- Email: [email protected]
Setting Time Affects In Vitro Biological Properties of Root Canal Sealers
Basic Research—Technology
Setting Time Affects In Vitro Biological Properties of Root Canal Sealers Carlos Henrique R. Camargo, DDS, PhD, Tatiana R. Oliveira, DDS, MSc, Gleyce O. Silva, DDS, MSc, Sylvia B. Rabelo, DDS, PhD, Marcia C. Valera, DDS, PhD, and Bruno N. Cavalcanti, DDS, PhD Abstract Introduction: Biocompatibility of root canal sealers is important because of the long-term contact of their eluates and/or degradation products with periapical tissues. The literature still lacks studies about the genotoxic effects of these materials and the influence of setting time on biological properties. The cytotoxicity and genotoxicity of an epoxy resin–based sealer (AH Plus), a single methacrylate-based sealer (EndoRez), and a silicone-based sealer (RoekoSeal) were assessed. Methods: Chinese hamster fibroblasts (V79) were cultured and exposed to different dilutions of extracts from the sealers that were left to set for 0, 12, and 24 hours before contact with culture medium. Cell viability was measured by the methyl-thiazol-diphenyltetrazolium assay. Genotoxicity was assessed by the comet assay. Data were statistically analyzed by Kruskal-Wallis and Dunn tests (P < .05). Results: Root canal sealers were statistically more cytotoxic than the untreated control group, except for the silicon-based sealer. Cell viability ranking was the following (from the most to the least cytotoxic): methacrylate-based > epoxy resin–based > silicone-based. The setting time influenced the epoxy resin–based sealer cytotoxicity (decreased at 12 hours) and the general genotoxicity (increased at 24 hours). DNA damage ranking was the following (from the most to the least genotoxic): methacrylate-based > siliconebased = epoxy resin–based. Conclusions: The setting time had influence on the cytotoxicity of the epoxy resin– based sealer and genotoxicity of all tested sealers. The methacrylate-based sealer was the most cytotoxic, and the silicone-based sealer was not cytotoxic. Genotoxicity was observed for all sealers. (J Endod 2014;40:530–533)
T
he development of endodontic sealers aims to achieve good mechanical properties and biocompatibility. This property is necessary because of the long-term contact of their eluates and/or degradation products with periapical tissues (1, 2). Toxic materials can damage periapical cells and affect the DNA, leading to carcinogenic transformations and/or genome instability (3). It is known that epoxy resin–based sealers could induce an initial mild inflammatory reaction on surrounding tissues, as well as cytotoxicity (4–6), with slight mutagenic capacity (7). Single methacrylate resin–based sealers were also studied. Whereas some authors reported them as well-tolerated by connective tissues (8), others observed an intense and long-lasting inflammatory reaction (9). A possible cause for this effect could be the presence of urethane dimethacrylate in the structure of the sealer (10). Previous studies also reported that high concentrations of methacrylate monomers might induce DNA damage (11). Recently, silicone-based sealers were introduced, with none or minimal cytotoxicity (12); however, there are no current studies regarding its genotoxic effects. There are a number of studies assessing the cytotoxicity of endodontic sealers, although only a few authors observed the effects of setting time on the genotoxicity of root canal sealers (5, 13–15). With this gap in the knowledge, we hypothesized that different setting times would change both cytotoxicity and genotoxicity of root canal sealers. The objective of this study was to evaluate the cytotoxic and genotoxic effects of 3 different endodontic sealers by using the methyl-thiazol-diphenyltetrazolium (MTT) assay and the comet assay in a setting time–dependent manner.
Materials and Methods Cell Culture Chinese hamster fibroblasts (V79) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (10,000 IU/ mL), and 1% streptomycin (10 mg/mL). Cultures were incubated under an atmosphere of 5% CO2 at 37 C until confluency. Cells were then detached from the flasks, seeded according to the assay (cytotoxicity or genotoxicity), and incubated again for 24 hours.
