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Original Articles

CYTOTOXICITY STUDY OF AH26 AND AMALGAM, IN VITRO, USING HUMAN PERIODONTAL LIGAMENT FIBROBLASTS
S. Al-Nazhan, BDS, MDS*, L. Spangberg, DDS**, PhD

The toxicity of two endodontic filling materials (AH26 and amalgam) were evaluated in vitro using the radiochromium release method and primary culture of human periodontal ligament (PDL) fibroblasts. The materials were tested fresh and when set after one day. Results showed a high 51Cr-release when the materials were tested fresh. The level of the 51Cr-release decreased when the materials were allowed to set for one day. The results indicated that the human PDL fibroblast is an appropriate cell to use when evaluating the cytotoxicity of endodontic filling materials, in vitro, since they are in close contact with the tissue of periodontal ligament in case of clinical situations.

Introduction The Federation Dentaire Internationale (FDI) and the American Dental Association (ADA) have recommended several methods to evaluate the biocompatibility of dental materials in vitro and in vivo 1, 2 In vitro, established cell lines such as L929 and HeLa cells have been the most commonly used. These cell lines have a heteropioid chromosome pattern and their response to toxic constituents may not reflect that of corresponding target cells in vivo, which have a normal diploid chromosome pattern. Thus, an alternative to the established cell line is to use primary culture of human cells of dental origin when evaluating the biocompatibility of dental materials, such as root canal filling materials and sealers or retrograde filling materials, such as amalgam. These materials are in direct contact with periodontal and alveolar
* Lecturer, Department of Restorative Dental Sciences, King Saud University College of Dentis tr y, P.O. Box 60169, Riyadh 11545, Saudi Arabia **Professor and Head, Department of Restorative Dentistry and Endodontology, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut, USA Address reprint requests to: Dr. S. Al-Nazhan

bone connective tissues. Therefore, primary cultures of cells derived from such connective tissue may be the cells of choice for the evaluation of endodontic materials in vitro. One of the more commonly used endodontic materials for orthograde filling is AH26. For retrograde fillings, silver amalgam is the material of choice. AH26 and silver amalgam have been evaluated in vitro and in vivo by many investigators using different techniques. 3-10 In vivo studies were done using polyethylene tubes filled with the material and implanted into muscle, bone or subcutaneous tissues of rabbits or guinea pigs. The tissue reaction to the specimens and degree of inflammation were evaluated after various time intervals. Materials which set slowly may cause substantial irritation at first. Silver amalgam has been evaluated in vivo by several investigators. 4,6,9 . They all agreed that silver amalgam is well tolerated by animal connective tissue. AH26 caused a severe initial tissue reaction when freshly implanted. As the observation time increased, the irritational effect of the AH26 decreased. 4,5,10 By using the above-mentioned in vivo methods, several methodological problems such as flow and frag-

The Saudi Dental Journal, Volume 2, Number 2,1990

CYTOTOXICITY STUDY OF AH26 AND AMALGAM, IN VITRO, mentation of the material into the tissue were found to alter the tissue reaction. This will influence the final evaluation of the material. 7,8 In an in vitro study, Spangberg and Langeland 11 used HeLa cells and radioactive chromium to study AH26. The study resulted in a high initial toxicity which decreased when the material was allowed to set before the experiment. Similar results were reported by Kettering and Torabinejad 12 using both HeLa cells, and human fetal diploid lung fibroblasts. The toxicity in vitro of amalgam has been studied using L929 cells and measuring cell multiplication, 13 by using the millipore filter method, 14 and with the radiochromium release method.15 They all found that the toxicity of amalgam was reduced as the setting of the material progressed. Amalgam, as has also been reported, inhibits the growth of NCTC 2544 cells in culture. 16 The toxicity of amalgam to cells in culture has been confirmed through biochemical metabolic studies. The in vitro method is a simple, rapid and inexpensive screening tool that can give meaningful information about the general toxicity of dental materiafs. Well-designed in vitro methodologies may, in certain situations, make in vivo screening tests superfluous. Most of the earlier studies had been using established or primary cultures of nondental origin for the study of biocompatibility of endodontic materials. The purpose of this investigation was to evaluate, in vitro, the cytotoxicity of AH26 and silver amalgam using primary culture of human periodontal ligament fibroblasts, assess the reaction pattern and its usefulness for future experiments. Materials and Methods Two materials were evaluated: Amalgam (Caulk Dentsply Milford, Delaware 19963. Bat. #100785) and AH26 (De Trey AG, Zurich, Switzerland. Bat. #840927). The materials were prepared and mixed according to the manufacturers directions. Freshly prepared materials were evaluated after setting at 37C and 100% humidity for one day. Primary cultures of human periodontal ligament fibroblast were used. The cells were obtained from a maxillary premolar tooth extracted for orthodontic reasons from a 14-year-old boy. The culture medium was changed every other day

