Cytotoxicity, and antimicrobial and physicochemical properties of sealers incorporated with Uncaria tomentosa

Abstract This study evaluated the cytotoxicity, the antimicrobial and physicochemical properties of root canal sealers incorporated with phytotherapic Uncaria tomentosa (UT). Unmodified AH Plus (Dentsply, DeTrey, Germany) and MTA Fillapex (Angelus, Londrina, Brazil) were used as controls. UT was incorporated into AH Plus and MTA Fillapex, at concentrations of 2% and 5% of the total weight of these sealers (w/w). Flowability, setting time, and solubility were evaluated following ISO requirements. The pH values were measured at periods of 12, 24, 48 hours, and 7 days. The antimicrobial activity of the sealers against Enterococcus faecalis was analyzed by both direct contact tests in freshly prepared sealers, and after 7 days. The cytotoxicity of the samples was evaluated by the MTT assay, to check Balb/c 3T3 cell viability. The statistical analysis was performed by one-way ANOVA and Tukey's test (p < 0.05). The incorporation of UT was associated with a decrease in flow, for both sealers, an increase in AH Plus setting time, increase in MTA Fillapex pH values, and solubility (after 14 days), for both sealers (p < 0.05). Regarding the antibacterial evaluation, bacterial reduction was reported after incorporation of UT into both AH Plus and MTA Fillapex, up to 7 days after handling of the material (P<0.05). UT incorporation decreased the cytotoxic effects of both AH Plus and MTA Fillapex sealers in a way directly proportional to their respective concentrations (p < 0.05). In conclusion, UT can be added to both sealers to reduce their cytotoxicity, and improve their antibacterial effects, without compromising their original physicochemical properties.

Physical-chemical properties and cytotoxicity analysis of 5 different endodontic sealers / Propriedades físico-químicas e análise da citotoxicidade de 5 diferentes cimentos endodônticos

The aim of this study was to analyze the radiopacity, setting time, flowability, pH, calcium ion release, solubility and cytotoxicity of bioceramic cements Totalfill BC Sealer and Totalfill BC RRM, and compare them with AH Plus, MTA Fillapex and MTA Angelus. The groups were divided and compared among them according to the filling and retro-filling cement functions. Totalfill BC Sealer was compared with AH Plus and MTA Fillapex; and Totalfill BC RRM retrofilling cement with MTA Angelus. For radiopacity analysis, specimens were placed in metal rings measuring 10x1 mm placed on occlusal film together with the aluminum scale. Digora 1.51 software was used to evaluate the digitized images and determine radiographic density. Setting time was tested in accordance with the American Society for Testing and Materials C266-08 standard specifications, but specimens were fabricated in accordance with the International Organization for Standardization 6876: 2001. Flow was tested in accordance with ANSI/ADA No.57/200 specifications. In total 30 acrylic teeth were filled with filling-cements and 20, with (retrograde cavity) retro-filling cements. All teeth were immersed in ultrapure water for pH and calcium ion release measurement (atomic absorption spectrophotometer) for time intervals of 1, 3, 24, 72, 168 and 360 hours. Solubility was tested by scanning and digitizing 50 acrylic teeth twice by Micro- CT, before and after immersion in ultrapure water for time intervals of 168, 360 and 720 hours. The images were reconstructed and volume (mm3) values of samples obtained by means of CTan software (CTan v1.11.10.0, SkyScan). The in vitro effects on cells were analyzed at concentrations of 100, 50, 10, 5, 1 mg/mL, and 0 mg / mLnegative control group and recorded in time intervals of 24, 48 and 72 hours by MTT reduction assay. The results were statistically analyzed by the ANOVA, Tukey, Kruskal-Wallis and Dunn tests (P<0.05). All radiopacity values according to ISO 6876/2001, AH Plus (7.86 mm Al) being the most radiopaque followed by Totalfill BC Sealer (4.84 mm Al), MTA Fillapex (3.41 mm Al), Totalfill BC RRM (6.8 mm Al), and MTA Angelus (6.7 mm Al). The following values were the initial and final setting time (in hours), respectively: AH Plus (8 and 15); Totalfill BC Sealer (11 and 24); MTA Fillapex (13 and 26); MTA Angelus (10 and 120 minutes) and Totalfill BC RRM (3 hours and 22 hours). In flow analysis, the cements behaved as follows: MTA Fillapex (47 mm), Totalfill BC Sealer (41.5 mm), Totalfill BC RRM (33.5 mm), AH Plus (33 mm) e MTA Angelus (17.5 mm) (p < 0.05). pH analysis showed in general the lowest values for AH Plus cement, followed by Totalfill BC RRM, MTA Angelus, MTA Fillapex and Totalfill BC Sealer. AH Plus showed the highest Ca2+ release in time interval 1 hour (1.38 mg/L); MTA Fillapex, in 360 hours (3.81 mg/L); MTA Angelus, 1 hour (1.38 mg/L); Totalfill BC Sealer, 360 hours (6.77 mg/L) and Totalfill BC RRM, 360 hours (3.81 mg/L). Almost all the sealers presented solubility lower than 3% in all periods, as recommended by ISO 6876/2001. Whereas, the MTA Fillapex solubility value was higher than 5% in all periods. Relative to cytotoxicity, all the cements were shown to be toxic at the concentration of 100 mg/mL, however, Totalfill BC Sealer and Totalfill BC RRM showed the best cell viability result compared with the other cements tested. We concluded that all root canal filling and root retro-filling complied with the requisites of radiopacity, setting time, flow, pH, calcium ion release, solubility and cytotoxicity. With the exception of the MTA Fillapex that not only fulfilled the requirement of solubility. Of the sealers, Totalfill BC Sealer was outstanding: it showed the highest pH and Ca2+ release, and lowest cytotoxicity. Among the retrofilling cements, Totalfill BC RRM maintained its high pH, higher Ca2+ release, and lower cytotoxicity. (AU)