Key Words Biocompatibility, cytotoxicity, endodontic sealer, genotoxicity From the Department of Restorative Dentistry, Institute of Science and Technology, Universidad Estadual Paulista (UNESP), Sao Jose dos Campos, Brazil. Address requests for reprints to Dr Carlos Henrique R. Camargo, Department of Restorative Dentistry, Institute of Science and Technology, Universidad Estadual Paulista, Av. Eng. Francisco Jose Longo, 777, Sao Jose dos Campos, SP, 12245-000 Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.08.009
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Preparation of Extracts Three sealers were tested, each one with a specific chemical base (Table 1). Sealers were manipulated following the manufacturers’ instructions, layered into 24-well plates, and covered by 2.5 mL cell culture medium after waiting 0, 12, and 24 hours for setting. Samples were then reincubated for 24 hours at 37 C. Original extracts (1:1) were serially diluted in the cell culture medium (1:2, 1:4, 1:8, 1:16, and 1:32). Cytotoxicity Analysis Cells seeded in 96-well plates (5 103 cells/well) were exposed to 200 mL/ well of the serial dilutions of the extracts. Untreated cells were used as controls. Cells under the experimental conditions were incubated at 5% CO2 37 C for 24 hours. The culture medium in each well was replaced by 100 mL MTT solution (Sigma
JOE — Volume 40, Number 4, April 2014
Basic Research—Technology TABLE 1. Main Components, Setting Time, and Manufacturer of Tested Sealers Sealer
Composition
AH Plus
Paste A: bisphenol-A epoxy resin, bisphenol-F epoxy resin, calcium tungstate, zirconium oxide, silica, iron oxide pigments; Paste B: dibenzyldiamine, aminoadamantane, tricyclodecane-diamine, calcium tungstate, zirconium oxide, silica, silicone oil RoekoSeal Polydimethysiloxane, silicone oil, paraffin-base oil, hexachloroplatinic acid (catalytic agent), zirconium dioxide EndoRez 30% urethane dimethacrylate, zinc oxide, barium sulfate, resins, pigments
Aldrich Co, Munich, Germany) and incubated for 1 hour. This solution was removed, and resulting formazan crystals were dissolved by the addition of 100 mL dimethyl sulfoxide (Sigma Aldrich). Plates were shaken for 10 minutes at room temperature and read at 570 nm in a spectrophotometer (ASYS Hitech GmbH, Eugendorf, Austria). Four wells were exposed to each dilution of the extracts in 3 independent experiments. Absorbance readings were normalized to the untreated control cultures and represent the inhibition of succinyl dehydrogenase activity (cellular metabolism). Statistical differences were analyzed by Kruskal-Wallis complemented by Dunn multiple comparisons test, both with significance of P < .05.
Genotoxicity Analysis The comet assay was performed following a standard protocol (16). Chemicals were obtained from Sigma (St Louis, MO). Dilutions that allowed cell viability of approximately 50% in the MTT assay were chosen for this test. The basic principle of the single-cell gel (comet) assay is the migration of DNA fragments in an agarose matrix under electrophoresis. Under a microscope, cells resemble a comet, with a nucleus and a tail containing DNA fragments or strands migrating toward the anode. The negative control group was treated with cell culture medium (Dulbecco modified Eagle medium), and positive control group was
Setting time
Manufacturer
8h
Dentsply De Trey GmbH, Konstanz, Germany
45–50 min
Coltene-Whaledent, Langenau, Germany
15–20 min
Ultradent, South Jordan, UT
established by using ethyl methane sulfonate at 5 mmol/L. Three slides were prepared per treatment. After incubation with the extracts, cells were suspended in culture medium (approximately 1 104 cells/ mL), and 10 mL of this suspension was added to 120 mL 0.5% low melting point agarose at 37 C. This mixture was layered onto a precoated slide with 1.5% regular agarose and covered with a coverslip. The agarose was gelled in a refrigerator, the coverslip was removed, and slides were immersed in lysis solution (2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris-HCl buffer pH 10, with 1% Triton X-100, and 10% dimethyl sulfoxide) for 2 hours at 4 C. After immersion in alkaline buffer (0.3 mmol/L NaOH, 1 mmol/L EDTA pH >13) for 20 minutes, slides were electrophoresed for another 20 minutes at 25 V (0.86 V/cm, 300 mA), neutralized in 0.4 mol/L Tris-HCl (pH 7.5) for 15 minutes, and fixed in absolute ethanol. Staining was performed with 300 mL DAPI solution (40 ,6-diamidino-2-phenylindole dihydrochloride) for 5 minutes. At least 50 randomly captured comets per treatment (25 cells from each slide) were examined at 400 magnification by using a fluorescence microscope (Leica Leitz, Wetzlar, Germany). Images were analyzed by software (Comet Assay IV v4.3; Perceptive Instruments Ltd, Bury St Edmunds, UK). Tail moment was calculated by the image analysis system as the product of the tail length (DNA migration) and the fraction of DNA in the comet tail (% DNA in the tail). At least 2 slides derived from independent experiments were analyzed, and the statistical differences were analyzed by Kruskal-
Figure 1. Graphical representation of the sealers’ cytotoxicity in V79 cells after exposure to extracts, according to dilution and setting time. Columns represent the median cell viability expressed as percentage. Bars represent 25% and 75% percentiles. Original extracts (1:1) were serially diluted in fresh medium. 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). Symbols (*, #) indicate the statistical differences among groups.
