In silico toxicity and immunological interactions of components of calcium silicate-based and epoxy resin-based endodontic sealers.

OBJECTIVES: The present study aimed to determine in silico toxicity predictions of test compounds from hydraulic calcium silicate-based sealers (HCSBS) and AH Plus and computationally simulate the interaction between these substances and mediators of periapical inflammation via molecular docking. MATERIALS AND METHODS: All chemical information of the test compounds was obtained from the PubChem site. Predictions for bioavailability and toxicity analyses were determined by the Molinspiration Cheminformatics, pkCSM, ProTox-II and OSIRIS Property Explorer platforms. Molecular docking was performed using the Autodock4 AMDock v.1.5.2 program to analyse interactions between proteins (IL-1ß, IL-6, IL-8, IL-10 and TNF-α) and ligands (calcium silicate hydrate, zirconium oxide, bisphenol-A epoxy resin, dibenzylamine, iron oxide and calcium tungstate) to establish the affinity and bonding mode between systems. RESULTS: Bisphenol-A epoxy resin had the lowest maximum dose tolerated in humans and was the test compound with the largest number of toxicological properties (hepatotoxicity, carcinogenicity and irritant). All systems had favourable molecular docking. However, the ligands bisphenol-A epoxy resin and dibenzylamine had the greatest affinity with the cytokines tested. CONCLUSION: In silico predictions and molecular docking pointed the higher toxicity and greater interaction with mediators of periapical inflammation of the main test compounds from AH Plus compared to those from HCSBS. CLINICAL RELEVANCE: This is the first in silico study involving endodontic materials and may serve as the basis for further research that can generate more data, producing knowledge on the interference of each chemical compound in the composition of different root canal sealers.

Analysis of the cytotoxicity and bioactivity of CeraSeal, BioRoot™ and AH Plus® sealers in pre-osteoblast lineage cells.

BACKGROUND: The objective of the present study was to evaluate in vitro the cytotoxicity and bioactivity of various endodontic sealers (CeraSeal, BioRoot™ and AH Plus®) in pre-osteoblast mouse cells (MC3T3 cells). METHODS: MC3T3 cells (ATCC CRL-2594) were plated in 1 × 104 cells/well in 96-well plates in contact with endodontic sealers at concentrations of 1:10 and 1:100. Cell viability was evaluated by MTT assay after 24 and 48 h. In addition, sealer bioactivity was measured by RT-PCR for mediator of inflammation (Tnf, Ptgs2) and mineralization (Runx2, Msx1, Ssp1 and Dmp1) after 24 h and by Alizarin Red S Assay of mineralization after 28 days. Data were analyzed using one-way ANOVA followed by the Tukey's post-test at a significance level of 5%. RESULTS: BioRoot™ presented 24-hour cytotoxicity (p < 0.05) at 1:10 concentration. In the period of 48 h, no endodontic cement was cytotoxic to the cells compared to the control (p > 0.05). TNF-α gene expression was induced by AH Plus® (p < 0.05), while Ptgs2 was induced by the CeraSeal and BioRoot™ (p < 0.05). The expression of Runx2 was stimulated by BioRoot™ and AH Plus® (p < 0.05). In contrast, the expression of Dmp-1 Dmp1 was higher for the CeraSeal and BioRoot™ (p < 0.05). Nonetheless, the sealers did not impact the formation of mineralization nodules (p > 0.05). CONCLUSION: CeraSeal, BioRoot™ and AH Plus® sealers were not cytotoxic to MC3T3 cells within 48 h, but differentially induced the expression of genes related to inflammation and mineralization without impacting biomineralization by the cells.

Cytotoxicity, biocompatibility and osteoinductive profile of an MTA-hydrogel-based cement: An in vitro and animal study.