O objetivo do presente estudo foi analisar a radiopacidade, tempo de presa, escoamento, pH, liberação de íons cálcio, solubilidade e citotoxicidade dos cimentos biocerâmicos Totalfill BC Sealer e Totalfill BC RRM e compará-los ao AH Plus, MTA Fillapex e MTA Angelus. Os grupos foram divididos e comparados entre si de acordo com as funções dos cimentos de obturação e retro-obturação. Comparamos o cimento obturador Totalfill BC Sealer com os cimentos AH Plus e MTA Filapex, e o cimento retrobturador Totalfill BC RRM com o cimento retrobturador MTA Angelus. Para análise da radiopacidade, os espécimes foram colocados em anéis metálicos medindo 10x1 mm, dispostos sobre um filme oclusal com uma escala de alumínio. O software Digora 1.51 foi utilizado para avaliar as imagens digitalizadas e determinar a densidade radiográfica. O tempo de presa foi realizado de acordo com as especificações da American Society for Testing and Materials C266-08 standard specifications, mas os espécimes foram feitos de acordo com a International Organization for Standardization 6876: 2001. O escoamento foi realizado de acordo com as especificações ANSI/ADA N0 57/2000. Trinta dentes acrílicos foram preenchidos com cimentos obturadores e vinte dentes de acrílico (com cavidade retrógrada) foram preenchidos com cimentos retro-obturadores e imersos em água ultrapura para mensuração do pH e liberação de íons cálcio (espectrofotômetro de absorção atômica) no período de 1, 3, 24, 72, 168 e 360 horas. Para o teste de solubilidade, foram escaneados 50 dentes acrílicos e digitalizados duas vezes pelo Micro-CT, antes e após a imersão em água ultrapura nos períodos de 168, 360 e 720 horas. As imagens foram reconstruídas e o volume (mm3) das amostras foi obtido usando o software CTan (CTan v1.11.10.0, SkyScan). Os efeitos celulares in vitro foram analisados nas concentrações de 100, 50, 10, 5, 1 mg/mL e 0 mg / mLgrupo controle negativo e registados nos períodos de 24, 48 e 72 horas através do ensaio de redução de MTT. Os resultados foram analisados estatisticamente pelos testes ANOVA, Tukey, Kruskal-Wallis e Dunn (p < 0.05). Todos os valores de radiopacidade estavam de acordo com a norma ISO 6876/2001, sendo o AH Plus (7.86 mm Al) o mais radiopaco seguido dos demais cimentos; Totalfill BC Sealer (4.84 mm Al), MTA Filapex (3.41 mm Al), Totalfill BC RRM (6,8 mm Al), MTA Angelus (6,7 mm Al). Os valores obtidos para o tempo de presa inicial e final foram respectivamente, AH Plus (8 e 15 horas), Totalfill BC Sealer (11 e 24 horas), MTA Filapex (13 e 26 horas), Totalfill BC RRM (3 horas e 22 horas) e MTA Angelus (10 e 120 minutos). Na análise de escoamento os cimentos se comportaram da seguinte forma: AH Plus (33 mm), MTA Filapex (47 mm), Totalfill BC Sealer (41,5 mm), Totalfill BC RRM (33,5 mm), e MTA Angelus (17,5 mm) (p < 0.05). A análise do pH mostrou que o cimento AH Plus de um modo geral, foi o que apresentou os menores valores, seguido do Totalfill BC RRM, MTA Angelus, MTA Filapex e Totalfill BC Sealer. A maior liberação de Ca2+ do AH Plus foi no período de 1 hora (1.38 mg/L), MTA Filapex foi em 360 horas (3.81 mg/L), Totalfill BC Sealer 360 horas (6.77 mg/L), Totalfill BC RRM 360 horas (3.81 mg/L) e MTA Angelus em 1 hora (1.38 mg/L). Todos os cimentos apresentaram solubilidade menor que 3% em todos os períodos, como recomendado pela ISO 6876/2001. Entretanto, os valores de solubilidade do MTA Fillapex excedeu mais que 5% em todos os períodos. Com relação à citotoxicidade, todos os cimentos mostraram-se tóxicos na concentração de 100 mg/mL, porém o Totalfill BC Sealer e Totalfill BC RRM apresentaram melhor resultado de viabilidade celular comparado aos demais cimentos testados. Concluiu-se que os cimentos de obturação e retro-obturação cumpriram os requisitos de radiopacidade, tempo de presa, escomento, pH, liberação de íons cálcio, solubilidade e citotoxicidade. Com exceção do MTA Fillapex que não cumpriu somente o requisito de solubilidade. Dos cimentos obturadores, o que melhor se portou foi o Totalfill BC Sealer, apresentando maior pH e liberação de íons cálcio e menor citotoxicidade. Dentre os cimentos retro-obturadores, Totalfill BC RRM foi o que melhor se destacou, mantendo seu pH elevado, possuindo maior liberação de Ca2+ e menor citotoxicidade. (AU)

