Cytotoxicity and Genotoxicity of Root Canal Sealers Based on Mineral Trioxide Aggregate

Cytotoxicity and Genotoxicity of Root Canal Sealers Based on Mineral Trioxide Aggregate

Basic Research—Technology Cytotoxicity and Genotoxicity of Root Canal Sealers Based on Mineral Trioxide Aggregate Claudia V. Bin, DDS, MSc, Marcia C...

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Basic Research—Technology

Cytotoxicity and Genotoxicity of Root Canal Sealers Based on Mineral Trioxide Aggregate Claudia V. Bin, DDS, MSc, Marcia C. Valera, DDS, PhD, Samira E.A. Camargo, DDS, PhD, Sylvia B. Rabelo, DDS, MSc, Gleyce O. Silva, DDS, MSc, Ivan Balducci, MSc, and Carlos Henrique R. Camargo, DDS, PhD Abstract Introduction: MTA has good biological properties, and it is a mineralization-inducing material with different indications in endodontics. Initially this material was not recommended as root canal sealer. However, a resin sealer based on mineral trioxide aggregate (MTA Fillapex) was recently released with this indication. Because MTA is in contact with the periodontal tissues, bone, and pulp, it is important to know its cytotoxic and genotoxic effects. The purpose of this study was to evaluate the cytotoxicity and genotoxicity of MTA canal sealer (Fillapex) compared with white MTA cement and AH Plus. Methods: Chinese hamster fibroblasts (V79) were placed in contact with different dilutions of culture media previously exposed to such materials. Cytotoxicity was evaluated by methol-thiazol-diphenyl tetrazolium assay in spectrophotometer to check the viability rate and cell survival. The genotoxicity was accessed by the micronucleus formation assay. Cell survival rate and micronuclei number were assessed before and after exposure to cement extracts, and the results were statistically analyzed by Kruskal-Wallis and Dunn tests (P < .05). Results: The results showed that the cell viability remained above 50% in white MTA group for all dilutions. AH Plus induced an intermediate cytotoxicity in a dilution-dependent manner, followed by Fillapex MTA. Conclusions: White MTA group was the less cytotoxic material in this study. Both AH Plus and Fillapex MTA sealer showed the lowest cell viability rates and caused an increased micronucleus formation when compared with control untreated group. (J Endod 2012;38:495–500)

Key Words Biocompatibility, cytotoxicity, genotoxicity, MTA Fillapex, White MTA

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ndodontic therapy aims at the elimination of residual pulp, tissue breakdown products, and microorganisms present inside the root canal system, followed by hermetic filling as possible (1). The seal must be carried out by using filling materials that do not interfere and, preferably, stimulate the process of apical and periapical repair. Tissue mineralization induction is another important request of endodontic sealers (2). AH Plus is an epoxy resin–based sealer widely used in endodontics. It presents good sealing, visible radiopacity, easy handling, good resistance, dimensional stability, high flow, and low solubility, besides good adhesion to root canal walls. It has satisfactory biocompatibility, inducing an initial mild inflammatory reaction on surrounding tissues. This feature might be related either to the release of formaldehyde during polymerization or to another proven cytotoxic component, bisphenol A, which is present in its composition (2, 3). Mineral trioxide aggregate (MTA) was introduced in endodontics in 1993 by Torabinejad. It has been used during retrofilling procedures, root and furcation perforations, in direct pulp capping, and also in teeth with incomplete root formation (4). Calcium oxide is one of MTA’s main components. When in presence of moisture, it promotes calcium hydroxide formation, leading to a high alkaline pH, which might explain the antimicrobial action of MTA (4–7). The alkaline pH has a destructive effect on protein structures of some microorganisms, promoting some cell membrane enzyme inactivation and loss of biological activity, followed by membrane integrity damage (5). In addition, the alkaline pH (10.2–12.5) might also be responsible for the formation of mineralized tissue when MTA is placed in contact with organic tissues (6). The high pH activates and stimulates the expression of the enzyme alkaline phosphatase, favoring the formation and deposition of mineralized tissue and then allowing the formation of a fibrous capsule, with lower inflammatory reaction (6, 8). In 1999, Holland et al (5) suggested the use of MTA cement as a filling material. However, all of the properties and activities described by them refer to white MTA cement, which has unsatisfactory physical characteristics when used as a root canal sealer. Recently, a new MTA root canal sealer has been introduced in the market, MTA Fillapex. It is important to know the cytotoxic and biological properties of this new material when it is compared with white MTA and with other root canal sealers. When assessing the biocompatibility of a sealer, in vitro cytotoxicity tests can be used. They might be readily reproducible, relevant, and appropriate for the evaluation of some aspects that are related to biological compatibility. Bromide (3-(4,5dimethylthiazol-2il)-2,5-diphenyl tetrazolium (MTT) test is a quick and accurate assay. This test indicates not only the number of viable cells in a sample but also the level of metabolic activity, because it is based on the activity of enzymes that are found in viable cells such as succinyl dehydrogenase (9).

