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The biocompatibility evaluation of epoxy resin-based root canal sealers in vitro
Biomaterials 23 (2002) 77–83
The biocompatibility evaluation of epoxy resin-based root canal sealers in vitro Tsui-Hsien Huanga, Jaw-Ji Yanga, Huei Lib, Chia-Tze Kaoc,* b
a Dental Department, Chung Shan Medical and Dental College, No.110, Section 1, Chien Kuo N Road, Taichung 402, Taiwan Institute of Toxicology, Chung Shan Medical and Dental College, No.110, Section 1, Chien Kuo N Road, Taichung 402, Taiwan c Institute of Stomatology, Chung Shan Medical and Dental College, 620 Shr Jeng Road, Taichung 402, Taiwan
Received 23 October 2000; accepted 19 February 2001
Abstract The cytotoxic and mutagenic effects of epoxy resin-based root canal sealer AH26 and AH-Plus were determined in vitro. Root canal sealers were eluted for 24 h in dimethyl sulfoxide (DMSO) and diluted in culture medium. Cytotoxic effects were assessed using the MTT [tetrazolium dye, 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide, C18H16N5SBr] assay for mitochondrial enzyme activity and also the cell viability. Genotoxicity assays were assessed using the alkaline single cell gel electrophoresis assay (comet assay) for DNA damage measurement. Result indicated that both the AH26 and AH-Plus sealers exhibited a dose-dependent increase in astrocyte toxic effects. Additionally, dose-dependent astrocyte DNA damage was also noted for both sealers. Therefore, these epoxy resin-based sealers, AH26 and AH-Plus demonstrated both cytotoxicity and genotoxicity in vitro. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biocompatibility of epoxy resin sealer; MTT assay; Alkaline single cell gel electrophoresis
1. Introduction Clearly, one of the principal requirements of an endodontic root canal sealer should be that it is noncytotoxic and immunologically compatible with peripheral tissue [1]. Sealer-elutable substances or the degradation or corrosion products from a root canal sealer may gain access to periodontal tissue through numerous pathways [2,3]. Root canal sealers and their diffusible components, therefore, need to be critically evaluated for their cytocompatibility and genotoxicity prior to their general clinical use. Numerous root canal sealers are available, based on various formulae and containing a variety of different and partly mutagenic components, such as epoxy resin sealers, e.g., AH26 and AH Plus, Ca(OH)2-based materials such as Sealapex and Apeixt, and ZnOeugenol cements, e.g., N2 and endomethasone [4]. The
*Corresponding author. Tel.: +886-4-2255-3715; fax: +886-4-22553713. E-mail address: [email protected] (C.-T. Kao).
biocompatiblity and antimicrobial activity of a specific root canal sealer remains one of the principal considerations for selecting an appropriate sealer for a dental restoration [4,5]. From the literature, it would appear that the side effects of the use of various root canal sealers have been previously studied to some extent [6–8]. It has been previously demonstrated that sealer material based upon zinc oxide-eugenol was moderately to severely toxic in implantation studies, the results of such research revealing severe inflammation being observed following short setting times when the sealers were implanted subcutaneously into rabbits [7]. Spangberg et al. [8] noted that the AH 26 release of formaldehyde following component mixing will reach a maximum rate two days post-mixing. Formaldehyde release from curing endodontic material has been recognized for many years [8], formaldehyde being reputed to act as a disinfectant [9]. The disinfective agent in AH26 is methenamin [10], which is hydrolyzed to ammonia and formaldehyde [11]. The efficacy of long-term disinfection of canal by formaldehyde released from a root canal sealer has previously been shown to be low [8].
0142-9612/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 0 8 1 - 3
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There have been case reports of adverse reactions such as paraesthesia of the inferior alveolar nerve attributed to the formaldehyde released from root canal sealers [12,13], although any nerve cell reaction to the formaldehyde released from the use of a root canal sealer has not been previously reported. Thus, it is of importance to evaluate the biocompatibility of root canal sea-lers and neurological cells. In the present study the astro-cyte was used as the target cell to evaluate the toxicity. Genotoxicity, mutagenicity and carcinogenicity are very important issues associated with the systemic compatibility of root canal sealers [14]. In vitro test systems can be differentiated into a variety of different testing procedures, including test systems using bacterial cells, e.g. the classic Ames test (mutation assay) and the newly developed umu test (genotoxicity assay), and the test systems using eukaryotic cells in culture, such as the hypoxanthine-guanine phosphoribosyltransferase test (HGPRT, mutation assay), the chromosomal aberration test (mutation assay) and the DNA synthesis inhibition test (DIT, genotoxicity assay) [4]. From a review of the literature, it would appear that the investigation of any genetic effects associated with the use of epoxy resin-based root canal sealer is rare. In a previous study employing the Ames test for the use of silver-free AH26, in vitro, some degree of mutagenicity was demonstrated [15], and, further, AH26 tested with the V79/HGPRT mammalian-cell assay revealed that this sealer material elicits some mutagenic effect 24 h subsequent to mixing and administration [16]. Recently, a new assay for assessing the mutagenic potential of various compounds has been developed and is known as the alkaline singlecell gel electrophoresis assay (comet assay) [17]. This alkaline single-cell gel electrophoresis assay is both a rapid and sensitive procedure for quantitating DNA lesions in mammalian cells, and may be used to detect specific DNA damage and also DNA repair [17]. The present study was going to use this method to evaluate the root canal sealers genotoxicity. There is a newly developed epoxy resin sealer AH-Plus. According to the manufacturer’s instruction AH Plus will not release formaldehyde. The purpose of this study was to analyze the biocompatibility of 1st and 2nd generation epoxy resin sealers, e.g. AH26 and AH Plus sealers, when treated on primary cultured rat cerebral astrocytes, since only very limited information regarding the genotoxicity of resin-based root canal sealers would appear to be available. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric test was used as a cytotoxicity assay and comet assay was used to evaluate the genotoxicity.