Human tooth germ stem cell response to calcium-silicate based endodontic cements

OBJECTIVE: The aim of this study was to compare the cytotoxic effects of endodontic cements on human tooth germ stem cells (hTGSCs). MTA Fillapex, a mineral trioxide aggregate (MTA)-based, salicylate resin containing root canal sealer, was compared with iRoot SP, a bioceramic sealer, and AH Plus Jet, an epoxy resin-based root canal sealer. MATERIAL AND METHODS: To evaluate cytotoxicity, all materials were packed into Teflon rings (4 mmµ3 mm) and co-cultured with hTGSCs with the aid of 24-well Transwell permeable supports, which had a pore size of 0.4 µm. Coverslips were coated with MTA Fillapex, iRoot SP and AH Plus Jet and each coverslip was placed onto the bottom of one well of a six-well plate for scanning electron microscopy (SEM) analysis. Before the cytotoxicity and SEM analysis, all samples were stored at 37ºC and at 95% humidity and 5% CO2 for 24 hours to set. The cellular viability was analyzed using MTS test (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium). The cytotoxic effects and SEM visualization of the tested materials were analyzed at 24-hour, 72-hour, one-week and two-week periods. RESULTS: On the 1st day, only MTA Fillapex caused cytotoxicity compared to negative control (NC) group (p<0.008). No significant difference was observed between the other tested materials at this period (p>0.05). After 14 days of incubation with the test materials, MTA Fillapex exhibited significantly higher cytotoxicity compared with iRoot SP, AH Plus Jet and the NC group (P<0.008). In the SEM analysis, the highest levels of cell attachment were observed for iRoot SP and the control group. After 24 hours, MTA Fillapex reduced the number of cells attached to the surface. CONCLUSIONS: Within the limitations of this study, sealers exerted different cytotoxic effects on hTGSCs. Although all materials ...

Long-term cytotoxic effects of contemporary root canal sealers

Objectives: The aim of the present study was to investigate the effects of root canal sealers on the cytotoxicity of 3T3 fibroblasts during a period of 5 weeks. Material and Methods: Fibroblasts (3T3, 1×105 cells per well) were incubated with elutes of fresh specimens from eight root canal sealers (AH Plus, Epiphany, Endomethasone N, EndoREZ, MTA Fillapex, Pulp Canal Sealer EWT, RoekoSeal and Sealapex) and with elutes of the same specimens for 5 succeeding weeks after immersing in simulated body fluid. The cytotoxicity of all root canal sealers was determined using the MTT assay. Data were analyzed using ANOVA and Tukey's test. Results: RoekoSeal was the only sealer that did not show any cytotoxic effects (p<0.05). All the other tested sealers exhibited severe toxicity initially (week 0). MTA Fillapex remained moderately cytotoxic after the end of experimental period. Toxicity of the other tested sealers decreased gradually over time. The evaluated root canal sealers presented varying degrees of cytotoxicity, mainly in fresh mode.Conclusions: RoekoSeal had no cytotoxic effect both freshly mixed and in the other tested time points. MTA Fillapex was associated with significantly less cell viability when compared to the other tested root canal sealers.