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Figure 2. DNA damage in V79 cells after exposure to sealers. (A) Representative photomicrographs for the comet assay, according to setting time and sealer tested. (B) Median percentage of tail moment (DNA damage), according to the tested groups. Bars represent 25% and 75% percentiles. Median DNA damage was also calculated for 5 mmol ethyl methane sulfonate (EMS). NC, negative control.
Wallis complemented by Dunn multiple comparisons test, both with significance of P < .05.
Results Cytotoxicity Assay The silicone-based sealer demonstrated cell viability close to 100% for all setting times. On the other hand, the methacrylate-based sealer was highly cytotoxic in more concentrated dilutions (1:1 and 1:2), independently of the setting time. The epoxy-based sealer was highly cytotoxic in lowest dilutions, but 12-hour samples presented low cytotoxicity (1:2 and 1:4 dilutions). At 1:8 dilution, it was highly cytotoxic only at 0 hours. None of the sealers were cytotoxic at highest dilutions (1:16 and 1:32). Cytotoxicity data are summarized in Figure 1. The ranking of cell viability from the most to the least cytotoxic material was the following: methacrylate-based > epoxy resin–based > siliconebased. Genotoxicity Assay Figure 2A shows representative DNA damage caused after exposure to the extracts. Percentage of tail moment has increased through 532
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setting times for all sealers, suggesting an influence of this factor on sealer genotoxicity. Statistical differences were observed for the methacrylate-based sealer at 24 hours (Fig. 2B). The ranking of DNA damage verified at the comet assay from the most to the least genotoxic was the following: methacrylate-based > silicone-based = epoxy resin– based.
Discussion In the present study, the setting time affected the cytotoxicity of an epoxy resin–based sealer, reducing its effects at 12 hours. In addition, as the most remarkable data, genotoxicity was increased through time for all sealers, with statistical differences for the methacrylate resin– based sealer at 24 hours. Cytotoxicity was evaluated by the MTT assay, which has been used to test the biocompatibility of endodontic sealers (7, 12, 17–19). Testing freshly mixed sealers is relevant, because sealers are clinically used right after mixing and incompletely set. However, evaluation in different periods of time is important, because the diffusion of eluates and subproducts into the tissues may lead to changes in cytotoxicity and genotoxic levels (5, 17, 19, 20). Our JOE — Volume 40, Number 4, April 2014
Basic Research—Technology study confirmed this hypothesis, because all tested sealers showed different levels of toxicity in different times. In this context, the use of Chinese hamster fibroblasts (V79) is also corroborated by others, because these cells have been extensively used for cytotoxicity and genotoxicity evaluation of biomaterials (7, 17), with similar results to those obtained with 2 primary human oral fibroblasts (21, 22). We observed that the epoxy resin–based sealer (AH Plus; Dentsply DeTrey GmbH, Konstanz, Germany) presented immediate high level of cytotoxicity, which is in disagreement with another study (13). According to the manufacturer, AH Plus does not release formaldehyde; however, other studies (5, 20, 23) reported that this sealer preserves amines to accelerate polymerization, which may be the reason for strong initial cytotoxicity. In addition, it is suggested that a cytotoxic by-product appears later during setting (4). This may be the reason for the decreased cytotoxicity at 12 hours for this sealer, with a recovery of the cytotoxic effect at 24 hours. The methacrylate-based sealer showed high cytotoxicity, probably because of the presence of urethane dimethacrylate in the structure (24). Other important data from our study are that the methacrylate-based sealer (EndoRez; Ultradent Products, South Jordan, UT) was not completely set, even following all the manufacturer’s instructions. This may explain its harmful biological effects on the cultures. On the other hand, the silicone-based sealer (RoekoSeal; Coltene-Whaledent, Langenau, Germany) was not cytotoxic at any of the tested times. This is in agreement with another study (19). The comet assay test has been used to determine the genotoxicity of the biomaterials. It is a rapid, simple, and reliable biochemical