From the Department of Restorative Dentistry, S~ao Paulo State University (UNESP), S~ao Jose dos Campos, S~ao Paulo, Brazil. This study was supported by CAPES - Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nıvel Superior, Brazil. Address requests for reprints to Dr Marcia C. Valera, Department of Restorative Dentistry, S~ao Paulo State University - UNESP, Av. Eng. Francisco Jose Longo, 777, 12245-000, S~ao Jose dos Campos, SP, Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2011.11.003

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Basic Research—Technology Even though the literature has many studies concerning the cytotoxicity of endodontic sealers, it appears that just a few of them assess the potential damage that these materials can cause on cellular DNA. Because the damage to the cell genome can significantly decrease the self-tissue repair potential or even can cause the development of a neoplasm in a long-term (10), further research still needs to be performed. In vitro genotoxicity tests are designed to detect products and components that induce damage to genetic material of cells such as DNA breaks, gene mutation, chromosomal breaks, and alterations in DNA repair ability, an important carcinogen indicator (10). The micronucleus assay (MNT) can be used to detect chromosomal mutations, clastogenicity, and aneugenicity (11), because chromosomes and their fragments might lead to micronuclei formation during interphase of the cell cycle (12). This study evaluates by MTT assay and MNT the cytotoxicity and genotoxicity of MTA-based sealer (MTA Fillapex; Angelus, Londrina, PR, Brazil), compared with white MTA cement (Angelus) and AH Plus (Dentsply, S~ao Paulo, SP, Brazil), on Chinese hamster fibroblasts (V79).

Materials and Methods Preparation of Extracts The following materials were used: white MTA (MTA Branco; Angelus), MTA Fillapex (Angelus), and AH Plus (Dentsply). The main components list of tested material is shown in Table 1. The sealer samples were prepared in 24-well plates (16.2 mm in diameter, 2 mm high), and they were incubated at 37 C for 12, 48, or 72 hours immediately after mixing. Specimens were then covered with 2.5 mL cell culture Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, penicillin, and streptomycin and incubated in the dark for 24 hours at 37 C. After incubation, these original extracts (1:1) were then serially diluted in cell culture medium before testing. Cytotoxicity Testing Chinese hamster fibroblasts (V79) were routinely cultivated in DMEM medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37 C and 5% CO2. Cells were seeded at 5  103 cells/well in 96-well plates and incubated for 24 hours at 37 C. Then the cells were exposed to 200 mL of tested materials original extracts and also to their serial dilutions. After 24 hours, the medium was removed. Cell survival was determined by using the MTT assay (Sigma-Aldrich, St Louis, MO). A volume of 100 mL of MTT solution was added to each well, and cells were incubated for an additional 1 hour. The resulting formazan crystals were dissolved when removing the culture medium and adding 100 mL of dimethyl sulfoxide solvent (Sigma-Aldrich) to each well. The plates were shaken at room temperature for 10 minutes to dissolve the crystals and were then taken to the reader. The enzyme inhibition quantification was made by using a spec-

trophotometer (Asys Hitech GmbH, Eugendorf, Austria) at 570 nm. Four replicate cell cultures were exposed to each of the extract serial dilutions in 3 independent experiments. The absorbance value readings were normalized to untreated control cultures (100%), and differences between median values were statistically analyzed by using KrsukalWallis test for comparison among groups and Dunn multiple comparison post test, with significance of P <.05.

MNT In Vitro First, 3  105 cells were cultivated on microscopic glass slides in 4 mL DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin for 24 hours at 37 C and 5% CO2. Cell cultures were then exposed to different dilutions of the sealer extracts White MTA, MTA Fillapex, and AH Plus for 24 hours and fixed in ethanol afterwards. The micronuclei were determined microscopically in 1000 cells/slide of 2 parallel cultures (slides) per concentration as described by Camargo et al (13) in 2009. Ethyl methanesulfonate (EMS) was used as a positive control. At least 4 slides derived from 2 independent experiments were analyzed, and differences between median values were statistically analyzed by using the Kruskal-Wallis test for comparisons among groups at the .05 level of significance.