Effect of time of extraction on the biocompatibility of endodontic sealers with primary human fibroblasts

The aim of this work was to evaluate the effects of different times of extraction on the cytotoxicity of six representatives of different root canal sealer groups-Real Seal SE, AH Plus, GuttaFlow, Sealapex, Roth 801, and ThermaSeal Plus-with human gingival fibroblasts. The materials were prepared according to manufacturers' specifications, and were incubated in culture medium (DMEM) at 37ºC for 1, 7, 14, 21, and 28 days, with daily washing, to simulate periodontal ligament clearance. Human fibroblasts were exposed to the final extracts at 24 hours, and cell viability was determined by MTT assay, with exposure to unconditioned DMEM as a negative control. Statistical analysis comparing cytotoxicities at each exposure time was performed by ANOVA with Scheffé adjustment for multiple comparisons at a 95% confidence level. Results indicated that GuttaFlow was significantly less cytotoxic than all other sealers (p < 0.05) at 1 day of extraction. After 7 days of extraction, cell viability for GuttaFlow was significantly increased as compared with that of all groups except sealer AH Plus. At day 14, cytotoxicity of Sealapex was significantly higher than that of all other sealers (p < 0.05). At days 21 and 28, there were no significant differences in cytotoxicity among sealer groups. All materials presented some level of cytotoxicity to fibroblasts, while GuttaFlow was the least cytotoxic sealer tested. However, the cytotoxicity of all materials seemed to decrease similarly in a time-dependent manner.

Biocompatibility of RealSeal, its primer and AH Plus implanted in subcutaneous connective tissue of rats

OBJECTIVE: This study tested rat connective tissue response to RealSeal, RealSeal primer or AH Plus after 7, 15, 30, 60 and 90 days of implantation. MATERIAL AND METHODS: Thirty Wistar rats had subcutaneous sockets created on their back and received four implants each of polyethylene tubes containing one of the materials tested according to the groups: AH (AH Plus Sealer); RS (RealSeal Sealer); RP (RealSeal Primer); CG (control group - empty tube). After histological processing, sections were analyzed to identify the presence of neutrophils, lymphocytes and plasma cells, eosinophils, macrophages and giant cells, as well as fibrous capsule and abscesses, by an examiner using light microscope. Kruskal-Wallis and multiple-comparisons test were used for statistical analysis. Significance level was set at 5 percent. RESULTS: Lymphoplasmacytic infiltrate scores significantly higher than those of the control group were observed at 14 and 60 days in AH group, and at 90 days in RS group (p<0.05). There were no differences in terms of presence of macrophages, giant cells, eosinophils, neutrophils or fibrosis. AH Plus group scored higher for abscesses at 7 days than after any other period (p=0.031). RP group scored higher for lymphoplasmacytic infiltrate at 14 days than at 90 days (p=0.04). CONCLUSION: The main contribution of this study was to demonstrate that issues involved with tissue tolerance of a Resilon-containing sealer, RealSeal Sealer, cannot be attributed to its primer content.

An endodontic sealer induces a pathological condition when associated with persistent tissue toxicity and presence of myofibroblasts

The aims of this study were to evaluate the ratio between inflammatory reactions induced by four endodontic sealers and the occurrence of fibrosis and the number of myofibroblasts with positivity to α-smooth-actin muscle (α-SMA). Polyethylene tubes were filled with a root canal sealer (Endofill, AH Plus, Acroseal and Epiphany) and inserted into 4 site at the dorsal region of 24 Wistar rats; 2 empty tubes (control) were grafted in 6 rats. After 7, 21, and 45 days, 8 animals were euthanized, providing 6 specimens per test group and 2 specimens from the control group. The fragments were subjected to histological processing and immunohistochemical analysis for anti α-SMA protein. All specimens, except those from the control group, presented severe inflammatory reaction on the 7th postoperative day, which also coincided with a large number of myofibroblasts. On the 21st and 45th days post-surgery, the inflammatory reaction induced by Endofill, AH Plus and Acroseal decreased significantly, which coincided with reduced presence of myofibroblasts and usual collagen deposition. In contrast, in the group filled with Epiphany, significant inflammatory cell infiltrate was present in all analyzed periods. The persistence of an inflammatory reaction induced by endodontic sealer may also induce the development of fibrosis in combination with presence of myofibroblasts.