2. Materials and methods 2.1. Sample preparation Epoxy resin sealers, AH26 and AH-Plus (De Trey Dentsply, Ballaigues, Switzerland; Table 1) were mixed according to the manufacturer’s instructions. The mixed material 100 mg was eluated in 2 ml dimethyl sulfoxide (DMSO) immediately after mixing at 378C for 24 h [18]. The solution was then centrifuged at 10,000g for 10 min. The supernatant 1 ml was added to 9 ml of Dulbecco’s modified Eagle’s medium (DMEM, SIGMA, USA) and serially diluted until the final concentration of eluate solution was 0.01, 0.02, 0.04, 0.08, and 0.10 mg/ml for use in the MTT assay, and the final concentration was 0.01, 0.05, and 0.25 mg/ml for use in the comet assay. For all culture media, the final concentration of DMSO in sealer was less than 0.1%. It is reported that DMSO at a concentration of 1% showed no toxicity to cell culture [19]. In cytotoxicity test, the pure medium without cell and sealer was used as control. In genotoxicity test, the DMSO 0.05% concentration was used as negative control and 4NQO 0.0003 mg/ml concentration was used as positive control. 2.2. Rat cerebral astrocyte culture Astrocytes were prepared as Amruthesh et al. [20] have previously reported. Briefly, cerebral cortices were isolated from one- to two-day-old rats, cleaned of white matter and meninges, minced, and trypsin-digested for 10 min. The tissue was then diluted into feeding medium, washed, triturated, counted, and placed into 75 cm2 flasks and cultured for 10–14 days at 378C in an air atmosphere containing 5% CO2. When confluent, the 1 105 cells were plated into 96 culture wells. To accomplish this, confluent cells were washed with 0.25% trypsin/0.02% EDTA in saline for exactly 1 min. The trypsin/EDTA was then aspirated, and the cell monolayer, which was still attached to the flask, was covered with 10 ml Dulbecco’s modified Eagle’s medium (DMEM) and incubated at 378C, in an air atomphosphere of 5% CO2 for approximately 10 min. The lifted
Table 1 The components of the AH26 and AH Plus sealers Product
Composition
AH26 (silver-free)
Powder: Bismuth (III) oxide, Methenamine Liquid: Bisphenol-A-diglycidyl ether
AH Plus
Paste A: Epoxy resin, Calcium tungstate, Zirconium oxide, Aerosil, Iron oxide Paste B: Adamantane amine N; N 0 -dibenzyl5-oxanonane-diamine-1,9,TCD-diamine calcium tungstate, Zirconium oxide, Aerosil, Silicone oil
T.-H. Huang et al. / Biomaterials 23 (2002) 77–83
cells were then transferred to a plastic tube, washed with DMEM, centrifuged, and counted, and 2 105 cells in 1 ml DMEM containing 10% fetal bovine serum (FBS, SIGMA, USA) were plated onto collagen-coated 25 mm-diameter silastic membranes, which are 2 mm thick and form the bottom surface of each well in a sixwell tissue culture flex plate. Utilizing a method previously reported by Amruthesh et al. [20], the cells were then characterized for purity. The cells were used for experiments at a total of four weeks after removal from the rat. 2.3. MTT assay A modified MTT assay based upon an original method described by Guigand et al. [21] was used. Briefly, a 5 mg/ml MTT solution (SIGMA, USA) was prepared in 378C warm DMEM immediately prior to use. A 500 : l aliquot of freshly prepared MTT solution was added to each well of each plate. The plates were incubated for 4 h at 378C in an air atmosphere containing 5% CO2. The medium was then aspirated from the wells and 500 : l of DMSO was added. The plates were shaken to maximize MTT formazan dissolution. Spectrophotometric absorbance was measured at 570 nm using an ELISA microplate reader using dimethyl sulfoxide as the blank. Mitochondrial dehydrogenase acitivity was calculated as survival %=[(the absorbance of treatment of samplethe absorbance of medium)/(the absorbance of DMSOthe absorbance of medium)] 100%. 2.4. Comet assay (alkaline single cell gel electrophoresis assay) 2.4.1. Slide preparation Logarithmic growth phase astrocytes in DMEM medium were treated with root canal sealers after mixing for 3 h at 378C in an air atmosphere containing 5% CO2 and subsequently renewed medium and incubated for 24 h prior to sampling. We followed the technique described by Singh et al. [22] for the quantitation of low levels of DNA damage in individual cells. 75 : l of 0.5% agarose (SIGMA, USA) was quickly layered onto a fully frosted glass slide and covered with another slide. The slides were placed on ice (5 min) to allow the agarose to gel, following which the top slide was removed and 50 : l of agarose containing the astrocytes (5 104 cells/50 : l) was quickly layered onto the agarose gel. Finally, 75 : l of 0.5% agarose was quickly layered on the previous layer. The cells were lysed immediately by immersion of the slide for 1 h in a lysis solution (pH=10) containing 2.5 m NaCl, 100 mm EDTA, 10 mm Trizma [Tris(hydroxymethyl)aminomethane] (SIGMA,USA), 1% Sarkosyl (SIGMA,
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USA), 10% DMSO, and 1% Triton X-100 (SIGMA, USA) maintained at 48C. 2.5. Unwinding and electrophoresis Following cell lysis, the slides were placed upon a horizontal gel electrophoresis platform and covered with an alkaline solution made up of 300 mm NaOH and 1 mm EDTA. The slides were left in the solution for 20 min to allow unwinding of the DNA and expression of alkali-labile sites. The power supply was set at 15 V and 250 mA, and the DNA was electrophoresed for 20 min, following which the slides were rinsed gently three times with 400 mm Trizma buffer (pH=7.5) to neutralize the excess alkali from the previous solution. Each slide was stained with 50 : l ethidium bromide (EthBr) and covered with a coverslip. 2.6. Examination of the cells and statistical analysis Fifty cells on one slide per treatment group were examined at 200 magnification using a fluorescence microscope equipped with an excitation filter of 515– 560 nm and a barrier filter of 590 nm which was connected through the CCD camera to an image analysis system (Matrox Inspector, Matrox Electronic systems, USA). If the cell DNA was damaged, the shape of the cell was like comet. The entire length of the comet (length) and the diameter of the head (diameter) were measured. The ‘‘shape factor’’ was calculated as the ratio of length to diameter. Migration (m) was calculated as the difference between length and diameter. The quantitative results from the MTT tests and comet assay were analyzed using the one way analysis of variance (ANOVA) to compare the various means.
