Endodontics

A Comparison of the Effect of Three Endodontic Sealers on Adherence of Mouse Peritoneal Macrophages

Ahmad Sadeghein, DDS, MS; Behnam Bolhari, DDS, MS; and A. Sarafnejad, PhD

Copyright 2001 Journal of the California Dental Association.

Leakage of sealer from root canals to periapical tissues during root canal obturation may occur. This study was designed to evaluate the possible effect of three root canal sealers (zinc oxide-eugenol, Grossman, and AH 26) on adherence of mouse macrophages. Macrophages were obtained from the peritoneal cavity of BALB/c mice and suspended in RPMI-1640 medium. Adherence capacity assays were carried out in Eppendorf tubes. Each sealer was tested four times after mixing (immediately, 12 hours, 24 hours and 48 hours after) and for three period of incubation (10, 20, and 30 minutes) with suspended cells. Cells were counted under the light microscope, and the adherence index was determined. Zinc oxide-eugenol and Grossman sealers killed all macrophages, and the adherence index was considered less than 1 for these sealers. AH 26 reduced the adherence index in all different periods after mixing and incubation times. But in sealer that had mixed 48 hours before the experiment and with 10 minutes of incubation, the adherence index was increased slightly, but the difference was not statistically significant (P<0.05).

One of the major reason for endodontic failure is incomplete obturation of the root canal. Usually, gutta percha cones and a sealer are used to obturate the root canal. A sealer should have at least some of the ideal characteristic as listed by Grossman; however, at this time there are no sealers that have all the ideal characteristics.¹

Root canal filling materials should be confined within the root canal system. However, leakage of the sealer from the root canal system to the periapical tissue during obturation may occur, which could affect treatment prognosis.^1,2

It has been demonstrated that inflamed periapical tissue contains a variety of immunocompetent cells, with macrophages predominating.^3,4 Macrophages are implicated in bone resorption⁵ and play an essential role in the pathogenesis of human periapical pathosis.⁶ They have a main role in healing by phagocytosis and debridement of wound areas.⁷ It is well-known that adherence is the first step in the phagocytic process of inflammatory macrophages.^7,8 Therefore, each material that can affect the adherence of macrophages will affect the phagocytosis.

This study was designed to evaluate possible effects of three root canal sealers -- zinc oxide-eugenol (Kemdent Works, Purton, Swindon, Wiltshire ), Grossman (Roth International Ltd., Chicago) and AH 26 (Dentrey Division Dentsply Ltd. Weybridge, Surrey, England) -- on macrophage adherence and, consequently, on repair.

Materials and Methods

Peritoneal macrophages were elicited from BALB/c mice aged 6 to 12 weeks. Each mouse was injected intraperitoneally with 5 ml of sterile thyoglycolate. Animals were sacrificed after two days, and the peritoneal cavity was washed with 10 ml of cold normal saline. After a two-minute massage, the cell exudate was removed with a syringe and centrifuged for five minutes at 250 x g at 4 degrees Celsius. Then, cells resuspended in RPMI-1640 medium were counted, adjusted in the same medium at 2-4 x 10⁶ macrophage /ml, and immediately tested. Eighty-five percent to 95 percent of these cells were macrophages by morphological criteria in Giemsa staining techniques. Viability as determined by trypan blue exclusion was always greater than 95 percent. The quantification of substrate adherence capacity was carried out according to the technique described by De la Fuenta and colleagues,⁹ with minor modification. Sealers were placed in Eppendorf tubes at a certain height (5 mm) on the wall and on the floor of the tubes. Each sealer was tested four times after being mixing and placed in the tubes (immediately, 12 hours, 24 hours, and 48 hours after). After these periods, cell suspension was added to the tubes. In the control group, cell suspension was added to empty tubes. Adherence assays were performed at 10, 20 and 30 minutes of incubation at 37 degrees Celsius in a humidified atmosphere of 5 percent CO₂ to provide a maximum adherence index.¹⁰ After these times, nonadherent cell aliquots of 10 μl from each sample were taken, and the number of nonadherent macrophage/ml was counted in Neubauer chambers. The adherence index was calculated according to the equation in Figure 1. The data were the evaluated statistically with the student’s t-test. A value of P>0.05 was considered statistically significant.