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and the day before an experiment. Cells were harvested with 0.02% trypsin in phosphate buffered saline (PBS). For the experiments, the cells were suspended in culture medium at a density of 5x10 s per milliliter. Eagles MEM with Earls BBS was used (Flow laboratory), supplemented with 10% (v/v) fetal calf serum, 2 mm L-giutamine and 2.2 mg sodium bicarbonate per ml. In addition, 100IU/ml penicillin and 50 ug/ml streptomycin was added to the culture medium. Cr was supplied as sodium chromate in a sterile isotonic solution. The radiochromium labeling procedure described by Spangberg 17 was followed. The labelled cells were harvested and washed four times in PBS before being suspended in culture medium. The experiments were performed in tissue culture clusters (Costar Cambridge, Mass. USA), each containing 24 wells with an inner diameter of 16 mm. The material to be evaluated was spread or condensed over the whole bottom surface of the culture well. For each material, ten experiments were done for each setting condition and each experimental time. Two ml of cell suspension was added to each culture well, and incubated at 37C. Two incubation periods of two hours, or four hours, were used. Cell suspension was added to culture wells with no test materials to provide negative controls. At the end of the incubation period, one ml of culture medium was withdrawn from each culture well and transferred into test tubes. They were centrifuged for 10 minutes at 500Xg after which 0.5 ml of the supernatant in each test tube was withdrawn and counted for 10 minutes in a gamma particle counter. During dispersion of the cells, samples were randomly withdrawn. They were used as reference for the calculation of the s1Cr- release in the experiments. The percentage of 51 Cr release was calculated on the basis of the total amount incorporated in the target cells. 17 Results The results are summarized in Table 1. Freshly prepared materials The spontaneous release was 13.2 0.3 per cent after two hours and 16.8 0.7 percent af ter four
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Table 1. Ef fect of test materials on periodontal ligament (PDL) fibroblasts, n = 10
5 ]