3. Results The relative cytotoxicity of the epoxy resin-based sealers that was estimated over a 24 h period was assessed using an MTT assay. A typical dose-response relationship between eluates of test samples that were eluted in culture medium and corresponding cell survival rates is presented in Tables 2 and 3. Resultant cytotoxic effects of AH26 upon astrocyte cells as assessed using an MTT assay is indicated in Table 2. When the exposure concentration of the sealer powder or liquid or the mixed material was increased, the astrocyte survival rate (%) dropped ðP50:05Þ. The cytotoxic effects of AH Plus upon astrocyte cells as assessed using an MTT assay is depicted in Table 3. When the concentration of paste A or paste B or mixed sealer was increased, the astrocyte survival rate (%) decreased ðP50:05Þ.
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Table 2 Cytotoxicity of AH26 in astrocyte cells evaluated by MTT assaya Concentration
Powder
Liquid
Absorbance (M SD) Control DMSO 0.01 mg/ml 0.02 mg/ml 0.04 mg/ml 0.08 mg/ml 0.10 mg/ml F value P50:05
Survival %
0.10 0.02 1.97 0.09 1.85 0.09 1.88 0.06 1.71 0.11 0.95 0.12 0.79 0.04
Mixed
Absorbance (M SD) 0.10 0.02 1.97 0.09 2.03 0.05 1.88 0.10 1.58 0.04 0.30 0.01 0.25 0.07
93.16 94.87 85.75 45.62 36.17
172.9 Yes
Survival %
Absorbance (M SD) 0.10 0.02 1.97 0.09 1.64 0.03 1.72 0.07 1.10 0.05 0.69 0.02 0.25 0.01
103.25 94.77 79.24 10.88 7.95
984.25 Yes
Survival %
82.01 86.28 53.30 31.75 7.95
1111.22 Yes
a
Survival %=[(the absorbance of treatment of sample the absorbance of medium)/(the absorbance of DMSO the absorbance of medium)] 100%. The sample size of each group is five. The absorbance (mean standard deviation) is analyzed by one way analysis of variance method. The pure culture medium is taken as control. The DMSO concentration is 0.05% of the medium.
Table 3 Cytotoxicity of AH Plus in astrocyte cells evaluated by MTT assaya Concentration
Paste A
Paste B
Absorbance Control DMSO 0.01 mg/ml 0.02 mg/ml 0.04 mg/ml 0.08 mg/ml 0.10 mg/ml F value P50:05
Survival %
0.10 0.02 1.97 0.09 2.02 0.11 2.01 0.08 1.20 0.10 0.64 0.08 0.63 0.07
Mixed
Absorbance 0.10 0.02 1.97 0.09 1.92 0.04 2.05 0.08 2.05 0.05 1.90 0.05 1.05 0.04
102.24 101.97 58.91 28.76 28.44
301.04 Yes
Survival %
Absorbance 0.10 0.02 1.97 0.09 2.04 0.05 1.88 0.11 0.76 0.10 0.35 0.03 0.29 0.01
96.95 104.10 103.89 53.09 50.74
304.67 Yes
Survival %
103.57 95.03 35.37 13.39 10.13
688.41 Yes
a Survival %=[(the absorbance of treatment of sample the absorbance of medium)/(the absorbance of DMSO the absorbance of medium)] 100%. The sample size of each group is five. The absorbance (mean standard deviation) is analyzed by a one way analysis of variance method. The pure culture medium is as control. The DMSO concentration is 0.05% of the medium.
Table 4 The inhibition dose (ID 50) of the AH26 and AH Plus sealers AH26
Concentration (mg/ml)
AH plus
Powder
Liquid
Mixed
Paste A
Paste B
Mixed
0.08
0.06
0.05
0.05
0.11
0.04
Cellular inhibition dose 50 data are based upon a 50% reduction in mitochondrial dehydrogenase activity. The inhibition dose (ID 50) of the AH26 and AH Plus sealers is referred to in Table 4. In AH26, the lowest ID 50 was the mixed group (0.05 mg/ml). The highest ID 50 was the powder group (0.08 mg/ml). In AH Plus, the lowest ID 50 was the mixed group (0.04 mg/ml). The highest ID50 was the paste B group (0.11 mg/ml) (Table 4). In genotoxicity test, a negative control (DMSO) and a positive control (4NQO) were included in each experi-
ment and their results are summarized in Table 5. Treatment with AH26 led to a noted dose-dependent increase in all measures of DNA damage. In addition, the AH-Plus resin was also able to induce DNA damage after treatment. The DNA migration of AH Plus at this concentration (0.25 mg/ml) is higher than that for the concentration of AH26 ðP50:05Þ, suggesting that the DNA damage elicited by AH Plus is more substantial than that for AH26 when applied at the same dose.
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T.-H. Huang et al. / Biomaterials 23 (2002) 77–83 Table 5 The comet assay of the AH26 and AH Plus sealers Migration (M SD) (lengthdiameter)
Shape factor (M SD) (length/diameter)
Condition
N
DMSO (Negative control) 4NQO (Positive control) AH26 (mg/ml) 0.01 0.05 0.25 AH Plus (mg/ml) 0.01 0.05 0.25
50
44.16 1.47
F ¼ 5:20
1.00 0
F ¼ 89:23
50
57.89 5.84
P ¼ 0:025a
2.75 0.19
P ¼ 0a
50 50 50
58.26 4.69 67.74 2.32 89.13 3.21
F ¼ 19:91 P ¼ 0a
2.62 0.17 3.63 0.14 3.54 0.12
F ¼ 14:90 P ¼ 0a
50 50 50
80.20 4.90 79.04 3.23 110.83 3.33
F ¼ 21:42 P ¼ 0a
3.89 0.27 3.73 0.14 4.97 0.17
F ¼ 11:24 P ¼ 0a
F value P50:05
29.2 a
48.44 a
a It represents that the comparison is statistically significant difference at P50:05. The entire length of the comet (including the head) is defined as its length and the diameter of the head is defined as diameter. Shape factor was calculated as the ratio of length to diameter. Migration (m) was calculated as the difference between length and diameter. The negative control: DMSO concentration is 0.05% of the medium. The positive control: 4NQO concentration is 0.0003 mg/ml.