technique for evaluating DNA damage in mammalian cells (16). It is important to emphasize that this experiment demands viable cells. Thus, the use of diluted extracts, besides presenting more comprehensive data on sealer cytotoxicity (for example, simulation of the diffusion of the sealer on different layers of tissue), provides an adequate concentration to be used on the cells for additional experiments. Our data demonstrate that the methacrylate-based sealer was able to induce DNA damage with significant genotoxicity at 24 hours. This suggests the possibility that the components of this sealer can induce a late toxic reaction, probably because of increased generation of reactive oxygen species, which may be responsible for genotoxic and mutagenic effects (25, 26). The epoxy resin–based sealer was shown to be genotoxic as the cytotoxicity increases or decreases. This was not found in another study, where no genotoxic evidence with EndoRez or AH Plus was found in contact with the cells (20). Despite not showing cytotoxic effects, the silicone-based sealer presented genotoxicity. It is important to emphasize that this is the first study to report data on the potential genotoxic effects of the silicone-based RoekoSeal. With all data exposed, it is clear that the setting time plays an important role in the biological effects of endodontic sealers. Further studies may evaluate longer time-dependent effects to present a wider picture on the cytotoxicity and/or genotoxicity of endodontic sealers. In addition, cytotoxicity and genotoxicity are not necessarily interdependent, and the cellular mechanisms involved in both processes may be different.
Conclusions The setting time has influenced the cytotoxicity of the epoxy resin– based sealer and genotoxicity of all sealers. The methacrylate-based sealer was the most cytotoxic, mainly in more concentrated dilutions, followed by the epoxy resin–based sealer, with the silicone-based sealer not cytotoxic at any concentration. Genotoxicity was observed for all sealers, again with harmful effects observed for the methacrylate-based sealer.
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Acknowledgments Supported by the Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP 2009/13285-8). The authors deny any conflicts of interest related to this study.
References 1. Zmener O, Pameijer CH, Kokubu GA, Grana DR. Subcutaneous connective tissue reaction to methacrylate resin-based and zinc oxide and eugenol sealers. J Endod 2010;36:1574–9. 2. Scarparo RK, Grecca FS, Fachin EV. Analysis of tissue reactions to methacrylate resin based, epoxy resin-based, and zinc oxide-eugenol endodontic sealers. J Endod 2009;35:229–32. 3. Bertram JS. The molecular biology of cancer. Mol Aspects Med 2000;21:167–223. 4. Leonardo MR, Bezerra da Silva LA, Filho MT, Santana da Silva R. Release of formaldehyde by 4 endodontic sealers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;88:221–5. 5. Miletic I, Devcic N, Anic I, et al. The cytotoxicity of RoekoSeal and AH plus compared during different setting periods. J Endod 2005;31:307–9. 6. Kim TG, Lee YH, Lee NH, et al. The antioxidant property of pachymic acid improves bone disturbance against AH Plus-induced inflammation in MC-3T3 E1 cells. J Endod 2013;39:461–6. 7. Camargo CH, Camargo SE, Valera MC, et al. The induction of cytotoxicity, oxidative stress, and genotoxicity by root canal sealers in mammalian cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:952–60. 8. Zmener O. Tissue response to a new methacrylate-based root canal sealer: preliminary observations in the subcutaneous connective tissue of rats. J Endod 2004;30: 348–51. 9. Sousa CJ, Montes CR, Pascon EA, et al. Comparison of the intraosseous biocompatibility of AH Plus, EndoREZ, and Epiphany root canal sealers. J Endod 2006;32: 656–62. 10. Al-Hiyasat AS, Tayyar M, Darmani H. Cytotoxicity evaluation of various resin based root canal sealers. Int Endod J 2010;43:148–53. 11. Urcan E, Scherthan H, Styllou M, et al. Induction of DNA double-strand breaks in primary gingival fibroblasts by exposure to dental resin composites. Biomaterials 2010;31:2010–4. 12. Ashraf H, Moradimajd N, Mozayeni MA, et al. Cytotoxicity evaluation of three resinbased sealers on an L929 cell line. Dent Res J (Isfahan) 2012;9:549–53. 13. Karapınar-Kazandag M, Bayrak OF, Yalvac¸ ME, et al. Cytotoxicity of 5 endodontic sealers on L929 cell line and human dental pulp cells. Int Endod J 2011;44:626–34. 