Results Cytotoxicity of Sealers The results obtained from a spectrophotometer indicate the activity of cellular metabolism. They represent the inhibition of succinyl dehydrogenase activity, which is caused by the contact between cells and sealer extracts serially diluted to 1:1 (original extract), 1:2, 1:4, 1:8, 1:16, and 1:32. The percentage of cell viability was considered optimal when the average value obtained was 50% or higher. It could be observed by the cell survival mean values that White MTA kept cell viability rates above 70% for all dilutions. Therefore, it was not classified as a cytotoxic material. On the other hand, MTA Fillapex was severely cytotoxic at highest concentrations (1:1, 1:2, and 1:4), and so was AH Plus (except the 1:4 dilution at 12 and 72 hours) (Fig. 1). Original extracts of MTA Fillapex reduced survival rates of V79 to 1.53%. In the 48-hour group, 77.14% of cells died after exposure to 1:2 dilution of the original extract. The average of the control group was expressed as percentage (100%), according to each evaluation period. When comparing the different setting times for each dilution of sealer extracts, it could be observed that there was no significant direct correlation between the dilutions. However, we can observe from the data that White MTA seems to play its best action within 48 hours after setting. MTA Fillapex showed lower levels of cell viability 12 hours after manipulation (1:2, 1:4, and 1:8 dilutions) when compared with 24hour and 72-hour groups at the same dilutions. AH Plus groups showed their highest cytotoxicity levels at the 48-hour group (1:2 and 1:4 dilutions). MTA Fillapex extracts were significantly more toxic to V79 cells than White MTA at the dilutions up to 1:8 (P < .001). White MTA was able to increase cell proliferation to values as high as the control

TABLE 1. Main Components of Tested Sealers Material

Components

White MTA (Angelus, Londrina, PR, Brazil)

Tricalcium silicate, dicalcium silicate, tricalcium aluminate, tetracalcium aluminoferrite, bismuth oxide, iron oxide, calcium oxide Natural resin, salicylate resin, diluting resin, bismuth trioxide, nanoparticulated silica, MTA, pigments Paste A: bisphenol-A epoxy resin, bisphenol-F epoxy resin, calcium tungstate, silica, zirconium oxide, iron oxide pigments. Paste B: dibenzyldiamine, aminoadamantane, tricyclodecanediamine, calcium tungstate, zirconium oxide, silica, silicone oil

Fillapex MTA (Angelus, Londrina, PR, Brazil) ~o Paulo, SP, Brazil) AH Plus (Dentsply, Sa

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Figure 1. Graphical representation (mean  standard deviation) (%) of the sealer’s cytotoxicity in V79 cells after exposure to extracts, according to type of dilution and setting time. Columns represent the mean cell viability expressed as percentage. Original extracts (1:1) were serially diluted in fresh media as indicated. Cell cultures were exposed for 24 hours, and cellular survival in treated and untreated cell cultures was determined in quadruplicate in 3 independent experiments (n = 12).

(shown in the 1:32 dilution) even when undiluted. In this study, AH Plus cytotoxicity was at an intermediate level; higher dilutions of White MTA (1:16 and 1:32) showed similar performance to their more concentrated extracts (1:1 and 1:2) and to 1:1 and 1:2 dilutions of MTA Fillapex.

Formation of Micronuclei Micronuclei formation in V79 cells exposed to sealers extracts was analyzed. EMS, which was used as a positive control, increased the number of micronuclei in treated cultures about 10-fold compared with those detected in untreated control. In contrast, no increase in the number of micronuclei was detected with White MTA extract dilutions tested here (P > .05). AH Plus and MTA Fillapex diluted extract specimens increased the number of micronuclei in V79 cell cultures

compared with untreated control. The 1:4 dilutions of AH Plus and MTA Fillapex (48 hours) were the most genotoxic materials, creating micronuclei 8 times higher than the untreated control and similar to EMS (P < .05). At 1:8 and 1:16 AH Plus dilutions (48 hours) the increase was similar to negative control (untreated) (Fig. 2). Higher concentrations of AH Plus and MTA Fillapex tested cytotoxic in V79 cells, and micronuclei could not be quantified.