O objetivo deste estudo foi avaliar a relação entre reação inflamatória induzida por quatro cimentos endodônticos e a presença de fibrose e quantidade de miofibroblastos que apresentam positividade para α-SMA. Tubos de polietileno foram preenchidos com o cimento (I: Endofill; II: AH Plus; III: Acroseal; IV: Epiphany) e inseridos em 4 regiões do dorso de 24 ratos Wistar, enquanto 2 tubos vazios (V - controle) foram inseridos em 6 ratos. Após 7, 21 e 45 dias, oito animais foram sacrificados obtendo 6 indivíduos por grupo e 2 para o grupo controle. Os fragmentos foram submetidos ao processamento histológico e à análise imuno-histoquímica para a proteína anti-α-SMA. Todos os grupos, exceto o controle, demonstraram notável reação inflamatória no 7º dia pós-operatório, que também coincidiu com uma grande quantidade de miofibroblastos. No 21º e 45º dia pós-operatório, a reação inflamatória induzida pelo Endofill, AH Plus e Acroseal diminuiu significativamente, o que coincidiu com reduzida presença de miofibroblastos e deposição de colágeno normal. Em contraste, no grupo Epiphany, infiltrado inflamatório significativo esteve presente em todos os períodos analisados. A persistência do infiltrado inflamatório induzido por cimento endodôntico pode também provocar uma fibrose associada com a presença de miofibroblastos.

Cytotoxicity evaluation of two root canal sealers and a commercial calcium hydroxide paste on THP1 cell line by Trypan Blue assay

OBJECTIVE: The aim of this investigation was to evaluate the cytotoxicity of two brands of root canal sealers, epoxy-resin based and zinc oxide-eugenol based, and one commercial calcium hydroxide paste on a monocyte cell line THP-1. MATERIAL AND METHODS: Undiluted (crude extract) and diluted extracts to 10 percent, 1 percent, 0.1 percent, 0.01 percent, 0.001 percent and 0.0001 percent of the sealers were tested for cytotoxicity to THP-1 cells using the trypan blue assay. Extracts were obtained according to ISO standard. Data were analyzed statistically by the Kruskal-Wallis and Mann-Whitney tests at 5 percent significance level. RESULTS: Crude extract of AH Plus and Fill Canal killed approximately 90 percent of THP-1 cells versus 36 percent of THP-1 cells killed by L&C crude extract (p<0.05). Ten-fold dilutions of L&C, Fill Canal and AH Plus killed 24, 35 and 61 percent of THP-1 cells (p<0.05), respectively. Dilutions lesser than 1 percent caused minimal cell death as compared to the control groups (p>0.05), except for L&C 1 percent extract. CONCLUSIONS: The results revealed that the L&C paste crude extract was less cytotoxic to THP-1 cells than AH Plus or Fill Canal crude extracts.

L929 cell response to root perforation repair cements: an in vitro cytotoxicity assay

This study compared the cytotoxicity of an experimental epoxy-resin and calcium hydroxide-based cement (MBPc), gray mineral trioxide aggregate (MTA) and white mineral trioxide aggregate (WMTA) using the agar overlay method with neutral red dye. L929 cells were seeded into 6-well culture plates where 48-h set test materials were placed on the agar overlay, in triplicate. Teflon and natural rubber served as negative and positive controls. After an incubation period of 24 h at 37ºC in a humidified atmosphere of 5 percent CO2 in air, a discolored area around the samples and the positive controls could be observed and measured per quadrant. The mean values were compared and converted into grades to classify the results according to the table of cytotoxicity grades according to the Standard Operating Procedures (SOP) of the Oswaldo Cruz Foundation, Brazil. The nonviable cell areas and the morphological changes in the cells were observed with an inverted microscope. The results showed grade 1 (slight) for the two types of MTA (p>0.05) and grade 2 (mild) for the MBPc (p<0.001). All samples met the requirements of the test as none of the cultures showed reactivity higher than grade 2.

