0099-2399/95/2109-0446503.00/0 JOURNALOF ENDODONTICS Copyright © 1995 by The American Association of Endodontists
Printed in U.S.A. VOL. 21, NO. 9, SEPTEMBER1995
Cytotoxicity Evaluation of Six Root Canal Sealers Roberto Gerosa, MD, DDS, Gianluca Menegazzi, MD, DDS, Marco Borin, DDS, and Giacomo Cavalleri, MD, DDS
Cell cultures of human gingival fibroblasts obtained from healthy patients were used to evaluate the toxicity of six different endodontic cements: AH-26, Pulp Canal Sealer, RocanaI-R2, RocanalR3, Bioseal, and Endomethasone. The toxicity was determined by measuring spectrophotometrically at 405 nm the colorimetric reaction of N-acetyl-/3hexosaminidase, an endogenous enzyme, with the chromogenic substrate [p-nitrophenoI-N-acetyl-/3D-glucosamide (NAG)]. Severe cytotoxicity was observed in the 1- and 2-wk test solutions of AH26. Pulp Canal Sealer and Endomethasone showed low cytotoxicity in the 1- and 2-wk test solutions at 24, 48, and 72 h. Moderate cytotoxicity was observed in the 1- and 2-wk test solutions of Bioseal, except at 48 and 72 h of 1-wk test solutions. RocanaI-R2 showed severe cytotoxicity in the 1-wk test solutions at 48 and 72 h, and in the 2-wk test solutions at 24, 48, and 72 h. Moderate cytotoxicity was seen in the 1- and 2-wk test solutions of RocanaI-R3 only at 24 h.
greater control over the experimental situation. The main disadvantage in that cellular reactions in the in vitro tests are extremely sensitive. We used human gingival fibroblasts, because in tissue culture studies they more closely resemble in vivo conditions than do other types of cells. To determine toxicity, N-acetyl-/3-hexosaminidase--an endogenous enzyme involved in cellular degradation-was used. The degradation enzyme reacting with the chromogenic substrate (NAG) produces a yellow color that determines the degree of toxicity (16) when measured spectrophotometrically at 405 nm.
MATERIALS AND METHODS
Preparation of Samples and Test Solutions The following six root canal filling materials were used: Pulp Canal Sealer (Kerr, Detroit, MI), AH-26 (De-Trey, Zurich, Switzerland), Rocanal-R2 and Rocanal-R3 (La Maison Dentaire, SA Balzers/Liechtenstein), Bioseal (Ogna Pharma, Milan, Italy), and Endomethasone (Sp6cialitrs, Septodont, France). The cements were mixed according to the manufacturer's instructions. Teflon tubes as recommended by the Federation Dentaire Interuationale (17) (3 mm internal diameter and 4 mm long) were filled with the cements to be tested. After setting, both rough ends of the tubes were smoothed to obtain a constant surface area (11). All samples were sterilized in an ethylene oxide gas chamber at 37°C for 48 h. Following sterilization, they were allowed to remain in the aeration chamber with forced circulation for another 72 h to ensure ,the complete removal of any gas. For each cement tested, 30 teflon tubes were filled and placed in 30 ml of phosphate-buffered saline (PBS) solution at 37°C and 100% humidity for 1 wk. At the end of the 1st wk, PBS was withdrawn and examined for cytotoxicity. Thirty ml of fresh PBS was added to the samples that were stored for another week. With this procedure, test solutions were obtained for each endodontic cement. A 20% test solution was transferred to cluster well cell culture plates (each containing 24 wells), with fibroblasts at a concentration of 10E+3 cell/ml/well (surface area cm2), and incubated at 37°C, 100% relative humidity for 24, 48, and 72 h (11).