AIM: This study aimed to evaluate the cytotoxicity, biocompatibility and osteoinductive profile of a mineral trioxide aggregate (MTA)-hydrogel-based material (MTA Flow) in comparison with MTA Angelus. METHODOLOGY: Cell viability was evaluated in human periodontal ligament stem cells (hPDLSCs) using the methyl-thiazol-tetrazolium (MTT) colourimetric assay. Polyethylene tubes containing the tested materials and empty polyethylene tubes (control) were implanted in the subcutaneous tissue of Wistar rats. Cellular (lymphocyte infiltration) and extracellular events (ECM; collagen fibres) were analysed in histological sections. Immunohistochemical (collagen I, osteopontin, bone sialoprotein, bone morphogenetic protein4) analyses were also performed. RESULTS: At 24, 48 and 72 h, all tested groups showed cell viability similar to control (p > .05). Regarding biocompatibility, all groups showed similar cellular events represented by a slight inflammatory reaction characterized by hyperaemia and a mild lymphocytic inflammatory infiltrate. The analysis of lymphocytes during the time showed a decrease in these cells in the control group and a significant interaction between MTA Angelus and control (p < .001), with MTA Angelus showing a more extensive inflammatory infiltrate. Regarding fibres, an increase in content was observed in all groups during the experimental time (7, 30 and 60 days), however, no difference was detected among the experimental groups (p = .063). After 60 days, the immunoexpression of bone matrix proteins in the MTA Flow group was similar to or higher than that observed in the MTA Angelus and in the control group. CONCLUSIONS: MTA Flow showed a non-cytotoxic behaviour, biocompatibility and ability to stimulate tissue mineralization.

The solubility, pH value, chemical structure, radiopacity, and cytotoxicity of four different root canal sealers: an in vitro study.

OBJECTIVE: The aim of this study was to investigate solubility, pH value, chemical structure, radiopacity, and cytotoxicity of AH Plus BC, TotalFill BC, AH Plus, and AH Plus Jet sealers. MATERIALS AND METHODS: Cytotoxicity analysis with direct and extraction tests at 3 different concentrations (1:1, 1:2, 1:4 v/v%) and time (24 h, 48 h, and 72 h) on Saos-2, PdLF, and THP-1 cell lines, chemical structure with scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) analysis, solubility, pH, and radiopacity values of AH Plus BC, TotalFill BC, AH Plus, and AH Plus Jet were evaluated. For statistical analyses of the groups, repeated measures, factorial, and one-way ANOVA tests were used. The statistical significance level was set at p < .05. RESULTS: Resin-based sealers showed higher cytotoxicity values than the bioceramic-based sealers (p < 0.05). Time and concentrations were effective on the cell viabilities for cell lines. Higher peaks of calcium were detected bioceramic-based sealers and higher amount of zirconium was detected in AH Plus BC (p < 0.05). AH Plus BC showed similar radiopacity value with AH Plus, AH Plus Jet, whereas TotalFill BC showed the lowest radiopacity (p < 0.05). Bioceramic-based sealers had higher pH values in all experiment periods, and the difference between resin- and bioceramic-based sealer groups was significant (p < 0.05). However, the solubility values of the tested root canal sealers revealed no differences (p > 0.05). CONCLUSIONS: The newly produced AH Plus BC Sealer showed similar properties with TotalFill BC, and their biological properties were better than AH Plus and AH Plus Jet. CLINICAL RELEVANCE: AH Plus BC could be a possible alternative to other bioceramic- or resin-based sealers.

A critical analysis of research methods and experimental models to study biocompatibility of endodontic materials.

Materials used for endodontics and with direct contact to tissues have a wide range of indications, from vital pulpal treatments to root filling materials and those used in endodontic surgery. In principle, interaction with dental materials may result in damage to tissues locally or systemically. Thus, a great variety of test methods are applied to evaluate a materials' potential risk of adverse biological effects to ensure their biocompatibility before commercialization. However, the results of biocompatibility evaluations are dependent on not only the tested materials but also the test methods due to the diversity of these effects and numerous variables involved. In addition, diverse biological effects require equally diverse assessments on a structured and planned approach. Such a structured assessment of the materials consists of four phases: general toxicity, local tissue irritation, pre-clinical tests and clinical evaluations. Various types of screening assays are available; it is imperative to understand their advantages and limitations to recognize their appropriateness and for an accurate interpretation of their results. Recent scientific advances are rapidly introducing new materials to endodontics including nanomaterials, gene therapy and tissue engineering biomaterials. These new modalities open a new era to restore and regenerate dental tissues; however, all these new technologies can also present new hazards to patients. Before any clinical usage, new materials must be proven to be safe and not hazardous to health. Certain international standards exist for safety evaluation of dental materials (ISO 10993 series, ISO 7405 and ISO 14155-1), but researchers often fail to follow these standards due to lack of access to standards, limitation of the guidelines and complexity of new experimental methods, which may cause technical errors. Moreover, many laboratories have developed their testing strategy for biocompatibility, which makes any comparison between findings more difficult. The purpose of this review was to discuss the concept of biocompatibility, structured test programmes and international standards for testing the biocompatibility of endodontic material biocompatibility. The text will further detail current test methods for evaluating the biocompatibility of endodontic materials, and their advantages and limitations.