S.AL-NAZHAN AND LSPANCBERC tigators. 18-20 Cells of periodontal ligament of different animals, monkey 21 and pig 22 , have been cultured successfully in vitro. The use of human PDL fibroblasts to evaluate the toxicity of endodontic filling materials in vitro has not yet been reported. The evaluation of the toxicity of dental materials using human PDL fibroblasts have only been reported in few instances. 23,24 Periodontal ligament fibroblasts, isolated from human periodontal ligament tissue, were successfully cultured in this study. They were introduced in this study since they represented an appropriate mode! for testing the cytotoxicity of endodontic filling materials. In addition, they were chosen with the rationale that they may respond more like PDL fibroblasts in vivo than would an established cell line. AH26 showed a higher level of toxicity than silver amalgam during all experimental conditions. The higher level of toxicity of freshly tested AH26 has been reported by other investigators using different methodology and cell lines [HeLa cell by Rappaport et al, 25 L 929 cell by Mohammad et al, 26 human fetal diploid lung cell (L-645) by Kettering and Torabinejad 12 ]. Spangberg and Langefand 11 using HeLa cell, and the same method used in this study, reported that when AH26 was allowed to set for 24 hours, the level of toxicity decreased. The degree of cell injury increased with observation time. Such findings were also observed in this study. This could be explained that AH26 has toxic components which passively diffuse from the materials if they get in contact with a liquid phase. The toxicity of silver amalgam was found to be low. It was also reported to decrease with time after trituration when tested in vitro using L 929 cell line 13,27 using human epithelial cells (NCTC 2544), Leirskar 28 reported an In vitro experiment studying cytotoxicity of silver and copper amalgam. He found that silver amalgam releases several metals into the medium in trace amounts including silver and mercury. It was unlikely that these metals were important cytotoxic components since they do not reach sufficient concentration in the medium. In an in vivo situation, it is also unlikely that a local accumulation of such slow released ions will ever occur as tissue fluid dilution will take place. Pure silver has also been reported to be nontoxic. 29 This might explain the low level of toxicity of silver amalgam when tested in vitro. The results

Per cent release of Materials Tested

Cr at indicate

Cell-material contact time 2 Hours 4 Hours XSE XSE

Fresh: Control Amalgam AH26 One day set: Control Amalgam AH26 12.1 0.8 14.91.1 19.5 + 0.7 15.1 1.2 29.1 0.6 49.70.3 13.2 0.3 22.0 1.5 77.31.0 16.8 + 0,7 30.30.7 81 30.3

(n) = Number of experiments.

hours of incubation. AH26 produced high 3l Cr release (77.3 1.0 per cent) at two hours cellmaterial contact. This 51 Cr release was further increased after four hours of cell-material contact. As indicated by radiochromium release, amalgam had a significantly (P<0.001) lower toxicity than AH26 af ter both two and four hours of contact time. The 51Cr release for amalgam was, however, significantly increased (P<0.001) at both times as compared to the controls. Set materials The spontaneous release was 12.1 0.8 percent in the two-hour experiments, and 15.1 1.2 per cent in the four-hour experiments. The effect on cells of both set materials after two-hour contact was relatively small and not significantly increased compared to the control. The 51 Cr release as a result of the effect of amalgam at four-hour contact {29.1 0.6 percent) was significantly (P<0.001) lower than that of AH26 (49.7 0.3 percent). The effect on the cells at four hours of both materials was consistent with significant (P<0.001) higher cell injury than the controls. Discussion Tissue culture of human periodontal ligament has been reported in detail by several inves-

The Saudi Dental Journal, Volume 2, Number 2,1990

CYTOTOXICITY STUDY OF AH26 AND AMALGAM, IN VITRO, of this study, in regard to silver amalgam, correspond to earlier report from this laboratory using the chromium release method with established cell line L 929. 15
11.