O objetivo deste estudo foi comparar a citotoxicidade de um cimento experimental à base de resina epóxica e hidróxido de cálcio (MBPc), do agregado trióxido mineral (MTA) cinza e do MTA branco, utilizando o ensaio de difusão em agar com o corante vermelho neutro. Células L929 foram semeadas em placas de 6 poços e sobre elas a camada de agar, onde foram colocados os materiais endurecidos por 48 h, em triplicata, além de teflon como controle negativo e látex como controle positivo. Após 24 h em estufa umidificada a 37ºC com 5 por cento CO2, um halo claro se formou ao redor das amostras e dos controles positivos. As medidas foram tomadas, por quadrante, e as médias foram comparadas e convertidas em graus para qualificar os resultados, de acordo com a tabela de grau de citotoxicidade do POP/FIOCRUZ. As zonas de inibição e as alterações da morfologia celular foram avaliadas sob microscópio invertido. Os resultados revelaram grau 1 (leve) para os dois tipos de MTA (p>0,05) e grau 2 (branda) para o MBPc (p<0,001). Todas as amostras foram consideradas satisfatórias, pois nenhuma cultura exposta aos cimentos revelou toxicidade superior ao grau 2.

Cytotoxicity evaluation of four endodontic sealers

This study evaluated in vitro the cytotoxicity of four root canal sealers (Topseal, EndoRez, TubliSeal and Kerr Pulp Canal Sealer E.W.T.) and their effects on reactive oxygen/nitrogen intermediate induction by mouse peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 mL 10 mM phosphate-buffered saline, washed twice and resuspended (106 cells/mL) in appropriate medium for each test. Cytotoxicity was determined by the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. Sealer suspensions were obtained in two different concentrations from each material: 18 mg/mL and 9 mg/mL, established according to compatibility parameters following MTT assay. Comparing the sealers, H2O2 release at concentrations of 9 mg/mL and 18 mg/mL was similar: Topseal > positive control (medium + cells + 5 mg/mL zimozan solution) > EndoRez > TubliSeal > Kerr Pulp E.W.T. > negative control (medium + cells). NO release at concentration of 9 mg/mL was: positive control (medium + cells + 10 µg/mL LPS solution) > Topseal > Kerr Pulp E.W.T. > TubliSeal = EndoRez > negative control (medium + cells); at concentration of 18 mg/mL was: positive control > Topseal > Kerr Pulp E.W.T > TubliSeal > EndoRez > negative control. Based on the results, it may be concluded that Topseal presented the highest cytotoxicity among the tested sealers, releasing higher concentrations of NO and H2O2 in macrophage culture.

Este estudo avaliou in vitro a citotoxicidade de quatro cimentos obturadores (Topseal, EndoRez, TubliSeal e Kerr Pulp Canal Sealer E.W.T) e seus efeitos na liberação de reativos intermediários do oxigênio e do nitrogênio em cultura de macrófagos peritoniais de ratos.Tioglicolato foi utlizado para se obter células peritoneias de camundongos. A cavidade peritoneal foi irrigada com 5 mL de solução salina 10 mM. As células foram lavadas duas vezes e foi feita uma suspensão (106 células/mL) em meio apropriado para cada um dos testes. A citotoxicidade dos cimentos foi determinada pela presença de peróxido de hidrogênio (H2O2) e óxido nítrico (NO) pela oxidação peroxidase-dependente do vermelho fenol e pela reação de Griess, respectivamente. Suspensões de cimento foram obtidas em duas diferentes concentrações para cada material: 18 mg/mL e 9 mg/mL, estabelecidas previamente pelo teste de viabilidade celular MTT. Comparando os cimentos, a liberação de H2O2 foi similar nas duas concentrações: Topseal > controle positivo (meio + células + Zimozan a 5mg/mL ) > EndoRez > TubliSeal > Kerr Pulp E.W.T. > controle negativo (meio + células). A liberação de NO na concentração de 9 mg/mL foi: de 9 mg/mL foi: controle positivo (meio + células + solução de LPS a 10 »g/mL) > Topseal > Kerr Pulp E.W.T. > TubliSeal = EndoRez > controle negativo (meio + células); e na concentração de 18 mg/mL; e na concentração de 18 mg/mL: controle positivo > Topseal > Kerr Pulp E.W.T > TubliSeal > EndoRez > controle negativo. Baseado nos resultados, pode-se concluir que o Topseal apresentou a maior citotoxicidade dentre os cimentos avaliados, liberando as mais altas concentrações de NO e H2O2 em cultura de macrófagos.