The evaluation of cytotoxicity and cytokine IL-6 production of root canal sealers with and without the incorporation of simvastatin: an invitro study.

BACKGROUND: Freshly mixed root canal sealers when proximate the periapical tissues, trigger varying degrees of cytotoxicity/inflammatory reactions. Simvastatin, a class of the drug statin, is a widely used cholesterol-lowering agent with additional anti-inflammatory activities. This study assessed the effects of simvastatin on cytotoxicity and the release of IL-6 (Interleukin-6) production when incorporated in zinc oxide eugenol and methacrylate resin-based sealers. METHODS: Experimental groups consisted of conventional zinc oxide eugenol and methacrylate based-EndoREZ sealers (ZE & ER respectively) and 0.5 mg/mL simvastatin incorporated sealers (ZES & ERS). L929 mouse fibroblast cells were exposed to freshly mixed experimental sealers and evaluated for cytotoxicity (MTT assay) and inflammation levels (inflammatory marker IL-6 for ELISA) at various time intervals (0h, 24h and 7th day). The values were compared to the cell control (CC; L929 cells alone) and solvent control (SC; L929 cells + DMSO) groups. All the experiments were conducted in triplicates and subjected to statistical analysis using IBM SPSS Statistics software. Non parametric tests were conducted using Kruskal-Wallis and Friedman tests for inter-group and intra-group comparisons respectively. Pairwise comparison was conducted by post hoc Dunn test followed by Bonferroni correction. P values < 0.05 were considered statistically significant. RESULTS: All the experimental groups (ZE, ER, ZES, ERS) exhibited varying degree of cytotoxicity and IL-6 expression compared to the control groups CC and SC. The cell viability for ZE and ER decreased on day 7 as compared to 24 h. ZES and ERS had higher viable cells (75.93% & 79.90%) compared to ZE and ER (54.39% & 57.84%) at all time periods. Increased expression of IL-6 was observed in ZE & ER (25.49 pg/mL & 23.14 pg/mL) when compared to simvastatin incorporated ZE & ER (ZES-12.70 pg/mL & ERS-14.68 pg/mL) at all time periods. Highest level of cytotoxicity and inflammation was observed in ZE compared to all the other groups on day 7. CONCLUSIONS: Addition of 0.5 mg/mL of simvastatin to the sealers (ZES and ERS) decreased the cytotoxicity in the freshly mixed state and reduces their inflammatory effect.

SHED's Response to Various Pulpotomy Materials: Cytotoxicity and Gene Expression Analysis.

PURPOSE: To examine the cytotoxicity and genetic expression of SHEDs cultured in eluates of various calcium silicate based pulpotomy materials. STUDY DESIGN: MTT assay, flow cytometry, alizarin red staining and scratch assay was used to assess the cellular viability, apoptosis, calcium matrix deposits and cell migration respectively. The gene expression of ALP, OCN and BMP -2, were measured with rtPCR. One way ANNOVA and Bonferroni post test was used for statistical analysis. RESULTS: MTT assay analysis reported that all the test specimen had no cytotoxic effects. The highest number of live cells [ % ] was found in RetroMTA. The highest percentage of cell migration was observed in SHEDs cultured in EndoCem Zr. The mean absorbance for calcium matrix deposition was higher or similar in all test specimens, when compared to control groups. The expression of BMP -2 and OCN were significantly higher in cells exposed to RetroMTA and NeoMTA respectively after 24 hrs of incubation. After 72 hrs of incubation the mRNA expression of ALP was significantly higher in MTA. CONCLUSIONS: SHEDs cultured in eluates of various calcium silicate based cements exhibited cytocompatibility and maintained odontogenic like phenotype differentiation in SHEDs.