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Spangberg L, Langeland K. Biological effect of dental materials. 1. Toxicity of root canal filling materials on HeLa cells in vitro. Oral Surg 1973;35:402-14. Kettering J, Torabinejad M. Cytotoxicity of root canal sealers: a study using HeLa cells and fibroblasts. Int Endodon J 1984;17:60-6. Kawahara H, Nakamura M, Yamagami A, Nakanishi T. Cellular responses to dental amalgam in vitro. J Dent Res 1975;54:394401. Tronstad L, Wennberg A. In vitro assessment of the toxicity of filling materials. Int Endodon J 1980;13:131-8. Al-Nazhan S, Sapounas G, Spangberg L. In vitro study of the toxicity of a composite resin, silver amalgam and cavit. J Endodon 1988;14:236-8. Helgeland K, Leirskar J. A further testing of the effect of dental materials on growth and adhesion of animal cells in vitro. Scand J Dent Res 1972;80:206-12. Spangberg L. Kinetic and quantitative evaluation of material cytotoxicity in vitro. Oral Surg 1973;35:389-401. Blomlof L, Otteskog P. Composition of human periodontal ligament cells in tissue culture. Scand J Dent Res 1981;89:43-6. Ragnarsson B, Carr G, Daniel D. Isolation and growth of human periodontal ligament cells in vitro. J Dent Res 1985;64:102630. Somerman M, Archer S, Imm G, Foster R. A comparative study of human perio- dontal ligament cells and gingival fibroblasts in vitro, J Dent Res 1988;67:66-70. Arnold L, Baram P. In vitro culture of periodontal ligament cells. J Dent Res 1972;51:953. Brunette D, Melcher A, Moe H. Culture and origin of epitheliallike and fibroblast-like cells from porcine periodontal ligament explants and cell suspensions. Arch Oral Biol 1976;21:393-400. Hanks C, Anderson H, Craig R. Cytotoxicity effects of dental cements on two cell culture systems. J Oral Path 1981;10:10112. Lindqwist L, Otteskog P. Eudenol: liberation from dental materiais and effect on human diploid fibroblasts. Scand J Dent Res 1981;89:552-6. Rappaport H, Lilly G, Kapsimalis P. Toxicity of endodontic filling materials. Oral Surg 1964;18:785-802. Mohammad A, Mincer H, Younis O, Dillingham E, Siskin M. Cytotoxicity evaluation of root canal sealers by the tissue cu!ture-agar overlay technique. Oral Surg 1978; 45:768-73. Kawahara H, Yamagami A, Nakamura M. Biological testing of dental materials by means of tissue culture. Int Dent J 1968;18:443-67. Leirskar J. On the mechanism of cytotoxicity of silver and copper amalgams in a cell culture system. Scand J Dent Res 1974;82:74-81. Spangberg L. Biologic effect of root canal filling materiais. (2) Effect in vitro of water soluble components of root canal filling materials on HeLa cells. Odonto Revy 1969;20:133-45.

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Conclusion
Human periodontal ligament fibroblasts have been introduced for the first time to the chromium release method for the evaluation of the cytotoxicity of endodontic filling materials. The results obtained with human periodontal ligament fibroblasts are comparable to those obtained with other established cell lines. Therefore, it is concluded that the PDL fibroblast is an appropriate cell to use when evaluating the cytotoxicity of endodontic filling materials in vitro.

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Acknowledgement
This study was financially supported by the College of Dentistry Research Center, Grant No. 1037, KingSaud University. References
1. American Dent al Association. Document No. 41 for recommended standard practices for biological evaluation of dental materials. J Am Dent Assoc 1979;99:697-8. FDI Technical Report No. 9. Recommended standard practices for biological evaluation of dental materials. 1980. Friend L, Browne R. Tissue reactions to some root filling materials. Brit Dent J 1968;125:291-8. Spangberg L Biological effects of root canal filling materials. 7. Reaction of bony tissue to implanted root canal filling material in guinea pigs. Odontol Tidskr 1969;77:501-27. Olsson B, Wennberg A. Early tissue reaction to endodontic filling materials. Endo Dent Traumatol 1985; 1:138-41. ZartnerR, James G, Burch B. Bone tissue response to zinc polycarboxylate cement and zinc-free amalgam. J Endodon 1976;2:203-8. Olsson B, Sliwkowski A, Langeland K. Intraosseous implantation for biological evaluation of endodontic materials. J Endodon 1981; 7:253-65. Olsson B, Sliwkowski A, Langeland K. Subcutaneous implantation for biological evaluation of endodontic materials. J Endodon 1981;7:355-69. Flanders D, James G, Burch B, Dockum N. Comparative histopathological study of zinc-free amalgam and cavit in connective tissue of the rat. J Endodon 1975;1:56-9. Orstavik D, Mjor I. Histopathology and X-ray micro-analysis of the subcutaneous tissue response to endodontic sealer. J Endodon 1988;14:13-22.

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