4. Discussion The MTT biological testing results of root canal sealers revealed a dose-dependent toxicity for AH26 and AH Plus, such results being in agreement with the observations of other workers applying AH26 to other cell culture systems [14,23]. The mixed group of AH26 sealer appears to be capable of inducing a greater degree of toxicity to astrocytes than is the case for either the pure powder or liquid form of AH26 ðP50:05Þ. Various in vitro and in vivo studies have shown that freshly prepared and cured specimens of the epoxy resinbased root canal sealer AH26 may induce strong cytotoxic effects [7,15,16,21]. These experimental observations have been confirmed by some clinical case reports [23–25]. It has been reported that the formaldehyde emanating from the curing sealer may be the main causative factor for the high cytotoxicity of AH26 during, particularly, the early setting period [8]. The liquid component of the AH26 is prepared using bisphenol-A-diglycidyl ether (Table 1). From our work, it is apparent that the survival rate of astrocytes treated with AH26 liquid (0.08 mg/ml) is lower than that for either powder or mixed formats ðP50:05Þ (Table 2). From the work of others, in vitro mammalian cell (V79/ hprt) mutation assay, it has been demonstrated that diglycidyl ether is the causative factor of toxicity [16], thus, clearly, it is dangerous if the mixing sealer is incomplete. There is a suggestion that the presence of uncured (liquid) sealer in the oral cavity may be a cause for health concern [16]. Thus, any remnant liquid sealer post-mixing may be more toxic than cured sealer. It is
recognized that during the setting process, AH26 may release formaldehyde [8], and that formaldehyde in the oral cavity is a toxic agent [26]. From our experiments, it is apparent that the cured AH26 sealer is toxic to astrocytes in a dose-dependent manner. The strongest cell inhibition elicited by the sealer mix occurs at a concentration of 0.10 mg/ml, at which concentration, both the liquid and mixed groups exhibit the same degree of toxicity, and, by contrast, the powder seems to be somewhat more compatible with astrocyte survival. The AH Plus product is a relatively new epoxy resinbased sealer, its manufacturer emphasizing that the cured resin will not release formaldehyde and is thus more biocompatible than AH26. From our experiments, the AH plus-induced astrocyte toxicity appears to be dose dependent, both pastes (independently) and the resin mixture groups demonstrating cytotoxicity, such results contrasting the results of Leyhausen et al. [13]. These authors suggest that minor or no cytotoxic effects were observed for this new epoxy resin-based sealer [13]. Schweikl et al. found that DMSO eluated of the mixed material, paste A and paste B clearly reduced the viability of V79 cells and was mutagenic in a dosedependent manner in V79 cells [18]. From the chemical composition of the AH Plus sealer (Table 1), it seems likely that numerous substances may be released from the material into the adjacent tissue as a result of its use in the oral cavity, and thus, there clearly exists a cogent need to undertake further investigation into the potential toxicity of the use of eluting material. The degradation of root canal sealers
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within the oral cavity, with time, may lead to component leakage into the adjacent tissue through the dentinal tubules or apex, such that the surrounding tissue of the tooth, bone or nerve, may be subsequently affected by resin-released compounds. The use of the astrocyte as target cell in this study is based upon its nerve origin. The other purpose of this study was to determine genotoxic effects due to dimethyl sulfoxide extracts of the AH26 and AH-Plus sealers, acknowledging that detectable chemical cytotoxic effects may not reflect as analogous gentoxic effects. There is only scant information regarding the mutagenicity of these root canal sealers. Schweikl et al. investigated the mutagenicity of AH26 in the V79/HGPRT mammalian cell assay [16]. They found that this material induces mutagenic effects 24 h after mixing which significantly decrease within 1 week. Stea et al. (Ames test) [27] and Heil et al. (umu, DIT) [28] found mutagenic substances even in the set material. The alkaline single cell gel electrophoresis assay (comet assay) is a sensitive method to investigate DNA breakage in individual cells as a consequence of their in vitro or in vivo exposure to genotoxic compounds [29]. In our experimental series, the mutagenic effects of AH26 and AH Plus demonstrated genotoxicity to astrocytes (Table 5). Resin-based sealers’ mutagenic potencies were noted to occur in a dosedependent manner; following exposure of astrocytes to these two compounds, an increased migration factor was noted. Observed DNA damage at a sealer concentration of 0.25 mg/ml of AH26 and AH Plus in culture medium revealed a more pronounced migration for the AH Plus group than for its analog, suggesting that the AH Plus sealer elicits more substantial DNA damage than is the case for the AH26 group. The effect of dying or dead cells upon the assay-derived data may be to influence the estimate of the positive response of resin-released chemicals, since dying or dead cells may increase DNA migration in this assay [17]. In our experiments, the AH Plus (ID 50=0.04 mg/ml) is more toxic than AH26 (ID 50=0.05 mg/ml) (Table 4); the migration of the AH Plus moiety is larger than that of its analog in the assay. There is a reduced possibility that dead cells participated in the positive responses of chemicals. In shape factor evaluation, the AH26 and AH-Plus sealers exert their influence dose-dependently, such results being similar to the results of the migration factor assessment. The study by Ersev et al. [15] indicated that mixed, silver-free AH26 elicited mutagenicity in eukaryotic and prokaryotic cells; they speculate that the mutagenic effect of AH26 may arise from the liquid component bisphenol-A-diglycidyl ether and also formaldehyde. Their experimental results were similar to those from our experiments that AH26 can elicit astrocyte DNA damage.
Our experimental series indicated that the epoxy resin-based sealers AH26 or AH Plus are not truly biocompatible [30,31]. From this work, we have demonstrated a direct dose-dependent in vitro relationship between the concentration of administered sealer and cytotoxic and mutagenic effects. In conclusion, in this study the epoxy resin-based sealers, AH26 and AHPlus demonstrated both cytotoxicity and genotoxicity in vitro.
Acknowledgements The authors acknowledge the Chung Shan Medical and Dental College (CSMC-88-OM-B-033) for supporting this project.