Discussion An MTA-based sealer (MTA Fillapex) was recently released to be used in root canal filling. However, when analyzing the chemical composition supplied by the manufacturer, it is worth emphasizing that this is a resin cement containing MTA. For that reason, it was expected that Fillapex MTA acted as a repairer.

Figure 2. Induction of micronuclei in V79 cells after exposure to sealers. The numbers of micronuclei caused by the materials were obtained from 2 treated cell cultures (n = 4), and bars represent medians (25% and 75% percentiles). Median numbers of micronuclei were also calculated for untreated controls (UC) and 5 mmol EMS. Statistically significant differences between untreated and treated cell cultures are indicated by asterisks.

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Basic Research—Technology In our study, the biocompatibility of MTA repairer (White MTA) and MTA sealer (Fillapex MTA) was assessed by the MTT cytotoxicity test that determines the number of viable cells as a function of their mitochondrial activity. Our results showed that White MTA was considered a biocompatible material because it has kept the cell viability rates above 50% for all tested dilutions. These data are consistent with those presented in previous studies (13, 14). The low cytotoxicity and good biocompatibility of this cement have been attributed to the main components present in White MTA, which are also the main components of dentin tissue, including tricalcium silicate, calcium oxide, oxide, silicate, and tricalcium aluminate (15). White MTA has shown statistical difference from the other cements in 3 dilutions (1:1, 1:2, and 1:32). All of the tested cements statistically differed among themselves at 1:4 and 1:8 dilutions. MTA sealer led to statistically inferior cell viable rates when compared with other materials at the 1:16 dilution. The data were not linear when comparing the setting times, preventing a significant correlation between the dilutions. However, White MTA seemed to play the best action during the 48-hour period, where V79 cells had shown the highest viability levels. Balto (16) found that human periodontal ligament fibroblasts showed altered morphology when in contact with a newly manipulated MTA (ProRoot MTA; Dentsply). Similar to positive control, the surface of the former cells had many vacuoles and a few bubbles, common characteristics of injured cells that were in the process of death. When in contact with ProRoot MTA cement before hardening time, fibroblasts showed varying morphology, with few cells adhered to the surface of the cement. On the other hand, when these fibroblasts were placed in direct contact with the same cement after complete hardening, a more homogeneous morphology could be noticed; this was similar to the one observed in the negative control group (which had lack of contact with any testing material). In the present study, no specific morphologic test was performed on V79 cells; however, during the reading of MNT slides we could see that fibroblasts that were exposed to White MTA exhibited similar morphology to the control group cells. Besides that, there is the finding that the rates of V79 fibroblast viability exposed to White MTA were high, even within the 12-hour group (the shortest after-manipulation period analyzed). In a similar study that also used the cement repairer MTA (White MTA-Angelus), Aranha et al (17) showed that the MDPC-23 cells exhibited odontoblast slightly elongated profile and thin cytoplasmic processes originating from the cell body, similar to those observed in the group in which the cells were kept in contact with DMEM culture medium. Balto (16) has verified that the setting time does not significantly influence the cytotoxicity of the material. Differences in the results showed by Balto in 2004 and by Aranha et al (17) in 2006 might have happened because of variable composition of tested materials. It is known that White MTA has a lower concentration of Fe2O3 (0.15%) when compared with ProRoot MTA (2.5%) (17) and thus might determine different cytotoxic effects. This study suggested that the greater cytotoxicity of recently manipulated MTA was due to the increased release of toxic components from the cement in aqueous solutions, which would have affected the cells’ morphology and their ability to adhere. Thus, the toxic effect of MTA cement repair before hardening can determine the minimum irritant material even when used in direct capping, because this cement is applied on the pulp immediately after manipulation with incomplete setting. Another important factor of sealer in vivo application is that the release of some components to the surrounding tissues might result in benefits to the healing process. In this context, it was shown that the calcium oxide present in the composition of various MTA-based cements can react with tissue fluids to form calcium hydroxide. Holland 498