MTT versus other cell viability assays to evaluate the biocompatibility of root canal filling materials: a systematic review.

OBJECTIVES: This systematic review aimed to compare the cytotoxicity of root canal filling materials (RCFMs) assessed using tetrazolium salt-based tests (TSBT), including the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, with those obtained using other cell viability assays. METHODS: A search was performed on PubMed, Scopus, Web of Science and OpenGrey up to March 2019, followed by a manual search. According to the Participants, Exposure, Comparator and Outcomes (PECO) criteria, in vitro studies that evaluated the cytotoxic effect of RCFMss on animal and/or human cells through TSBT and at least one other viability assay were compared. The methodological quality of selected papers was assessed using ToxRTool® and SciRAP® . Data were analysed using Wilcoxon's signed-rank test for paired samples and linear weighting kappa. RESULTS: A total of 230 non-duplicated records were identified. After applying the eligibility criteria, 55 studies were selected for methodological evaluation, seven were selected by manual searching, 22 were excluded for methodological reasons, and 40 were included. A total of 410 comparisons were performed between TSBT and distinct cell viability tests (DCVT). MTT had moderate concordance with DCVT using human cells (n = 138 samples) (P = 0.507; k = 0.4225) and animal cells (n = 122 samples) (P = 0.124; k = 0.5775). XTT had good concordance using human (n = 110 samples) (P = 0.507; k = 0.6336) and animal cells (n = 12 samples) (P = 0.564; k = 0.6604). MTT, XTT, WST and MTS assays showed moderate concordance with DCVT (n = 410 samples) (P = 0.375; k = 0.5138) and complete agreement in 226 samples. DISCUSSION: The included studies had methodological heterogeneity that was minimized by the systematic review methodology. CONCLUSIONS: MTT and XTT do not cause over- or underestimation of cell viability during cytotoxicity screening of root canal filling materials, implying that these assays can be considered reliable for this purpose. Nonetheless, the development of protocols for the cytotoxic screening of these materials on 3D tissue-like cultures aiming to improve their predictability in the clinical scenario is suggested.

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).

Comparative Evaluation of Cytotoxicity of Root Canal Sealers on Cultured Human Periodontal Fibroblasts: In vitro Study.

AIM: To evaluate and compare the cytotoxic effects of different types of root canal. MATERIALS AND METHODS: The sealers were eluted with culture medium for 1 hour, 7 days, and 14 days. Cell viability was estimated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and trypan blue exclusion method on human periodontal ligament (PDL) fibroblast cells. Sealers used are mineral trioxide aggregate (MTA)-based sealer (MTA Fillapex, Angelus), calcium hydroxide-based sealer (Apexit Plus, Ivoclar Vivadent), resin-based sealer (AH Plus, Dentsply), and zinc oxide eugenol-based sealer (Tubli Seal, SybronEndo). RESULTS: The order of cytotoxicity through MTT assay, at the end of the second week, was observed as MTA Fillapex> Tubli Seal> Apexit Plus > AH Plus. The percentage cell viability obtained after trypan blue exclusion method decreased in the order of Apexit Plus> Tubli Seal> AH Plus> MTA Fillapex, which was similar to the reported cytotoxicity from the MTT assay after 1 hour. CONCLUSION: Each type of sealer showed moderate-to-severe cytotoxic response when compared with the control. The MTA Fillapex was found to be the most cytotoxic sealer. Use of resin-based material as a root canal sealer may result in a more favorable response to PDL fibroblasts. CLINICAL SIGNIFICANCE: Having knowledge of the cytotoxicity of various sealers will help in increasing patient's comfort.

Comparative Evaluation of Cytotoxicity and Genotoxicity of Two Bioceramic Sealers on Fibroblast Cell Line: An in vitro Study.