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The biocompatibility evaluation of epoxy resin-based root canal sealers in vitro Tsui-Hsien Huanga, Jaw-Ji Yanga, Huei Lib, Chia-Tze Kaoc,* b
a Dental Department, Chung Shan Medical and Dental College, No.110, Section 1, Chien Kuo N Road, Taichung 402, Taiwan Institute of Toxicology, Chung Shan Medical and Dental College, No.110, Section 1, Chien Kuo N Road, Taichung 402, Taiwan c Institute of Stomatology, Chung Shan Medical and Dental College, 620 Shr Jeng Road, Taichung 402, Taiwan
Received 23 October 2000; accepted 19 February 2001
Abstract The cytotoxic and mutagenic effects of epoxy resin-based root canal sealer AH26 and AH-Plus were determined in vitro. Root canal sealers were eluted for 24 h in dimethyl sulfoxide (DMSO) and diluted in culture medium. Cytotoxic effects were assessed using the MTT [tetrazolium dye, 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide, C18H16N5SBr] assay for mitochondrial enzyme activity and also the cell viability. Genotoxicity assays were assessed using the alkaline single cell gel electrophoresis assay (comet assay) for DNA damage measurement. Result indicated that both the AH26 and AH-Plus sealers exhibited a dose-dependent increase in astrocyte toxic effects. Additionally, dose-dependent astrocyte DNA damage was also noted for both sealers. Therefore, these epoxy resin-based sealers, AH26 and AH-Plus demonstrated both cytotoxicity and genotoxicity in vitro. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biocompatibility of epoxy resin sealer; MTT assay; Alkaline single cell gel electrophoresis
1. Introduction Clearly, one of the principal requirements of an endodontic root canal sealer should be that it is noncytotoxic and immunologically compatible with peripheral tissue [1]. Sealer-elutable substances or the degradation or corrosion products from a root canal sealer may gain access to periodontal tissue through numerous pathways [2,3]. Root canal sealers and their diffusible components, therefore, need to be critically evaluated for their cytocompatibility and genotoxicity prior to their general clinical use. Numerous root canal sealers are available, based on various formulae and containing a variety of different and partly mutagenic components, such as epoxy resin sealers, e.g., AH26 and AH Plus, Ca(OH)2-based materials such as Sealapex and Apeixt, and ZnOeugenol cements, e.g., N2 and endomethasone [4]. The
*Corresponding author. Tel.: +886-4-2255-3715; fax: +886-4-22553713. E-mail address: [email protected] (C.-T. Kao).
biocompatiblity and antimicrobial activity of a specific root canal sealer remains one of the principal considerations for selecting an appropriate sealer for a dental restoration [4,5]. From the literature, it would appear that the side effects of the use of various root canal sealers have been previously studied to some extent [6–8]. It has been previously demonstrated that sealer material based upon zinc oxide-eugenol was moderately to severely toxic in implantation studies, the results of such research revealing severe inflammation being observed following short setting times when the sealers were implanted subcutaneously into rabbits [7]. Spangberg et al. [8] noted that the AH 26 release of formaldehyde following component mixing will reach a maximum rate two days post-mixing. Formaldehyde release from curing endodontic material has been recognized for many years [8], formaldehyde being reputed to act as a disinfectant [9]. The disinfective agent in AH26 is methenamin [10], which is hydrolyzed to ammonia and formaldehyde [11]. The efficacy of long-term disinfection of canal by formaldehyde released from a root canal sealer has previously been shown to be low [8].
0142-9612/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 0 8 1 - 3
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There have been case reports of adverse reactions such as paraesthesia of the inferior alveolar nerve attributed to the formaldehyde released from root canal sealers [12,13], although any nerve cell reaction to the formaldehyde released from the use of a root canal sealer has not been previously reported. Thus, it is of importance to evaluate the biocompatibility of root canal sea-lers and neurological cells. In the present study the astro-cyte was used as the target cell to evaluate the toxicity. Genotoxicity, mutagenicity and carcinogenicity are very important issues associated with the systemic compatibility of root canal sealers [14]. In vitro test systems can be differentiated into a variety of different testing procedures, including test systems using bacterial cells, e.g. the classic Ames test (mutation assay) and the newly developed umu test (genotoxicity assay), and the test systems using eukaryotic cells in culture, such as the hypoxanthine-guanine phosphoribosyltransferase test (HGPRT, mutation assay), the chromosomal aberration test (mutation assay) and the DNA synthesis inhibition test (DIT, genotoxicity assay) [4]. From a review of the literature, it would appear that the investigation of any genetic effects associated with the use of epoxy resin-based root canal sealer is rare. In a previous study employing the Ames test for the use of silver-free AH26, in vitro, some degree of mutagenicity was demonstrated [15], and, further, AH26 tested with the V79/HGPRT mammalian-cell assay revealed that this sealer material elicits some mutagenic effect 24 h subsequent to mixing and administration [16]. Recently, a new assay for assessing the mutagenic potential of various compounds has been developed and is known as the alkaline singlecell gel electrophoresis assay (comet assay) [17]. This alkaline single-cell gel electrophoresis assay is both a rapid and sensitive procedure for quantitating DNA lesions in mammalian cells, and may be used to detect specific DNA damage and also DNA repair [17]. The present study was going to use this method to evaluate the root canal sealers genotoxicity. There is a newly developed epoxy resin sealer AH-Plus. According to the manufacturer’s instruction AH Plus will not release formaldehyde. The purpose of this study was to analyze the biocompatibility of 1st and 2nd generation epoxy resin sealers, e.g. AH26 and AH Plus sealers, when treated on primary cultured rat cerebral astrocytes, since only very limited information regarding the genotoxicity of resin-based root canal sealers would appear to be available. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric test was used as a cytotoxicity assay and comet assay was used to evaluate the genotoxicity.