14. Brackett MG, Lewis JB, Kious AR, et al. Cytotoxicity of endodontic sealers after one year of aging in vitro. Journal Biomed Mater Res 2012;100B:1729–35. 15. Brzovic V, Miletic I, Zeljezic D, et al. In vitro genotoxicity of root canal sealers. Int Endod J 2009;42:253–63. 16. Tice RR, Agurell E, Anderson D, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000;35: 206–21. 17. Bin CV, Valera MC, Camargo SE, et al. Cytotoxicity and genotoxicity of root canal sealers based on mineral trioxide aggregate. J Endod 2012;38:495–500. 18. Yilmaz Z, Dogan AL, Ozdemir O, Serper A. Evaluation of the cytotoxicity of different root canal sealers on L929 cell line by MTT assay. Dent Mater J 2012;31:1028–32. 19. Silva EJ, Santos CC, Zaia AA. Long-term cytotoxic effects of contemporary root canal sealers. J Appl Oral Sci 2013;21:43–7. 20. Van Landuyt KL, Geebelen B, Shehata M, et al. No evidence for DNA double-strand breaks caused by endodontic sealers. J Endod 2012;38:636–41. 21. Tai KW, Huang FM, Chang YC. Cytotoxic evaluation of root canal filling materials on primary human oral fibroblast cultures and a permanent hamster cell line. J Endod 2001;27:571–3. 22. Huang FM, Tai KW, Chou MY, Chang YC. Cytotoxicity of resin-, zinc oxide-eugenol-, and calcium hydroxide-based root canal sealers on human periodontal ligament cells and permanent V79 cells. Int Endod J 2002;35:153–8. 23. Lodiene G, Morisbak E, Bruzell E, Ørstavik D. Toxicity evaluation of root canal sealers in vitro. Int Endod J 2008;41:72–7. 24. Volk J, Engelmann J, Leyhausen G, Geurtsen W. Effects of three resin monomers on the cellular glutathione concentration of cultured human gingival fibroblasts. Dent Mater 2006;22:499–505. 25. Schweikl H, Spagnuolo G, Schmalz G. Genetic and cellular toxicology of dental resin monomers. J Dent Res 2006;85:870–7. 26. Krifka S, Spagnuolo G, Schmalz G, Schweikl H. A review of adaptive mechanisms in cell responses towards oxidative stress caused by dental resin monomers. Biomaterials 2013;34:4555–63.
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Setting Time Affects In Vitro Biological Properties of Root Canal Sealers Carlos Henrique R. Camargo, DDS, PhD, Tatiana R. Oliveira, DDS, MSc, Gleyce O. Silva, DDS, MSc, Sylvia B. Rabelo, DDS, PhD, Marcia C. Valera, DDS, PhD, and Bruno N. Cavalcanti, DDS, PhD Abstract Introduction: Biocompatibility of root canal sealers is important because of the long-term contact of their eluates and/or degradation products with periapical tissues. The literature still lacks studies about the genotoxic effects of these materials and the influence of setting time on biological properties. The cytotoxicity and genotoxicity of an epoxy resin–based sealer (AH Plus), a single methacrylate-based sealer (EndoRez), and a silicone-based sealer (RoekoSeal) were assessed. Methods: Chinese hamster fibroblasts (V79) were cultured and exposed to different dilutions of extracts from the sealers that were left to set for 0, 12, and 24 hours before contact with culture medium. Cell viability was measured by the methyl-thiazol-diphenyltetrazolium assay. Genotoxicity was assessed by the comet assay. Data were statistically analyzed by Kruskal-Wallis and Dunn tests (P < .05). Results: Root canal sealers were statistically more cytotoxic than the untreated control group, except for the silicon-based sealer. Cell viability ranking was the following (from the most to the least cytotoxic): methacrylate-based > epoxy resin–based > silicone-based. The setting time influenced the epoxy resin–based sealer cytotoxicity (decreased at 12 hours) and the general genotoxicity (increased at 24 hours). DNA damage ranking was the following (from the most to the least genotoxic): methacrylate-based > siliconebased = epoxy resin–based. Conclusions: The setting time had influence on the cytotoxicity of the epoxy resin– based sealer and genotoxicity of all tested sealers. The methacrylate-based sealer was the most cytotoxic, and the silicone-based sealer was not cytotoxic. Genotoxicity was observed for all sealers. (J Endod 2014;40:530–533)
T