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et al (5) and Yaltirik et al (18) observed the presence of dystrophic calcification in connective tissue adjacent to the cement after implantation of ProRoot MTA in the dorsal subcutaneous tissue of rats, suggesting the potential for hard tissue formation by this material. When trying to determine the composition of MTA salt dissolved in water, FridLand and Rosado (19) have identified calcium ions and OH being released as main component; the latter is responsible for the MTA’s high alkalinity (pH from 11.94 to 11.99). After manipulation MTA suffers a hydration reaction that results in the formation of calcium hydroxide and subsequent ionic dissociation in Ca+2 ions and hydroxyl (OH ), which is responsible for the increase of pH and elevated calcium concentration in medium (7). The lower the pH of the cement, the smaller layer of superficial necrosis was formed when it was placed in contact with the tissue. However, the alkaline pH induces the expression of alkaline phosphatase activity by fibroblasts, which is associated with the mineralization process (18). By using a cytotoxicity test that uses the agar diffusion test with the neutral red dye, Torabinejad et al (20) reported an area of lysing cells around the MTA sample both after handling and after the material setting. Saidon et al (21) also found areas of denatured proteins and dead cells just below the MTA and cells with morphologic changes and a large amount of viable cells present on most cards. These findings are consistent with the formation of a necrosis tissue layer located just below the MTA repairer (22). According to Perez et al (23), a lower cell growth was observed in the presence of White MTA, even though it has been proved to be biocompatible. However, Camargo et al (13) showed that there were no statistically significant differences between Gray MTA and White MTA; the latter held a slight reaction on pulp cells during exposure to these original extracts (1:1) and to the dilution of 1:2 when compared with White MTA. According to our study, the most cytotoxic cement was MTA Fillapex, which has severely reduced the cell survival rates, even when they were exposed to an intermediate dilution (1:4). However, it was observed that this cement developed the best behavior after 48 hours even at less diluted concentrations of 1:2 and 1:4, where we could see an increasing level of cell viability. It has been difficult to discuss our results obtained with the tested materials because of the lack of previous studies. It is a newly released product in the market and is not yet widespread. However, these results suggest that the cytotoxic behavior presented by MTA Fillapex might be a consequence of resin components such as salicylate present in its composition. A previous study that evaluated the effect of resin salicylate on human fibrosarcoma cell line (HT-1080) by using MTT assay showed 25% of cellular apoptosis after 24 hours of exposure. On the other hand, there are findings where the cells on histologic examination presented clear signs of apoptosis such as cell rounding, shrinkage, presence of vacuoles and fragments of genetic material in the cytoplasm (24, 25). The mechanism by which this apoptosis occurs is still unclear, but this study showed that the higher the concentration of salicylate, the higher is the cell death rate (25). This finding is consistent with the results of our study, because the most concentrated dilutions of MTA Fillapex levels have caused evident cytotoxicity and cell death. In this study, AH Plus cement cytotoxicity was at an intermediate level; higher dilutions (1:16 and 1:32) showed similar performance to concentrated White MTA dilutions (1:1 and 1:2) and to less diluted (1:1 and 1:2) MTA extracts. This result is in agreement with previous studies (3) that have also observed a severe cytotoxic effect of AH Plus within 48 hours after setting (1:2 and 1:4 dilutions). The authors also observed that AH Plus was very cytotoxic immediately after handling JOE — Volume 38, Number 4, April 2012