The aim of this study was to evaluate the toxicity of six different endodontic sealers, most frequently used in our clinical practice. Methods for evaluating the biocompatibility of endodontic sealer materials are traditionally described as in vitro and in vivo. The in vivo tests (1-6) are acute systemic reaction responses following implantation and response over a long period of time (1). The preferred test is the implantation test, because it evaluates tissue reaction to endodontic cements. With the in vivo method, several factors such as bias control, experimental trauma, variations and errors in preoperative diagnosis, uncontrolled or undetected infections, and host response often interfere with the interpretation of the results (4, 5). With in vitro methods (7-11), it is possible to study the cytotoxicity of endodontic materials with reliability and reproducibility. The cells most frequently used are HeLa cells (1-3, 6, 7, 9), but transformed cells have different biological properties from those of normal diploid cells (12, 13). Gingival fibroblasts are easily reproducible in vitro (14), have a common connective tissue origin of periodontal membrane fibroblasts, and react much in the same way (15) as periodontal membrane fibroblasts in tissue cultures. The main advantage of these tests is a
Cell Culture Human gingival fibroblasts were obtained from the biopsy of normal attached gingival tissue from the lower molar region. The tissue was stored in 250 ml plastic culture flasks containing Dul-
Vol. 21, No. 9, September 1995
3O 2O 10 0
FiG 1. Cgtotoxicity of six endodontic cements at 1 wk expressed in percentage of viable cells compared with a control group. Hours refer to the duration of the cgtotoxicity experiment (24, 48, and 72 h).
FIG 2. Cytotoxicity of six endodontic cements at 2 wk expressed in percentage of viable cells compared with the control group. Hours refer to the duration of the cytotoxicity experiment (24, 48, and 72 h).
becco's modified Eagle's medium (DME). After 7 to 10 days, the biopsy tissue showed the growth of fibroblasts containing DME as a medium, as well as other cells. Fibroblasts were detached by trypsin and selected during the course of the same cellular detachment process. After seven passages of cellular expansion, the fibroblasts were transferred onto cluster well cell culture plates, each containing 24 wells, at a concentration of 10E+3 cell/ml/well (surface area 2 cm) and stored at 37°C and 100% humidity for 24, 48, and 72 h (7-11).
93.02% at 72 h. The 1-wk test solutions of Endomethasone had lower values: 85.2% at 24 h, 73.3% at 48 h, and 69.1% at 72 h. The 2-wk test solutions had between 100 and 86.9% viable cells at 24, 48, and 72 h. The test solutions of AH-26 had between 5.1% and 0.16% viable ceils at 24, 48, and 72 h. The 2-wk test solutions of Rocanal-R2 had 8.8% viable cells at 24 h, 1.5% at 48 h, and 2.2% at 72 h. The 1-wk test solutions of Rocanal-R3 had between 89.4% and 21.3% viable cells at 24, 48, and 72 h. The 1-wk test solutions of Bioseal had 88.7% viable cells at 24 h, 47.7% at 48 h, and 14.6% at 72 h.
Cytotoxicity Evaluation To determine the cytotoxicity, we compared the number of viable cells and control cells. We used the colorimetric reaction of N-acetyl-/3-hexosaminidase (EC 184.108.40.206). This enzyme is involved in the cellular degradation of the glycosylated constituents. For the chromogenic substrate solution, we used 3.75 mM of p-nitrophenol-N-acetyl-/3-D-glucosamide (NAG) in 0.05 M citrate buffer containing 0.25% Triton X-100 (pH 5). This solution turned yellowish in an alkaline environment and could be spectrophotometrically measured at 405 nm. After 24, 48, and 72 h of incubation at 37°C, 5% CO2, and 100% humidity, the cells were washed three times with PBS to remove the nonviable ceils. Then, 400/~1 of NAG substrate was added. After 3 h of incubation at 37°C and 100% humidity, 100 /~1 of buffer solution containing 250 mM glycine (pH 10.5), 10 mM EDTA was added. Absorption was measured using a Bio-Rad 450 microplate reader with a wavelength of 405 nm (16). This procedure was performed on the test solution from the 1st and 2nd wks. The reaction between the enzyme and the substrate produced a yellow coloration that, measured as optical density, gave the percentage of viable cells as compared with control cells. Therefore, the absorption is directly proportional to the percentage of viable cells as compared with control cells. The coefficient of variation for our method is 8.9%.