he development of endodontic sealers aims to achieve good mechanical properties and biocompatibility. This property is necessary because of the long-term contact of their eluates and/or degradation products with periapical tissues (1, 2). Toxic materials can damage periapical cells and affect the DNA, leading to carcinogenic transformations and/or genome instability (3). It is known that epoxy resin–based sealers could induce an initial mild inflammatory reaction on surrounding tissues, as well as cytotoxicity (4–6), with slight mutagenic capacity (7). Single methacrylate resin–based sealers were also studied. Whereas some authors reported them as well-tolerated by connective tissues (8), others observed an intense and long-lasting inflammatory reaction (9). A possible cause for this effect could be the presence of urethane dimethacrylate in the structure of the sealer (10). Previous studies also reported that high concentrations of methacrylate monomers might induce DNA damage (11). Recently, silicone-based sealers were introduced, with none or minimal cytotoxicity (12); however, there are no current studies regarding its genotoxic effects. There are a number of studies assessing the cytotoxicity of endodontic sealers, although only a few authors observed the effects of setting time on the genotoxicity of root canal sealers (5, 13–15). With this gap in the knowledge, we hypothesized that different setting times would change both cytotoxicity and genotoxicity of root canal sealers. The objective of this study was to evaluate the cytotoxic and genotoxic effects of 3 different endodontic sealers by using the methyl-thiazol-diphenyltetrazolium (MTT) assay and the comet assay in a setting time–dependent manner.
Materials and Methods Cell Culture Chinese hamster fibroblasts (V79) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (10,000 IU/ mL), and 1% streptomycin (10 mg/mL). Cultures were incubated under an atmosphere of 5% CO2 at 37 C until confluency. Cells were then detached from the flasks, seeded according to the assay (cytotoxicity or genotoxicity), and incubated again for 24 hours.
Key Words Biocompatibility, cytotoxicity, endodontic sealer, genotoxicity From the Department of Restorative Dentistry, Institute of Science and Technology, Universidad Estadual Paulista (UNESP), Sao Jose dos Campos, Brazil. Address requests for reprints to Dr Carlos Henrique R. Camargo, Department of Restorative Dentistry, Institute of Science and Technology, Universidad Estadual Paulista, Av. Eng. Francisco Jose Longo, 777, Sao Jose dos Campos, SP, 12245-000 Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.08.009
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Preparation of Extracts Three sealers were tested, each one with a specific chemical base (Table 1). Sealers were manipulated following the manufacturers’ instructions, layered into 24-well plates, and covered by 2.5 mL cell culture medium after waiting 0, 12, and 24 hours for setting. Samples were then reincubated for 24 hours at 37 C. Original extracts (1:1) were serially diluted in the cell culture medium (1:2, 1:4, 1:8, 1:16, and 1:32). Cytotoxicity Analysis Cells seeded in 96-well plates (5 103 cells/well) were exposed to 200 mL/ well of the serial dilutions of the extracts. Untreated cells were used as controls. Cells under the experimental conditions were incubated at 5% CO2 37 C for 24 hours. The culture medium in each well was replaced by 100 mL MTT solution (Sigma
JOE — Volume 40, Number 4, April 2014
Basic Research—Technology TABLE 1. Main Components, Setting Time, and Manufacturer of Tested Sealers Sealer
Composition
AH Plus
Paste A: bisphenol-A epoxy resin, bisphenol-F epoxy resin, calcium tungstate, zirconium oxide, silica, iron oxide pigments; Paste B: dibenzyldiamine, aminoadamantane, tricyclodecane-diamine, calcium tungstate, zirconium oxide, silica, silicone oil RoekoSeal Polydimethysiloxane, silicone oil, paraffin-base oil, hexachloroplatinic acid (catalytic agent), zirconium dioxide EndoRez 30% urethane dimethacrylate, zinc oxide, barium sulfate, resins, pigments
Aldrich Co, Munich, Germany) and incubated for 1 hour. This solution was removed, and resulting formazan crystals were dissolved by the addition of 100 mL dimethyl sulfoxide (Sigma Aldrich). Plates were shaken for 10 minutes at room temperature and read at 570 nm in a spectrophotometer (ASYS Hitech GmbH, Eugendorf, Austria). Four wells were exposed to each dilution of the extracts in 3 independent experiments. Absorbance readings were normalized to the untreated control cultures and represent the inhibition of succinyl dehydrogenase activity (cellular metabolism). Statistical differences were analyzed by Kruskal-Wallis complemented by Dunn multiple comparisons test, both with significance of P < .05.
