and Biocompatibility of New Resin Epoxy-based Endodontic Sealer Containing Calcium Hydroxide.

INTRODUCTION: Many endodontic sealers are available, but the search for the ideal sealer continues. This study evaluated the cytotoxicity and biocompatibility of Sealer Plus, a new resin epoxy-based endodontic sealer containing calcium hydroxide. AH Plus, Endofill, and SimpliSeal endodontics sealers were used for comparison. METHODS: L929 fibroblasts were cultured, and an MTT assay was used to determine the cytotoxicity of the sealer extracts at 6, 24, 48, and 72 hours. Tubes containing materials or empty tubes for control were inserted into the subcutaneous tissues of 20 rats. After 7 and 30 days, the rats were killed, and the tubes were removed with the surrounding tissues for histologic analysis. The data were submitted to statistical tests (P < .05). RESULTS: Undiluted Sealer Plus exhibited less cytotoxicity compared with other undiluted extracts at 6 hours (P < .05), and cell viability was higher for all Sealer Plus extracts after 24 hours (P < .05). At 48 hours, the undiluted and ½ Sealer Plus dilution were the extracts with less cytotoxicity (P < .05). At 72 hours, cell viability was higher for the undiluted and ½ Sealer Plus dilution compared with the other sealers (P < .05). At 7 days, Endofill and SimpliSeal had higher inflammation compared with the control and Sealer Plus (P < .05); AH Plus had moderate inflammation (P > .05). At 30 days, control, Sealer Plus, and AH Plus had less inflammation (P < .05). The fibrous capsule was thick at 7 days and thin at 30 days, except for SimpliSeal. CONCLUSIONS: In general, Sealer Plus promoted greater cell viability and was more biocompatible compared with the other sealers.

Cytotoxicity, Biocompatibility, and Biomineralization of the New High-plasticity MTA Material.

INTRODUCTION: Mineral trioxide aggregate (MTA) has excellent biological properties, but its handling properties have been criticized for both ProRoot MTA (Tulsa Dental Products, Tulsa, OK) and white MTA-Angelus (MTA-Ang; Angelus Indústria de Produtos Odontológicos S/A, Londrina, PR, Brazil). Angelus MTA HP (high plasticity) (Angelus Indústria de Produtos Odontológicos S/A) has been introduced recently. Considering the importance of biological properties of materials that will be in contact with the tissues, this study evaluated the cytotoxicity, biocompatibility, and biomineralization of MTA HP compared with white MTA-Ang. METHODS: L929 fibroblast cell lines were cultured, and cell viability was assessed at 6, 24, 48, and 72 hours using the alamar Blue assay (Thermo Fisher Scientific, Waltham, MA). A subcutaneous implant test was performed with polyethylene tubes containing 1 of the materials or empty tubes (control) using 20 Wistar rats. After 7 and 30 days of implantation, the tubes with surrounding tissues were removed for analysis using hematoxylin-eosin or von Kossa stain or they remained unstained for observation under polarized light. The results were statistically analyzed (P < .05). RESULTS: A significant increase in cell viability for MTA HP was observed after 24, 48, and 72 hours compared with the control (P < .05). At 72 hours, MTA HP exhibited a higher viability compared with white MTA-Ang (P < .05). Histologic analysis performed at 7 days showed moderate inflammation and a thick fibrous capsule in all groups (P > .05). At 30 days, mild inflammation and a thin fibrous capsule were observed in all groups (P > .05). All materials had structures positive for von Kossa and birefringent to polarized light. CONCLUSIONS: MTA HP showed biocompatibility and biomineralization similar to MTA-Ang. In addition, MTA HP showed increased fibroblast cell viability compared with white MTA-Ang after a longer period.

Hypertension Undermines Mineralization-inducing Capacity of and Tissue Response to Mineral Trioxide Aggregate Endodontic Cement.

INTRODUCTION: This study evaluated the effect of hypertension on tissue response to and mineralization capacity of white and gray mineral trioxide aggregate (MTA) (Angelus Industry Ontological Products, Londrina, Brazil), an endodontic reparative cement. METHODS: Polyethylene tubes containing gray MTA, white MTA, or intermediate restorative material (positive control) or an empty tube (negative control) were implanted into the dorsal connective tissue of spontaneous hypertensive and Wistar rats (n = 12 each). Six rats in each group were sacrificed after 7 days, and the remainder after 30 days. Tubes with surrounding tissue were removed, and a histologic analysis was performed using hematoxylin-eosin and von Kossa staining and examination by polarized light microscopy. RESULTS: The inflammatory response to all materials was greater in hypertensive compared with normotensive rats (P < .05). Positive von Kossa staining and birefringent structures in polarized light were observed for both gray and white MTA (P > .05), but these were more pronounced in normotensive rats (P < .05). Necrotic areas with positive von Kossa staining were observed for intermediate restorative material. CONCLUSIONS: Hypertension undermines tissue repair and mineralization, which can negatively affect treatment outcome. Nonetheless, mineralization in response to MTA was observed even under hypertensive conditions.