RESULTS The 1-wk experimental results are shown in Fig. 1. The 2-wk results are represented in Fig. 2. The 1- and 2-wk test solutions of the Teflon tubes and control group (PBS solutions) had 100% viable cells. The 1-wk test solutions of Pulp Canal Sealer had 100% viable cells at 24 h, 88.4% at 48 h, and 73.9% at 72 h. The 2-wk test solutions had 100% viable cells at 24 and 48 h, and
DISCUSSION The percentage of viable cells represents the level of cytotoxicity of the test materials. In this study, to determine the cytotoxicity, we compared the number of viable cells with the control cells. The choice of these six specific sealers was based on the positive results we have had in our clinical practice over 5 yr and on the manufacturers' indications of their low toxicity. The cellular behavior in cultures containing 1- and 2-wk test solutions and PBS showed that the insertion of unfilled test tubes and PBS had no influence on human gingival fibroblasts. Low cytotoxicity (100% to 73.9%) appeared in the 1- and 2-wk test solutions of Pulp Canal Sealer. Similar results were obtained in the studies of zinc oxideeugenol cements by Sp~inberg and AI-Nazhan (8), and by Arenholt-Bindslev and Hfrsted-Bindslev (9), both using cultures. Safavi et al. (10) also showed that, by using cultures, moderate cytotoxicity was observed in all experimental periods, except immediately after the mixing of Pulp Canal Sealer. The cause of this initial cytotoxicity may be free eugenol or zinc ions remaining in the zinc oxide and eugenol mixture. Moderate cytotoxicity was observed during all experimental periods in the 1- and 2-wk test solutions of Endomethasone and Bioseal. Endomethasone contains hydrocortisone, which can contribute to its toxicity (18). These results were in accordance with the work of Fonzi et al. (6). They reported that Bioseal exhibited a low cytotoxicity in a hemolysis test and a mild tissue reaction in a subcutaneous implantation. Severe cytotoxicity (8.8% to 2.2%) was found in the 2-wk test solutions of Rocanal R2, which contains O-phenilphenol and is definitely toxic (19). Severe cytotoxicity of AH-26 was also observed under all experimental conditions in our study. The fact that AH26 contains toxic epoxide bisphenol resin and that it supposedly releases formaldehyde during the setting stage may explain, in part, the results of our study. These results were in accordance with the
Journal of Endodontics
Cavalleri et al.
work of Nakamura et al. (11), who used cultures, and with the work of Arenholt-Bindslev and H6rsted-Bindslev (9), who also used cell cultures. The present study differs from the studies by Cotton (2), who used histopathological techniques. He showed that AH-26 contained a nontoxic hardener and was well-tolerated. Moderate toxicity of AH-26 was described in the study by Wennberg (3), who used histopathological techniques as well as the Millipore filter method. The 2-wk test solutions of all materials examined, with the exception of Rocanal-R2, showed higher viable cells counts. This may be due to the fact that, because the PBS solutions were changed weekly, the 1-wk test solutions contained less cytotoxic substances than the 2-wk test solutions. All the cements in our study, with the exception of AH-26, contain eugenol. No positive correlation was found between eugenol release and cytotoxicity of the root canal filling material (20). Moreover, each cell culture was examined under an inversion microscope to observe the morphological changes of the cells and the cytotoxic effects of the various endodontic cements. In the cultures exposed 24 h to a 1-wk test solution of Pulp Canal Sealer, all the fibroblasts were alive at 24 h. The fibroblast culture exposed 24 h to a 1-wk test solutions of Bioseal showed a moderate reduction of viable cells. In those exposed 24 h to a 1-wk test solution of Rocanal-R2 diluted to 50%, the cultures showed pseudosyncytial formations that are a sign of morphological cellular alteration and evident cytotoxicity caused by the components of the cements. Finally, fibroblast cultures exposed 24 h to a 1-wk test solution of AH-26 caused the death of all the human gingival fibroblasts. When in vitro techniques are used, the purpose is to study the toxicity, and not the response of the tissue. The sensitivity of in vitro methods is markedly influenced by the technique, the cells, the medium used, the serum content of the medium, pH and incubation temperatures, nutrients, and the surface area of the test specimen. It is obvious that other factors may influence the toxic effect of the material. Tissue response also depends on several factors, of which toxicity is often the most important. Biocompatibility of tissue depends mainly on the histopathological techniques used, the use of different experimental animals, the implantation site, the control of the operator, experimental trauma, variations and errors in preoperative diagnosis, uncontrolled or undetected infections, and host response. Discrepancies in the results of these published experiments are difficult to analyze. We must remember that in vitro toxicity data are relative and not directly comparable to an in vivo situation, although in vitro models are useful in the selection of the filling materials to be used. As demonstrated in this and other related studies, cultures of human oral fibroblasts are easily grown and propagated under standardized conditions. Our model of human gingival fibroblasts is simple, relatively cheap, and can be reproduced in any laboratory that routinely handles cells. This method can also be used to evaluate the same material sample in different conditions, and the cell cultures can be observed under a microscope. Advances in the study of the cytotoxicity of endodontic cements
as determined by in vitro methods are a step toward producing inert endodontic sealers. Drs. Cavalleri, Gerosa, Menegazzi, and Borin are affiliated with the Department of Endodontics and Restorative Dentistry, University of Verona, Verona, Italy. Address requests for reprints to Dr. Giacomo Cavalleri, Clinlca Odontoiatrica, Ospedale Policlinico, University of Verona, Via Detle Menegone, 37134, Verona, ltaty.