Genotoxicity Analysis The comet assay was performed following a standard protocol (16). Chemicals were obtained from Sigma (St Louis, MO). Dilutions that allowed cell viability of approximately 50% in the MTT assay were chosen for this test. The basic principle of the single-cell gel (comet) assay is the migration of DNA fragments in an agarose matrix under electrophoresis. Under a microscope, cells resemble a comet, with a nucleus and a tail containing DNA fragments or strands migrating toward the anode. The negative control group was treated with cell culture medium (Dulbecco modified Eagle medium), and positive control group was
Setting time
Manufacturer
8h
Dentsply De Trey GmbH, Konstanz, Germany
45–50 min
Coltene-Whaledent, Langenau, Germany
15–20 min
Ultradent, South Jordan, UT
established by using ethyl methane sulfonate at 5 mmol/L. Three slides were prepared per treatment. After incubation with the extracts, cells were suspended in culture medium (approximately 1 104 cells/ mL), and 10 mL of this suspension was added to 120 mL 0.5% low melting point agarose at 37 C. This mixture was layered onto a precoated slide with 1.5% regular agarose and covered with a coverslip. The agarose was gelled in a refrigerator, the coverslip was removed, and slides were immersed in lysis solution (2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris-HCl buffer pH 10, with 1% Triton X-100, and 10% dimethyl sulfoxide) for 2 hours at 4 C. After immersion in alkaline buffer (0.3 mmol/L NaOH, 1 mmol/L EDTA pH >13) for 20 minutes, slides were electrophoresed for another 20 minutes at 25 V (0.86 V/cm, 300 mA), neutralized in 0.4 mol/L Tris-HCl (pH 7.5) for 15 minutes, and fixed in absolute ethanol. Staining was performed with 300 mL DAPI solution (40 ,6-diamidino-2-phenylindole dihydrochloride) for 5 minutes. At least 50 randomly captured comets per treatment (25 cells from each slide) were examined at 400 magnification by using a fluorescence microscope (Leica Leitz, Wetzlar, Germany). Images were analyzed by software (Comet Assay IV v4.3; Perceptive Instruments Ltd, Bury St Edmunds, UK). Tail moment was calculated by the image analysis system as the product of the tail length (DNA migration) and the fraction of DNA in the comet tail (% DNA in the tail). At least 2 slides derived from independent experiments were analyzed, and the statistical differences were analyzed by Kruskal-
Figure 1. Graphical representation of the sealers’ cytotoxicity in V79 cells after exposure to extracts, according to dilution and setting time. Columns represent the median cell viability expressed as percentage. Bars represent 25% and 75% percentiles. Original extracts (1:1) were serially diluted in fresh medium. 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). Symbols (*, #) indicate the statistical differences among groups.
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Figure 2. DNA damage in V79 cells after exposure to sealers. (A) Representative photomicrographs for the comet assay, according to setting time and sealer tested. (B) Median percentage of tail moment (DNA damage), according to the tested groups. Bars represent 25% and 75% percentiles. Median DNA damage was also calculated for 5 mmol ethyl methane sulfonate (EMS). NC, negative control.
Wallis complemented by Dunn multiple comparisons test, both with significance of P < .05.
Results Cytotoxicity Assay The silicone-based sealer demonstrated cell viability close to 100% for all setting times. On the other hand, the methacrylate-based sealer was highly cytotoxic in more concentrated dilutions (1:1 and 1:2), independently of the setting time. The epoxy-based sealer was highly cytotoxic in lowest dilutions, but 12-hour samples presented low cytotoxicity (1:2 and 1:4 dilutions). At 1:8 dilution, it was highly cytotoxic only at 0 hours. None of the sealers were cytotoxic at highest dilutions (1:16 and 1:32). Cytotoxicity data are summarized in Figure 1. The ranking of cell viability from the most to the least cytotoxic material was the following: methacrylate-based > epoxy resin–based > siliconebased. Genotoxicity Assay Figure 2A shows representative DNA damage caused after exposure to the extracts. Percentage of tail moment has increased through 532
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setting times for all sealers, suggesting an influence of this factor on sealer genotoxicity. Statistical differences were observed for the methacrylate-based sealer at 24 hours (Fig. 2B). The ranking of DNA damage verified at the comet assay from the most to the least genotoxic was the following: methacrylate-based > silicone-based = epoxy resin– based.