AIM: The purpose of this study was to evaluate and compare the cytotoxicity and genotoxicity of two bioceramic root canal sealers: EndoSequence BC and iRoot SP with zinc oxide eugenol sealers on fibroblast cell line. MATERIALS AND METHODS: The sealers tested were zinc oxide eugenol, EndoSequence BC, and iRoot SP. Each material was mixed according to the manufacturer's instructions and mounted into sterile polyethylene color-coded rings, for cytotoxicity and genotoxicity evaluation. After 48 hours, the set materials were transferred to previously marked wells and cytotoxicity evaluation to L929 murine fibroblast cells was done by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The percentages of viable cells were then calculated and values were statistically analyzed by Kruskal-Wallis test. The evaluation of genotoxicity of the materials to L929 murine fibroblast cells was carried out by Comet assay. To quantify deoxyribonucleic acid (DNA) damage, the following comet parameters were evaluated in the assay using Comet scoring software: tail length, tail moment, and Olive moment. The values were statistically analyzed using Kruskal-Wallis test with a significance value set to p < 0.05. RESULTS: The results of the study showed that both cytotoxicity and genotoxicity evaluation by MTT assay and Comet assay can be done on L929 murine fibroblast cell line. Among the three tested materials, zinc oxide eugenol showed maximum cytotoxicity to the cells (30.64% viable cells), followed by EndoSequence BC (71.33% viable cells) and iRoot SP (75.11% viable cells). The evaluation of DNA damage by genotoxicity assessment showed iRoot SP to be least genotoxic followed closely by EndoSequence BC. Zinc oxide eugenol was genotoxic and induced more DNA damage on the fibroblast cell line studied. The statistical analyses for both the assays were nonsignificant. CONCLUSION: All the three tested sealers showed varying degrees of cytotoxicity and genotoxicity while using fibro-blast cell line. Zinc oxide eugenol was most toxic in both the assays and iRoot SP showed least toxicity, followed closely by EndoSequence BC.

Cytocompatibility of calcium silicate-based sealers in a three-dimensional cell culture model.

OBJECTIVES: The aim of the present study was to evaluate cytotoxic effects and cytokine production of calcium silicate-based sealers (EndoSeal, EndoSequence BC Sealer, and MTA Fillapex) using an in vitro root canal filling model and three-dimensional (3D) cell culture. AH Plus as a reference was compared to contemporary calcium silicate cements regarding cell viability and cytokine production. MATERIAL AND METHODS: Root canals of 30 human maxillary incisors were prepared using a single-file reciprocating technique. The samples were randomly distributed and canals filled with either AH Plus, EndoSeal, EndoSequence BC Sealer, and MTA Fillapex (n = 6). In the negative control group, the root canal remained unfilled. Sealers were placed into the canals along with a gutta-percha cone placed to working length. Balb/c 3T3 fibroblasts, cultured in a type I collagen 3D scaffold, were exposed to filling material and the respective root apex for 24 h. Cytocompatibility of the materials was evaluated using the methyl-thiazoldiphenyl-tetrazolium (MTT) assay. The production of IL-1ß, IL-6, and IL-8 was analyzed using enzyme-linked immunosorbent assay (ELISA). One-way analysis of variance was performed, and when the F-ratios were significant, data were compared by Duncan's multiple-range test. The alpha-type error was set at 0.05. RESULTS: EndoSeal, Endosequence BC Sealer and AH Plus showed cell viability that was similar to the negative control group (P > 0.05), while MTA Fillapex sealer was cytotoxic (P < 0.05). Varying production of IL-1ß, IL-6, and IL-8 was detected in all samples. CONCLUSIONS: In an in vitro root canal filling model with 3D cell culture, AH Plus, EndoSeal, and EndoSequence BC Sealer were cytocompatible. CLINICAL RELEVANCE: These results may suggest that AH Plus, EndoSeal and EndoSequence BC Sealer may achieve better biological response when compared to MTA Fillapex.

Evaluation of the genotoxicity and cytotoxicity of filling pastes used for pulp therapy on deciduous teeth using the micronucleus test on bone marrow from mice (Mus musculus).