2. Materials and methods 2.1. Sample preparation Epoxy resin sealers, AH26 and AH-Plus (De Trey Dentsply, Ballaigues, Switzerland; Table 1) were mixed according to the manufacturer’s instructions. The mixed material 100 mg was eluated in 2 ml dimethyl sulfoxide (DMSO) immediately after mixing at 378C for 24 h [18]. The solution was then centrifuged at 10,000g for 10 min. The supernatant 1 ml was added to 9 ml of Dulbecco’s modified Eagle’s medium (DMEM, SIGMA, USA) and serially diluted until the final concentration of eluate solution was 0.01, 0.02, 0.04, 0.08, and 0.10 mg/ml for use in the MTT assay, and the final concentration was 0.01, 0.05, and 0.25 mg/ml for use in the comet assay. For all culture media, the final concentration of DMSO in sealer was less than 0.1%. It is reported that DMSO at a concentration of 1% showed no toxicity to cell culture [19]. In cytotoxicity test, the pure medium without cell and sealer was used as control. In genotoxicity test, the DMSO 0.05% concentration was used as negative control and 4NQO 0.0003 mg/ml concentration was used as positive control. 2.2. Rat cerebral astrocyte culture Astrocytes were prepared as Amruthesh et al. [20] have previously reported. Briefly, cerebral cortices were isolated from one- to two-day-old rats, cleaned of white matter and meninges, minced, and trypsin-digested for 10 min. The tissue was then diluted into feeding medium, washed, triturated, counted, and placed into 75 cm2 flasks and cultured for 10–14 days at 378C in an air atmosphere containing 5% CO2. When confluent, the 1 105 cells were plated into 96 culture wells. To accomplish this, confluent cells were washed with 0.25% trypsin/0.02% EDTA in saline for exactly 1 min. The trypsin/EDTA was then aspirated, and the cell monolayer, which was still attached to the flask, was covered with 10 ml Dulbecco’s modified Eagle’s medium (DMEM) and incubated at 378C, in an air atomphosphere of 5% CO2 for approximately 10 min. The lifted
Table 1 The components of the AH26 and AH Plus sealers Product
Composition
AH26 (silver-free)
Powder: Bismuth (III) oxide, Methenamine Liquid: Bisphenol-A-diglycidyl ether
AH Plus
Paste A: Epoxy resin, Calcium tungstate, Zirconium oxide, Aerosil, Iron oxide Paste B: Adamantane amine N; N 0 -dibenzyl5-oxanonane-diamine-1,9,TCD-diamine calcium tungstate, Zirconium oxide, Aerosil, Silicone oil
T.-H. Huang et al. / Biomaterials 23 (2002) 77–83
cells were then transferred to a plastic tube, washed with DMEM, centrifuged, and counted, and 2 105 cells in 1 ml DMEM containing 10% fetal bovine serum (FBS, SIGMA, USA) were plated onto collagen-coated 25 mm-diameter silastic membranes, which are 2 mm thick and form the bottom surface of each well in a sixwell tissue culture flex plate. Utilizing a method previously reported by Amruthesh et al. [20], the cells were then characterized for purity. The cells were used for experiments at a total of four weeks after removal from the rat. 2.3. MTT assay A modified MTT assay based upon an original method described by Guigand et al. [21] was used. Briefly, a 5 mg/ml MTT solution (SIGMA, USA) was prepared in 378C warm DMEM immediately prior to use. A 500 : l aliquot of freshly prepared MTT solution was added to each well of each plate. The plates were incubated for 4 h at 378C in an air atmosphere containing 5% CO2. The medium was then aspirated from the wells and 500 : l of DMSO was added. The plates were shaken to maximize MTT formazan dissolution. Spectrophotometric absorbance was measured at 570 nm using an ELISA microplate reader using dimethyl sulfoxide as the blank. Mitochondrial dehydrogenase acitivity was calculated as survival %=[(the absorbance of treatment of samplethe absorbance of medium)/(the absorbance of DMSOthe absorbance of medium)] 100%. 2.4. Comet assay (alkaline single cell gel electrophoresis assay) 2.4.1. Slide preparation Logarithmic growth phase astrocytes in DMEM medium were treated with root canal sealers after mixing for 3 h at 378C in an air atmosphere containing 5% CO2 and subsequently renewed medium and incubated for 24 h prior to sampling. We followed the technique described by Singh et al. [22] for the quantitation of low levels of DNA damage in individual cells. 75 : l of 0.5% agarose (SIGMA, USA) was quickly layered onto a fully frosted glass slide and covered with another slide. The slides were placed on ice (5 min) to allow the agarose to gel, following which the top slide was removed and 50 : l of agarose containing the astrocytes (5 104 cells/50 : l) was quickly layered onto the agarose gel. Finally, 75 : l of 0.5% agarose was quickly layered on the previous layer. The cells were lysed immediately by immersion of the slide for 1 h in a lysis solution (pH=10) containing 2.5 m NaCl, 100 mm EDTA, 10 mm Trizma [Tris(hydroxymethyl)aminomethane] (SIGMA,USA), 1% Sarkosyl (SIGMA,
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USA), 10% DMSO, and 1% Triton X-100 (SIGMA, USA) maintained at 48C. 2.5. Unwinding and electrophoresis Following cell lysis, the slides were placed upon a horizontal gel electrophoresis platform and covered with an alkaline solution made up of 300 mm NaOH and 1 mm EDTA. The slides were left in the solution for 20 min to allow unwinding of the DNA and expression of alkali-labile sites. The power supply was set at 15 V and 250 mA, and the DNA was electrophoresed for 20 min, following which the slides were rinsed gently three times with 400 mm Trizma buffer (pH=7.5) to neutralize the excess alkali from the previous solution. Each slide was stained with 50 : l ethidium bromide (EthBr) and covered with a coverslip. 2.6. Examination of the cells and statistical analysis Fifty cells on one slide per treatment group were examined at 200 magnification using a fluorescence microscope equipped with an excitation filter of 515– 560 nm and a barrier filter of 590 nm which was connected through the CCD camera to an image analysis system (Matrox Inspector, Matrox Electronic systems, USA). If the cell DNA was damaged, the shape of the cell was like comet. The entire length of the comet (length) and the diameter of the head (diameter) were measured. The ‘‘shape factor’’ was calculated as the ratio of length to diameter. Migration (m) was calculated as the difference between length and diameter. The quantitative results from the MTT tests and comet assay were analyzed using the one way analysis of variance (ANOVA) to compare the various means.