Discussion In the present study, the setting time affected the cytotoxicity of an epoxy resin–based sealer, reducing its effects at 12 hours. In addition, as the most remarkable data, genotoxicity was increased through time for all sealers, with statistical differences for the methacrylate resin– based sealer at 24 hours. Cytotoxicity was evaluated by the MTT assay, which has been used to test the biocompatibility of endodontic sealers (7, 12, 17–19). Testing freshly mixed sealers is relevant, because sealers are clinically used right after mixing and incompletely set. However, evaluation in different periods of time is important, because the diffusion of eluates and subproducts into the tissues may lead to changes in cytotoxicity and genotoxic levels (5, 17, 19, 20). Our JOE — Volume 40, Number 4, April 2014
Basic Research—Technology study confirmed this hypothesis, because all tested sealers showed different levels of toxicity in different times. In this context, the use of Chinese hamster fibroblasts (V79) is also corroborated by others, because these cells have been extensively used for cytotoxicity and genotoxicity evaluation of biomaterials (7, 17), with similar results to those obtained with 2 primary human oral fibroblasts (21, 22). We observed that the epoxy resin–based sealer (AH Plus; Dentsply DeTrey GmbH, Konstanz, Germany) presented immediate high level of cytotoxicity, which is in disagreement with another study (13). According to the manufacturer, AH Plus does not release formaldehyde; however, other studies (5, 20, 23) reported that this sealer preserves amines to accelerate polymerization, which may be the reason for strong initial cytotoxicity. In addition, it is suggested that a cytotoxic by-product appears later during setting (4). This may be the reason for the decreased cytotoxicity at 12 hours for this sealer, with a recovery of the cytotoxic effect at 24 hours. The methacrylate-based sealer showed high cytotoxicity, probably because of the presence of urethane dimethacrylate in the structure (24). Other important data from our study are that the methacrylate-based sealer (EndoRez; Ultradent Products, South Jordan, UT) was not completely set, even following all the manufacturer’s instructions. This may explain its harmful biological effects on the cultures. On the other hand, the silicone-based sealer (RoekoSeal; Coltene-Whaledent, Langenau, Germany) was not cytotoxic at any of the tested times. This is in agreement with another study (19). The comet assay test has been used to determine the genotoxicity of the biomaterials. It is a rapid, simple, and reliable biochemical technique for evaluating DNA damage in mammalian cells (16). It is important to emphasize that this experiment demands viable cells. Thus, the use of diluted extracts, besides presenting more comprehensive data on sealer cytotoxicity (for example, simulation of the diffusion of the sealer on different layers of tissue), provides an adequate concentration to be used on the cells for additional experiments. Our data demonstrate that the methacrylate-based sealer was able to induce DNA damage with significant genotoxicity at 24 hours. This suggests the possibility that the components of this sealer can induce a late toxic reaction, probably because of increased generation of reactive oxygen species, which may be responsible for genotoxic and mutagenic effects (25, 26). The epoxy resin–based sealer was shown to be genotoxic as the cytotoxicity increases or decreases. This was not found in another study, where no genotoxic evidence with EndoRez or AH Plus was found in contact with the cells (20). Despite not showing cytotoxic effects, the silicone-based sealer presented genotoxicity. It is important to emphasize that this is the first study to report data on the potential genotoxic effects of the silicone-based RoekoSeal. With all data exposed, it is clear that the setting time plays an important role in the biological effects of endodontic sealers. Further studies may evaluate longer time-dependent effects to present a wider picture on the cytotoxicity and/or genotoxicity of endodontic sealers. In addition, cytotoxicity and genotoxicity are not necessarily interdependent, and the cellular mechanisms involved in both processes may be different.
Conclusions The setting time has influenced the cytotoxicity of the epoxy resin– based sealer and genotoxicity of all sealers. The methacrylate-based sealer was the most cytotoxic, mainly in more concentrated dilutions, followed by the epoxy resin–based sealer, with the silicone-based sealer not cytotoxic at any concentration. Genotoxicity was observed for all sealers, again with harmful effects observed for the methacrylate-based sealer.
JOE — Volume 40, Number 4, April 2014
Acknowledgments Supported by the Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP 2009/13285-8). The authors deny any conflicts of interest related to this study.
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