Basic Research—Technology (0 hour) and 24 and 48 hours after setting. However, after 7 and 30 days of hardening, there was no decrease in cell proliferation after direct exposure to the cement. The cytotoxic effects were also reported for AH 26 and AH Plus after a 24-hour hardening period (26). On the other hand, Pinna et al (27) observed after 72 hours of exposure that the toxicity has increased over time. However, AH Plus cement was more cytotoxic on different cell lines according to Tai et al (28). This can be explained by the fact that AH Plus still presents in its composition a mutagenic substance, the epoxy resin, which also displays cytotoxic profile, especially at slightly diluted concentrations (29). Although the manufacturer of cement-based epoxy resin AH Plus claims that this material does not release formaldehyde as its precursor AH 26, their cytotoxicity can be attributed to the minimum amount of the amine component added to the composition to accelerate curing epoxy (26). The results of AH Plus can also be explained by the variation in the hardening time, which in turn is influenced by factors such as humidity and temperature conditions as well as the removal of specimens. According to the results of this research, the release of resin constituents should occur mainly during the 12 hours after the hardening of cement, where we recorded the lowest cell viability rates (for the dilutions 1:2, 1:4, and 1:8). The use of genotoxicity tests is essential for assessing the risks caused by toxic materials on human genetic material (30). This study used MNT, which is based on loss of entire chromosomes or their fragments during cell mitosis that are not reinstated by the nucleus after cell division and therefore are transformed into smaller nuclei or micronuclei. EMS was used as a positive control because of its high genotoxic potential and its ability of intensely stimulating the formation of micronuclei in V79 cells (12, 29). Some dilutions of tested materials (AH Plus, 1:4, 1:8, and 1:16; MTA Fillapex, 1:8 and 1:16; White MTA, 1:1) were chosen according to the results of a preliminary study. The genotoxicity test was performed only with AH Plus and MTA on their highest cytotoxic concentrations. White MTA was excluded from the genotoxic assays because of high rates of cell viability reached by this cement in MTT test (at all tested dilutions and times). However, the 1:1 dilution (12 hours) was used as a comparison standard. In this study, White MTA was the one that has induced less micronucleus formation on V79 cells. Even at the 1:1 dilution it slightly stimulated the formation of micronuclei (from 11 to 17 micronuclei), similarly to untreated control group, where the number of micronuclei was between 7–10 micronuclei. Several studies have indicated the lack of genotoxicity of various concentrations (1–1000 mg/mL) of both Portland (gray and white) and ProRoot MTA cement (Gray and White) (10, 11). It might have happened because of their similar composition, because most of their constituents were common (31). Other studies have reported that exposure of human lymphocytes to MTA and Portland cement (White and Gray) have not caused genetic damage in these cells at all tested concentrations (21, 22). It might be because both Portland cements have similar chemical composition (31), except the lower iron content in the white cement that characterizes its color. In this study, AH Plus and MTA Fillapex, both at 1:4 dilution and hardening of 48 hours, were able to increase 8-fold the formation of micronuclei in V79 cell culture, compared with the nontreated control group. These data enabled the observation that the 48-hour postmanipulation time for both cements at 1:4 was not favorable, allowing the release of components that are capable of stimulating genetic damage to cellular DNA. Under the conditions of the experiment, the rest of the tested dilutions had no genotoxic effects, showing a behavior that JOE — Volume 38, Number 4, April 2012

was similar to the control group. White MTA genotoxic rates were way below all of the groups mentioned above. The genotoxic effects observed in this study might be related to the release of resinous compounds present in the cement composition as salicylate. This component has stimulated the process of apoptosis in human fibrosarcoma cells and has caused the fragmentation of cell genetic material, determining its precipitation in the cytoplasm (25). The same component might be the explanation for the biological behavior of the resin-based cement AH Plus. In addition, there is evidence from genetic mutation analysis with bacteria and eukaryotic cells that shows the epoxy resin present in AH Plus sealer as a mutagen, which can cause breaks in the chain of cellular DNA (3, 32, 33). However, Miletic et al (3) have reported that AH Plus sealer did not induce chromosomal aberrations or micronucleus in any trial period to evaluate the micronucleus on cultures of human lymphocytes. Lodiene et al (34) observed severe genotoxicity and cytotoxicity of AH Plus immediately after handling. However, in this study, AH Plus was cytotoxic in longer periods at 48 and 72 hours, especially in higher concentrations (1:1 and 1:2), precluding an analysis of the genotoxicity of these dilutions because of widespread cell death caused by samples mentioned. More tests must be carried out for a more detailed trial of sealers’ genotoxic potential. Given the diversity of root canal sealers on the market, it becomes difficult to know what the best indication is. The correct choice, however, should consider not only the biological behavior but also the joint evaluation of other parameters such as antimicrobial and physical properties and chemical.

Conclusions White MTA cement kept the rate of cell viability above 70% for all dilutions and all the assessed periods. MTA Fillapex sealer was cytotoxic at the dilutions 1:1, 1:2, 1:4, and 1:8 (12-hour group); it showed the highest cytotoxicity in this study. AH Plus was cytotoxic at the dilutions 1:1, 1:2, and 1:4 (48-hour group). White MTA cement stimulated the lower rates of micronuclei formation, even in the highest dilution (1:1); MTA Fillapex and AH Plus stimulated the formation of micronuclei to 8-fold compared with untreated control group, both of them in the 1:4 dilution groups at 48 hours. White MTA was considered the less cytotoxic and genotoxic material used in this study.

Acknowledgments The authors deny any conflicts of interest related to this study.

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JOE — Volume 38, Number 4, April 2012