Results

Results showed that zinc oxide-eugenol and Grossman sealers killed all macrophages in all tests situations, and the adherence index was considered less than 1 for these sealers. AH 26 reduced the adherence index in all different mixing and incubation periods, with the exception of 10 minute incubation in sealer that had been mixed 48 hours before the test. In that situation, the adherence index was increased slightly but this difference was not statistically significant (P<0.05) (Figures 2 through 5).

The difference in the adherence index in groups immediately and 12 hours after mixing was significant in 20- and 30-minute periods of incubation, and the difference in groups 24 hours after mixing was significant only in 30-minute periods of incubation (P>0.05).

Discussion

Gutta percha and sealer are essential for a successful root canal obturation. However, any sealer may extrude into periapical tissue, which could affect the healing process.^1,2,11 Macrophages play an important role in the immune response of the host to noxious stimuli, as well as in the reparative process as a scavenger.^7,12 At the level of periapical tissues, macrophages, with phagocytosis and antigen presentation, have a central role in repair of chronic apical periodontitis.^3,6,13 It is well-known that adherence is the first step in the phagocytic process of inflammatory macrophages.^7,8 Therefore, each material that might affect the adherence of macrophages will affect phagocytosis and perhaps repair and inflammatory responses in periapical tissues.

Macrophages with adherence capacity were needed for this study. These cells should be obtained from either an animal or human source. Since a lot of human blood is needed for human macrophages, animal models were used for this study. According to previous studies, the peritoneal cavity of mice is a good source of macrophages,^14-18 and mouse macrophages have similar characteristics to human macrophages.¹² Intraperitoneal injection of thyoglycolate was used for eliciting macrophages, per a technique by Valizadeh and colleagues.¹⁹ Substrate adherence capacity assays were carried out in Eppendorf tubes because the phagocytic cell adherence to a smooth plastic surface is comparable to that of animal tissues.^9,20 For this experiment, three root canal sealers were selected (zinc oxide-eugenol, Grossman and AH 26). Zinc oxide-eugenol is the base of most sealers; Grossman sealer is a zinc oxide-eugenol-based sealer with the most ideal characteristics; and AH 26 is a conventional root canal sealer with an epoxy resin base.

To evaluate macrophage adherence, the cell must be contacted only with sealer. For this reason, Eppendorf tubes were coated with sealer, and cell suspension was added to them. Therefore, these cells were surrounded with sealer. Results showed that zinc oxide-eugenol and Grossman sealers were very toxic to peritoneal macrophages. Some authors believe that the toxicity of these sealers is related to eugenol.^21-25 Becker and colleagues stated that eugenol is released from zinc oxide-eugenol up to one year after mixing.²⁶ Some authors, however, believe that the toxicity of zinc oxide eugenol results from zinc ions. Maseki and colleagues²⁷ observed the toxic effect of zn2+ ions that decrease the ruffling borders of cell surfaces. AH 26 results showed that this sealer had less toxic effect than zinc oxide-eugenol and Grossman sealers on macrophages. However, a gradual increase in the number of dead cells after a 30-minutes incubation period could indicate that the gradual release of formaldehyde in the presence of moisture has a toxic effect on these cells. AH 26 decreased the adherence index in all tests, with the exception of 10 minutes incubation in sealer that had mixed 48 hours before the test. In that case there, was a slight increase of adherence index, which might be related to sealer projection after setting.

In this test, the wall of Eppendorf tubes were coated with sealer, and cell suspension was placed on the center of the coated walls; but in vivo there is no such condition, and apical extrusion of these sealers during root canal therapy could modify macrophage functions by modulating reparative mechanisms. Results indicate that peritoneal macrophages are very sensitive cells when they come into contact with the sealers used in this study. Because AH 26 didn’t kill all the cells, it can be concluded that AH 26 is the least-toxic sealer tested on macrophages.

Authors

Ahmad Sadeghein, DDS , MS, is a dentist in Tehran, Iran.