3. Results The relative cytotoxicity of the epoxy resin-based sealers that was estimated over a 24 h period was assessed using an MTT assay. A typical dose-response relationship between eluates of test samples that were eluted in culture medium and corresponding cell survival rates is presented in Tables 2 and 3. Resultant cytotoxic effects of AH26 upon astrocyte cells as assessed using an MTT assay is indicated in Table 2. When the exposure concentration of the sealer powder or liquid or the mixed material was increased, the astrocyte survival rate (%) dropped ðP50:05Þ. The cytotoxic effects of AH Plus upon astrocyte cells as assessed using an MTT assay is depicted in Table 3. When the concentration of paste A or paste B or mixed sealer was increased, the astrocyte survival rate (%) decreased ðP50:05Þ.
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Table 2 Cytotoxicity of AH26 in astrocyte cells evaluated by MTT assaya Concentration
Powder
Liquid
Absorbance (M SD) Control DMSO 0.01 mg/ml 0.02 mg/ml 0.04 mg/ml 0.08 mg/ml 0.10 mg/ml F value P50:05
Survival %
0.10 0.02 1.97 0.09 1.85 0.09 1.88 0.06 1.71 0.11 0.95 0.12 0.79 0.04
Mixed
Absorbance (M SD) 0.10 0.02 1.97 0.09 2.03 0.05 1.88 0.10 1.58 0.04 0.30 0.01 0.25 0.07
93.16 94.87 85.75 45.62 36.17
172.9 Yes
Survival %
Absorbance (M SD) 0.10 0.02 1.97 0.09 1.64 0.03 1.72 0.07 1.10 0.05 0.69 0.02 0.25 0.01
103.25 94.77 79.24 10.88 7.95
984.25 Yes
Survival %
82.01 86.28 53.30 31.75 7.95
1111.22 Yes
a
Survival %=[(the absorbance of treatment of sample the absorbance of medium)/(the absorbance of DMSO the absorbance of medium)] 100%. The sample size of each group is five. The absorbance (mean standard deviation) is analyzed by one way analysis of variance method. The pure culture medium is taken as control. The DMSO concentration is 0.05% of the medium.
Table 3 Cytotoxicity of AH Plus in astrocyte cells evaluated by MTT assaya Concentration
Paste A
Paste B
Absorbance Control DMSO 0.01 mg/ml 0.02 mg/ml 0.04 mg/ml 0.08 mg/ml 0.10 mg/ml F value P50:05
Survival %
0.10 0.02 1.97 0.09 2.02 0.11 2.01 0.08 1.20 0.10 0.64 0.08 0.63 0.07
Mixed
Absorbance 0.10 0.02 1.97 0.09 1.92 0.04 2.05 0.08 2.05 0.05 1.90 0.05 1.05 0.04
102.24 101.97 58.91 28.76 28.44
301.04 Yes
Survival %
Absorbance 0.10 0.02 1.97 0.09 2.04 0.05 1.88 0.11 0.76 0.10 0.35 0.03 0.29 0.01
96.95 104.10 103.89 53.09 50.74
304.67 Yes
Survival %
103.57 95.03 35.37 13.39 10.13
688.41 Yes
a Survival %=[(the absorbance of treatment of sample the absorbance of medium)/(the absorbance of DMSO the absorbance of medium)] 100%. The sample size of each group is five. The absorbance (mean standard deviation) is analyzed by a one way analysis of variance method. The pure culture medium is as control. The DMSO concentration is 0.05% of the medium.
Table 4 The inhibition dose (ID 50) of the AH26 and AH Plus sealers AH26
Concentration (mg/ml)
AH plus
Powder
Liquid
Mixed
Paste A
Paste B
Mixed
0.08
0.06
0.05
0.05
0.11
0.04
Cellular inhibition dose 50 data are based upon a 50% reduction in mitochondrial dehydrogenase activity. The inhibition dose (ID 50) of the AH26 and AH Plus sealers is referred to in Table 4. In AH26, the lowest ID 50 was the mixed group (0.05 mg/ml). The highest ID 50 was the powder group (0.08 mg/ml). In AH Plus, the lowest ID 50 was the mixed group (0.04 mg/ml). The highest ID50 was the paste B group (0.11 mg/ml) (Table 4). In genotoxicity test, a negative control (DMSO) and a positive control (4NQO) were included in each experi-
ment and their results are summarized in Table 5. Treatment with AH26 led to a noted dose-dependent increase in all measures of DNA damage. In addition, the AH-Plus resin was also able to induce DNA damage after treatment. The DNA migration of AH Plus at this concentration (0.25 mg/ml) is higher than that for the concentration of AH26 ðP50:05Þ, suggesting that the DNA damage elicited by AH Plus is more substantial than that for AH26 when applied at the same dose.
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T.-H. Huang et al. / Biomaterials 23 (2002) 77–83 Table 5 The comet assay of the AH26 and AH Plus sealers Migration (M SD) (lengthdiameter)
Shape factor (M SD) (length/diameter)
Condition
N
DMSO (Negative control) 4NQO (Positive control) AH26 (mg/ml) 0.01 0.05 0.25 AH Plus (mg/ml) 0.01 0.05 0.25
50
44.16 1.47
F ¼ 5:20
1.00 0
F ¼ 89:23
50
57.89 5.84
P ¼ 0:025a
2.75 0.19
P ¼ 0a
50 50 50
58.26 4.69 67.74 2.32 89.13 3.21
F ¼ 19:91 P ¼ 0a
2.62 0.17 3.63 0.14 3.54 0.12
F ¼ 14:90 P ¼ 0a
50 50 50
80.20 4.90 79.04 3.23 110.83 3.33
F ¼ 21:42 P ¼ 0a
3.89 0.27 3.73 0.14 4.97 0.17
F ¼ 11:24 P ¼ 0a
F value P50:05
29.2 a
48.44 a
a It represents that the comparison is statistically significant difference at P50:05. The entire length of the comet (including the head) is defined as its length and the diameter of the head is defined as diameter. Shape factor was calculated as the ratio of length to diameter. Migration (m) was calculated as the difference between length and diameter. The negative control: DMSO concentration is 0.05% of the medium. The positive control: 4NQO concentration is 0.0003 mg/ml.