Pulp therapy is the last resort for preserving deciduous teeth. However, the genotoxic and cytotoxic effects of many products used in this therapy are not well established. The aim of this study was to use the micronucleus test on bone marrow from mice to evaluate the genotoxic and cytotoxic effects of four filling pastes: zinc oxide, calcium hydroxide P.A., mineral trioxide aggregate and an iodoform paste (iodoform + camphorated + paramonochlorophenol + rifamycin + prednisolone). Male Swiss mice were divided into 4 groups of 10 animals, each exposed to one of the pastes, and were subdivided according to the dilutions tested: 1/10, 1/50, 1/500 and 1/1000 administered intraperitoneally (0.1ml/10g of weight). Cyclophosphamide was the positive control. The negative controls were dimethylsulfoxide and buffered saline solution. Five animals were killed 24h and five 48h after the treatment. The material was processed in accordance with Schmid (1976) and micronuclei were counted in 1000 polychromatic erythrocytes (PCE), under an optical microscope in a blinded test. Cytotoxicity was evaluated using the PCE/normochromatic erythrocyte (NCE) ratio in 200 erythrocytes. The micronucleus analysis results were evaluated using the conditional test for comparing proportions in situations of rare events. Analysis of variance and Tukey's test were used to evaluate the PCE/NCE ratio. There was significantly greater occurrence of micronuclei in the animals treated with iodoform paste at all the dilutions tested, at both sacrifice times. Greater occurrence of micronuclei was observed among the animals treated with zinc oxide and sacrificed 48h after the treatment, at the dilutions 1:50; 1:500 and 1:1000. Calcium hydroxide P.A. and mineral trioxide aggregate did not present any genotoxic or cytotoxic effects. The genotoxicity and cytotoxicity of zinc oxide and iodoform paste revealed here constitute an initial step towards their contraindication, but additional studies will be necessary in order to securely establish the risks involved in their use.

DNA double-strand breaks caused by new and contemporary endodontic sealers.

AIM: To investigate the cytotoxicity and genotoxicity of a new silicate-based BioRoot RCS® sealer in comparison with contemporary sealers. METHODOLOGY: A periodontal ligament cell line using lentiviral gene transfer of human telomerase reverse transcriptase (hTERT) was used and exposed to subtoxic concentrations of 24-h eluates from two epoxy resin-based (AH Plus Jet® and Acroseal® ), four various methacrylate-based endodontic sealers (EndoREZ® , RealSeal® , RealSeal SE® and Hybrid Root SEAL® ) and three silicate-based sealers (BioRoot RCS® , iRootSP® and MTA Fillapex® ). The XTT-based cell viability assay was used for cytotoxicity screening of materials. The γ-H2AX assay was used for genotoxicity screening. In the γ-H2AX immunofluorescence assay, PDL-hTERT cells were exposed to eluates of the substances for 6 h and DNA double-strand breaks (DSB) were detected microscopically. Induced foci represented DSBs, which can induce ATM-dependent phosphorylation of the histone H2AX. The statistical significance of the differences between the experimental groups was compared using the Student's t-test (P < 0.05). RESULTS: The cytotoxicity of the 24-h eluates could be ranked in the following order: Hybrid Root SEAL® >RealSeal® >Acroseal® >RealSeal SE® ≥ AH Plus Jet® > EndoREZ® >MTA Fillapex® > iRoot SP® >BioRoot RCS® . In negative controls (cells which received medium only) 4.08 ± 0.53 DSB foci (mean ± SEM) whilst in positive controls 10.76 ± 4.05 DSB foci/cell were found. BioRoot RCS® and RealSeal SE® exhibited significant differences in foci formation at 1/3 EC50 compared with their 1/10 EC50 concentration (P < 0.05). Both concentrations (1/10 and 1/3 of EC50) of AH Plus Jet® , Acroseal® , RealSeal® and MTA Fillapex® sealers were not significantly different when compared with the medium control (P < 0.05). CONCLUSIONS: New BioRoot RCS® was not toxic whilst Hybrid Root SEAL® demonstrated more toxicity and DNA double-strand breaks when compared with other resin- and silicate-based root canal sealers.

Cytotoxicity and genotoxicity of natural resin-based experimental endodontic sealers.

OBJECTIVES: The development of endodontic sealers based on natural resins seems to be promising, given their improved biological properties. This study evaluated the cytotoxic and genotoxic effects of two experimental root canal sealers, based on extracts from Copaifera multijuga and Ricinus communis (castor oil polymer), comparing them to synthetic resin-based sealers: a single methacrylate-based, a multi-methacrylate-based, and an epoxy resin-based sealers. MATERIALS AND METHODS: Sealers were prepared, set, and exposed to cell culture medium for 24 h at 37 °C with CO2. V79 cells were exposed to serial dilutions of the extracts of each sealer for 24 h. Cell viability was measured by the MTT assay and genotoxicity was assessed by the formation of micronuclei. RESULTS: The single methacrylate-based sealer had the most cytotoxic effects, with significant reduction in cell viability in all dilutions of the extract. The castor oil polymer-based sealer was, on the other hand, the most biocompatible sealer, with no cytotoxic effects at any concentration. All tested sealers were not genotoxic, excepting the single methacrylate-based sealer. CONCLUSIONS: The tested natural resin-based sealers presented low cytotoxic and no genotoxic effects on cell cultures. CLINICAL RELEVANCE: These results may suggest a good alternative to develop new endodontic sealers, in order to achieve better biological response and healing, when compared to commercially available sealers.