References 1. Cohen S, Burns RC. Pathways of the pulp. 24th ed. St. Louis: CV Mosby, 1985:364. 2. Cotton WR. Symposium: methodology and criteria in the evaluation of biologic effects of dental materials. Introductory remarks. J Endodon 1978; 10:295-315. 3. Wennberg A. Biological evaluation of root canal sealers using in vitro and in vivo methods. J Endodon 1980;10:784-7. 4. Olsson B, Sliwkowski A, Langeland K. Introsseous implantation for biological evaluation of endodontic materials. J Endodon 1981 ;6:253-65. 5. Stern M, Mackler BF, Dreizen S. A quantitative method for the analysis of human periapical inflammation. J Enclodon 1981 ;2:70-4. 6. Fonzi L, Gasparoni A, Capezzuoli L, Carboncini S, Belli M, Kaitas V. Considerazioni su prove di biocompatibilita "in vitro" ed "in vivo" di alcuni cementi endodonticL Giorn Ital Endod 1991;3:70-8. 7. Briseno BM, Willershausen B. Root canal sealer cytotoxicity on human gingival fibroblast. I. Zinc oxide-eugenol-based sealers. J Endodon 1990;8: 383-6. 8. Sp&ngberg LSW, AI-Nazhan SA. The radiochromium release method for evaluation of cytotoxicity in vitro. Int J Endodon 1988;21:72-8. 9. Arenholt-Bindslev D, HSrsted-Bindslev P. A simple model for evaluating relative toxicity of root filling materials in cultures of human oral fibroblasts. Endod Dent Traumatol 1989;5:219-26. 10. Safavi KE, Sp&ngberg LSW, Costa NS, Sapounas G. An in vitro method for longitudinal evaluation of toxicity of endodontic sealers. J Endodon 1989;10:484-6. 11. Nakamura H, Sakakibara F, Matsumoto Y, Hirano S, Hayakawa H, Sakai K, Yip M. Study on the cytotoxicity of root canal filling materials. J Endodon 1986;4:156-60. 12. Hanks CT, Anderson M, Craig RG. Cytotoxic effects of dental cements on two cell culture systems_ J Oral Pathol 1981;10:101-12. 13. Feigal RJ, Yesilsoy C, Messer HH, Jeison J. Differential sensitivity of normal human pulp and transformed mouse fibroblast to cytotoxic challenge. Arch Oral Biol 1985;30:609-13. 14. Arvidson K, Cart G, Daniel JC. Isolation and growth of human periodontal ligament cells in vitro. J Dent Res 1985;64:1026-30. 15. Lange DE, Knolle G, Herforth A, Fesseler A. Empfehlungen der Deutschen Gesellschaft fLir Paradontologie zur Planung, Ausf0hrung, Ver£~ffentlichung von Untersuchungen zum Wirksamkeitnachweis von Medikamenten bei arodontalen Erkrankungen. (Recommendations by the German Association of Periodontology for planing, conducting and publishing of research concerning the proof of efficacy of drugs in periodontal disease.) Zahn&rzt Mitt 1988;1509-14. 16. Federation Dentaire Internationale. Technical report no. 9-1980: recommended standard practices for biological evaluation of dental materials. Inf Dent J 1974;24:235-50. 17. Landegren U. Measurement of cell number by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J Immunol Methods 1984;67:379-88. 18. Radostina AI, Zumangi N, Malomu U-UF. Changes in the ultrastructure and flbrogenic activity of rat dermal fibrobiasts as affected by various doses of hydrocortisone. Arkh Anat Gistol Embriol 1989;96:52-8. 19. Nakagawa Y, Tayama S, Moore G, Moldeus P. Cytotoxic effects of biphenyl and hydroxybiphenyls on isolated rat hepatocytes. Biochem Pharmacol 1993;45:1959-65. 20. Maseki T, Nakata K, Kohsaka T, Kobayashi F, Hirano S, Nakamura H. Lack of correlation between the amount of eugenol released from zinc oxideeugenol sealer and cytotoxicity of the sealer. J Endodon 1991;17:76-79.