4. Discussion The MTT biological testing results of root canal sealers revealed a dose-dependent toxicity for AH26 and AH Plus, such results being in agreement with the observations of other workers applying AH26 to other cell culture systems [14,23]. The mixed group of AH26 sealer appears to be capable of inducing a greater degree of toxicity to astrocytes than is the case for either the pure powder or liquid form of AH26 ðP50:05Þ. Various in vitro and in vivo studies have shown that freshly prepared and cured specimens of the epoxy resinbased root canal sealer AH26 may induce strong cytotoxic effects [7,15,16,21]. These experimental observations have been confirmed by some clinical case reports [23–25]. It has been reported that the formaldehyde emanating from the curing sealer may be the main causative factor for the high cytotoxicity of AH26 during, particularly, the early setting period [8]. The liquid component of the AH26 is prepared using bisphenol-A-diglycidyl ether (Table 1). From our work, it is apparent that the survival rate of astrocytes treated with AH26 liquid (0.08 mg/ml) is lower than that for either powder or mixed formats ðP50:05Þ (Table 2). From the work of others, in vitro mammalian cell (V79/ hprt) mutation assay, it has been demonstrated that diglycidyl ether is the causative factor of toxicity [16], thus, clearly, it is dangerous if the mixing sealer is incomplete. There is a suggestion that the presence of uncured (liquid) sealer in the oral cavity may be a cause for health concern [16]. Thus, any remnant liquid sealer post-mixing may be more toxic than cured sealer. It is
recognized that during the setting process, AH26 may release formaldehyde [8], and that formaldehyde in the oral cavity is a toxic agent [26]. From our experiments, it is apparent that the cured AH26 sealer is toxic to astrocytes in a dose-dependent manner. The strongest cell inhibition elicited by the sealer mix occurs at a concentration of 0.10 mg/ml, at which concentration, both the liquid and mixed groups exhibit the same degree of toxicity, and, by contrast, the powder seems to be somewhat more compatible with astrocyte survival. The AH Plus product is a relatively new epoxy resinbased sealer, its manufacturer emphasizing that the cured resin will not release formaldehyde and is thus more biocompatible than AH26. From our experiments, the AH plus-induced astrocyte toxicity appears to be dose dependent, both pastes (independently) and the resin mixture groups demonstrating cytotoxicity, such results contrasting the results of Leyhausen et al. [13]. These authors suggest that minor or no cytotoxic effects were observed for this new epoxy resin-based sealer [13]. Schweikl et al. found that DMSO eluated of the mixed material, paste A and paste B clearly reduced the viability of V79 cells and was mutagenic in a dosedependent manner in V79 cells [18]. From the chemical composition of the AH Plus sealer (Table 1), it seems likely that numerous substances may be released from the material into the adjacent tissue as a result of its use in the oral cavity, and thus, there clearly exists a cogent need to undertake further investigation into the potential toxicity of the use of eluting material. The degradation of root canal sealers
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within the oral cavity, with time, may lead to component leakage into the adjacent tissue through the dentinal tubules or apex, such that the surrounding tissue of the tooth, bone or nerve, may be subsequently affected by resin-released compounds. The use of the astrocyte as target cell in this study is based upon its nerve origin. The other purpose of this study was to determine genotoxic effects due to dimethyl sulfoxide extracts of the AH26 and AH-Plus sealers, acknowledging that detectable chemical cytotoxic effects may not reflect as analogous gentoxic effects. There is only scant information regarding the mutagenicity of these root canal sealers. Schweikl et al. investigated the mutagenicity of AH26 in the V79/HGPRT mammalian cell assay [16]. They found that this material induces mutagenic effects 24 h after mixing which significantly decrease within 1 week. Stea et al. (Ames test) [27] and Heil et al. (umu, DIT) [28] found mutagenic substances even in the set material. The alkaline single cell gel electrophoresis assay (comet assay) is a sensitive method to investigate DNA breakage in individual cells as a consequence of their in vitro or in vivo exposure to genotoxic compounds [29]. In our experimental series, the mutagenic effects of AH26 and AH Plus demonstrated genotoxicity to astrocytes (Table 5). Resin-based sealers’ mutagenic potencies were noted to occur in a dosedependent manner; following exposure of astrocytes to these two compounds, an increased migration factor was noted. Observed DNA damage at a sealer concentration of 0.25 mg/ml of AH26 and AH Plus in culture medium revealed a more pronounced migration for the AH Plus group than for its analog, suggesting that the AH Plus sealer elicits more substantial DNA damage than is the case for the AH26 group. The effect of dying or dead cells upon the assay-derived data may be to influence the estimate of the positive response of resin-released chemicals, since dying or dead cells may increase DNA migration in this assay [17]. In our experiments, the AH Plus (ID 50=0.04 mg/ml) is more toxic than AH26 (ID 50=0.05 mg/ml) (Table 4); the migration of the AH Plus moiety is larger than that of its analog in the assay. There is a reduced possibility that dead cells participated in the positive responses of chemicals. In shape factor evaluation, the AH26 and AH-Plus sealers exert their influence dose-dependently, such results being similar to the results of the migration factor assessment. The study by Ersev et al. [15] indicated that mixed, silver-free AH26 elicited mutagenicity in eukaryotic and prokaryotic cells; they speculate that the mutagenic effect of AH26 may arise from the liquid component bisphenol-A-diglycidyl ether and also formaldehyde. Their experimental results were similar to those from our experiments that AH26 can elicit astrocyte DNA damage.
Our experimental series indicated that the epoxy resin-based sealers AH26 or AH Plus are not truly biocompatible [30,31]. From this work, we have demonstrated a direct dose-dependent in vitro relationship between the concentration of administered sealer and cytotoxic and mutagenic effects. In conclusion, in this study the epoxy resin-based sealers, AH26 and AHPlus demonstrated both cytotoxicity and genotoxicity in vitro.
Acknowledgements The authors acknowledge the Chung Shan Medical and Dental College (CSMC-88-OM-B-033) for supporting this project.
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