[Effect of root canal sealers on biocompatibility of human periodontal ligament cells].

OBJECTIVE: To compare the effects of three root canal sealers with respect to time on biocompatibility of human periodontal ligament cells (hPDLCs).The sealers included zinc oxide and eugenol based sealers (ZOE), epoxy resin sealers (ERS) and silicone based sealers (SBS). METHODS: hPDLCs were primarily cultured,with the method combining of tissue explant and enzymatic digestion. The cells were then exposed to different extract fluids: (1) ZOE extracted for 24 h group ;(2) ZOE extracted for 1 week group;(3)ZOE extracted for 2 weeks group;(4) ERS extracted after 24 h group; (5) ERS extracted after 1 week group; (6) ERS extracted after 2 weeks group; (7) SBS extracted after 24 h group; (8) SBS extracted after 1 week group; (9) SBS extracted after 2 weeks group; (10) Dulbecco modified Eagle's medium/F12 (DMEM/F12) as negative control group. Cell morphology was observed under an inverted microscope.Cell proliferation was measured by methyl-thiazol-diphenyltetrazolium (MTT) assay. ALP assay kit was used for measuring alkaline phosphatase (ALP) activity. Sealers of 2 weeks' setting time were then immersed in an osteogenic medium for examination of mineral nodules and calcium deposits. RESULTS: Considering the relative growth rate(RGR),ZOE was severely to moderately cytotoxic(RGR:13.6%-39.9%), while ERS was slightly or not cytotoxic (RGR: 87.6%-95.3%).Only SBS did not show any cytotoxicity after setting (RGR: 91.8%-106.7%). The setting time influenced the cytotoxicity of ERS which decreased after 1 week. Considering the ALP activity,there was no difference between SBS group and control group (F=3.397,P=0.053). According to the results of calcium deposits, ZOE:D562 nm=0.180±0.050,ERS: D562 nm=2.968±0.201,SBS:D562 nm=3.623±0.039,Control:D562 nm=3.477±0.102,the ranking of ALP activity and calcium deposits was as follows: ZOE

Evaluation of Inflammatory Response to Endodontic Sealers in a Bone Defect Animal Model.

AIM: The aim of this study was to evaluate the inflammatory response to MTA Fillapex, AH Plus, and Pulp Canal Sealer Extensive Work Time (EWT), in a murine bone defect grafting model. MATERIALS AND METHODS: Bilateral mandibular critical defects were produced in 45 Wistar rats with a trephine bur#2 and filled with the endodontic sealers. After 7, 14, and 28 days, the rats were euthanized and their jaws were histologically prepared. RESULTS: For the 7-day group, no statistical significance was observed among all studied groups (p > 0.05), and high levels of inflammatory infiltrate were detected. After 14 and 28 days, Pulp Canal Sealer EWT showed statistically lower inflammatory response in comparison to other sealers (p < 0.05) except for the control group (no sealers). CONCLUSION: Pulp Canal Sealer EWT presented the lowest levels of inflammatory response. The critical defect grafting model was an effective method to detect differences among differences on the biological response to endodontic sealers. CLINICAL SIGNIFICANCE: Knowing the biocompatibility of endodontics sealers that will be used in filling the root canal.

Biocompatibility of new nanostructural materials based on active silicate systems and hydroxyapatite: in vitro and in vivo study.