Evaluation of photodynamic therapy on fibroblast viability and cytokine production.

BACKGROUND: The aim of this study was to evaluate the effects of photodynamic therapy with curcumin (PDT) comparatively to 5% sodium hypochlorite (NaOCl) and saline solution on cell viability and cytokine (IL-1ß and IL-6) production by mouse fibroblasts. METHODS: Sixty seconds of pre-irradiation time with curcumin 500mg/L and Led wavelength (λ) 480nm, 72Jcm(2), for 300s was used for PDT. Solutions were diluted in culture medium DMEM (1×10(4) cells) and placed into 24-well cell culture plates with mouse fibroblasts L-929. Culture medium was used as control. After 6, 24 and 48h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) was used to evaluate the cell viability and the supernatant was collected for cytokine evaluation using enzyme-linked immunosorbent assay (ELISA). The results were statistically analyzed by ANOVA and BonFerroni correction (p<0.05) for MTT and Kruskal-Wallis test and Dunn (p<0.05) for ELISA. RESULTS: PDT and saline solution presented low cytotoxic effect similar to the control group (p>0.05) while 5% NaOCl was more cytotoxic than PDT (p<0.05) in all periods of time. All materials similarly expressed IL-1ß and IL-6 regardless to the experimental period (p<0.05). CONCLUSIONS: PDT with curcumin was not cytotoxic to L929 fibroblasts differently from 5% NaOCl. In all groups occurred similar expression of IL-1ß and IL-6.

Influence of diabetes mellitus on tissue response to MTA and its ability to stimulate mineralization.

AIM: To evaluate the influence of diabetes mellitus on the tissue response to mineral trioxide aggregate (MTA) and its ability to stimulate mineralization. METHODS: Twenty-four male Wistar rats were divided into a non-diabetic control group and another with Alloxan-induced diabetes. Two polyethylene tubes, one kept empty as a control and the other containing Angelus MTA(®) , were implanted into the dorsal connective tissue of all rats for 30 days. Animals in each group received injections of calcein, alizarin, and oxytetracycline on day 7, 14, and 21, respectively. Animals were killed after 30 days; specimens were prepared by staining with hematoxylin and eosin (HE) and von Kossa technique as well as for examination of unstained sections with polarized light and fluorescence microscopy. RESULTS: The inflammatory reaction to the implanted tubes was equally mild in both groups. Structures staining with von Kossa were seen in response to Angelus MTA(®) , as were birefringent structures visualized on polarized light analysis; these had no relation to diabetic condition (P < 0.05). Fluorescence intensity was not changed in diabetic rats either (P < 0.05). CONCLUSION: Diabetes mellitus did not influence the tissue response to Angelus MTA(®) or the mineralization stimulated by it.

The use of NaOCl in combination with CHX produces cytotoxic product.

OBJECTIVES: The combination of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) yields a "precipitate potentially toxic" (PPT). The aim of this study was to evaluate the tissue response to implanted polyethylene tubes filled with PPT-soaked fibrin sponge. METHODS: Forty rats received four polyethylene tubes each; each tube was filled with fibrin sponge soaked by 2.5 % NaOCl, 2.0 % CHX, PPT (2.5 % NaOCl plus 2.0 % CHX), or not soaked (control). The observation time points were 7, 15, 30, 60, and 90 days. At each time point, eight animals were killed, and the tubes and surrounding tissues were removed, fixed, and prepared for light microscopic analysis by performing glycol methacrylate embedding, serial cutting into 3-µm sections, and hematoxylin-eosin staining. Qualitative and quantitative evaluations of the reactions were performed. Results were statistically analyzed by Kruskal-Wallis test (p < 0.05). RESULTS: All chemical solutions caused moderate reactions at 7 days. On day 30, PPT group was more cytotoxic than the control group and the CHX group (p < 0.05). On days 15 and 60, PPT group was more cytotoxic than the control group (p < 0.05). On day 90, there was no statistically significant difference between the different groups. CONCLUSION: PPT is more cytotoxic than NaOCl and CHX alone, particularly in the short term. CLINICAL SIGNIFICANCE: Protocols which suggest the use of CHX and NaOCl must be revised because this mixture produces cytotoxic product.