AIM: To evaluate in vitro cytotoxicity and in vivo inflammatory response to new nanostructural materials based on active calcium silicate systems (CS) and hydroxyapatite (HA-CS). METHODOLOGY: Cytotoxicity of eluates of new nanostructural noncommercial materials CS and HA-CS, and MTA (White MTA, Angelus(®) Soluções Odontológicas, Londrina, Brazil) as a control, were tested using the MTT assay on MRC-5 cells. Eluates of set materials were tested in 100% and 50% concentrations, 24 h, 7 days and 21 days post-elution. The pH values were determined for undiluted eluates of set materials. Polyethylene tubes containing the test materials (CS, HA-CS, MTA) were implanted in subcutaneous tissue of Wistar rats. Histopathological examinations were conducted at 7, 15, 30 and 60 days after the implantation. Data were statistically analyzed using three-way and one-way anova Tukey's post hoc test as well as Kruskall-Wallis test with Dunn's post hoc test at α = 0.05. RESULTS: All materials significantly reduced cell viability; especially when undiluted eluates were used (P < 0.001). After 24 h elution, cell viability was 10 ± 1.8%, 49.5 ± 4.2% and 61 ± 7.4%, for MTA, and HA-CS, respectively. However, CS and HA-CS were significantly less toxic than the control material MTA (P < 0.05). Cytotoxicity could be at least partially attributed to pH kinetics over time. Dilution of eluates of all tested materials resulted in better cell survival. Histopathological examination indicated similar inflammatory reaction, vascular congestion and connective tissue integrity associated with CS, HA-CS and MTA at each observation period (P > 0.05). The only significant difference was found for capsule thickness, that is thicker capsule was associated with HA-CS compared to MTA at 60 days (P = 0.0039). HA-CS induced moderately thick capsules (median score 3, score range 2-3), whereas MTA resulted in thin capsule formation (median score 2, score range 1-3). CONCLUSIONS: Evaluation of cytotoxicity and inflammatory response indicated better biocompatibility of CS and HA-CS, in comparison with MTA (White MTA, Angelus(®) Soluções Odontológicas, Londrina, Brazil).

Comparative cell attachment, cytotoxicity and antibacterial activity of radiopaque dicalcium silicate cement and white-coloured mineral trioxide aggregate.

AIM: To comparatively examine the cell attachment, cytotoxicity, and antibacterial activity of radiopaque dicalcium silicate cement (RDSC) and ProRoot white-coloured mineral trioxide aggregate (WMTA). METHODOLOGY: AlamarBlue was used for real-time and repeated monitoring of MG63 cell attachment on freshly mixed and set cements. The pH changes in the growth medium at different time-points were also measured. Cytotoxicity evaluation was performed according to ISO 10993-5 specifications. The antibacterial activity of the cement specimens was evaluated using Enterococcus faecalis. RESULTS: There were no significant differences (P > 0.05) between the two cements for cell attachment either in the fresh groups or in the set groups at all culture times. Neither freshly mixed group nor set groups had significant pH differences. In the case of cytotoxicity, RDSC was significantly (P < 0.05) superior to WMTA at 12 and 24 h of incubation. RDSC and WMTA possessed similar antimicrobial activity, substantiated by the formation of growth inhibition zones and bacteriostasis ratio in E. faecalis strains. CONCLUSIONS: The cell attachment, cytotoxicity and antibacterial efficacy of RDSC were comparable to those reported for ProRoot WMTA. The results of the current study suggest that this RDSC could be used as a root-end filling material and root sealer.

Assessment of the cytotoxicity of a mineral trioxide aggregate-based sealer with respect to macrophage activity.

AIM: To assess the influence of co-culture with mineral trioxide aggregate (MTA) and MTA Fillapex (FLPX) on the viability, adherence, and phagocytosis activity of peritoneal macrophages from two mouse strains. METHODOLOGY: Cellular viability, adherence, and phagocytosis of Saccharomyces boulardii were assayed in the presence of capillaries containing MTA and MTA Fillapex. The data were analyzed using parametric (Student's t) and non-parametric (Mann-Whitney) tests. RESULTS: FLPX was severely cytotoxic and decreased cell viability, adherence, and phagocytic activity of both macrophage subtypes. Cells that were treated with MTA Fillapex remained viable (>80%) for only 4 h after stimulation. Macrophages from C57BL/6 mice presented higher adherence and higher phagocytic activity compared with macrophages from BALB/c mice. CONCLUSION: Comparison of MTA and FLPX effects upon macrophages indicates that FLPX may impair macrophage activity and viability, while MTA seems to increase phagocytic activity.