Behnam Bolhari, DDS, MS, is a dentist in Tehran, Iran.

A. Sarafnejad, PhD, is with the Department of Immunology at Tehran Medical University in Iran.

References

1. Ingle J, Bakland L, Endodontics, 4th ed. William & Wilkins, Baltimore, 1994.

2. Cohen S, Burns R, Pathways of the Pulp, 7th ed. Mosby, St Louis, 1998.

3. Weine FS, Endodontic Therapy, 5th ed. Mosby, St Louis, 1995.

4. Stern MH, Dreizen S, et al, Isolation and characterization of inflammatory cells from the human periapical granuloma. J Dent Res 61:1408-12, 1982.

5. Horton JE , Oppenheim JJ, et al, Macrophage-lymphocyte synergy in the production of osteoclasts activating factor. J Immunol 113:1278-87, 1974.

6. Torabinejad M, Eby WC, Naidorf IJ, Inflammatory and immunological aspects of the pathogenesis of human periapical lesions. J Endod 11(11):479-88, 1985.

7. Trowbridge HO, Inflammation, 5th ed. Quintessence, Chicago, 1997.

8. Male D, Champion B, Cooke A, Advanced Immunology, 2nd ed. JB Lippincott, Philadelphia, 1989.

9. De la Fuente M, Del Rio M, et al, Modulation of phagocytic function in murine peritoneal macrophages by bombesin, gastrin-releasing peptide and neuromedin C. Immunology 73(2):205-11, 1991.

10. Segura JJ, Guerrero JM, et al, Vasoactive intestinal peptide (VIP) inhibits substrate adherence capacity of rat peritoneal macrophages by a mechanism that involves cAMP. Cell Adhes Commun 1(3):210-21, 1993.

11. Seltzer S, Endodontology: Biologic Considerations in Endodontic Procedure, 2nd ed. Lee & Febiger, 1988.

12. Lewis CE, Mc Gee J, The Natural Immune System: The Macrophage, 1st ed. Oxford University Press, New York, 1992.

13. Segura JJ, Jimenez Rubio, Gomez Millan, Macrophage, 1st ed. Quintessence, Chicago, 1996.

14. Jimenez-Rubio A, Segura JJ, et al, In vitro study of the effect of sodium hypochlorite and glutaraldehyde on substrate adherence capacity of macrophages. J Endod 23(9):562-4, 1997.

15. Jimenez-Rubio A and Segura JJ, The effect of the bleaching agent sodium perborate on macrophage adhesion in vitro: implications in external cervical root resorption. J Endod 24(4):229-32, 1998.

16. Segura JJ, Calvo JR, et al, EDTA inhibits in vitro substrate adherence capacity of macrophages: endodontic implications J Endod 23(4):205-8, 1997.

17. Segura JJ, Liamas R, et al, Calcium hydroxide inhibits substrate adherence capacity of macrophages. J Endod 23(7):444-7, 1997.

18. Segura JJ, Jimenez-Rubio AJ, et al, Effect in vitro of Tifell (formocresol-eugenol) on macrophage adhesion. Int Endod J 31(2):112-6, 1998.

19. Valizadeh M, Effect of Ithraconazole & ketoconazole on major lyschmania in cell culture. Hygiene faculty of Tehran University, Thesis, 1992 .

20. Noga SJ, Norman SJ, Weiner RS, Method in laboratory investigation: Isolation of guinea pigs monocytes and Kurloff cells. Characterization on monocyte subsets by morphology, cytochemistry and adherence. Lab Invest 51:244-52, 1984.

21. Biggs JT, Kaminki EJ, Osetek EM, Rat macrophage response to implanted sealer cements. J Endod 11(1):30-6, 1985.

22. Arenholt-Bindslev D, Horsted-Bindslev P, A simple model for evaluating relative toxicity of root canal filling materials in cultures of human oral fibroblasts. Endod Dent Traumatol 5(5):219-26, 1989.

23. Briseno BM and Willershausen B, Root canal sealer cytotoxicity on human gingival fibroblasts: 2. Silicone- and resin-based sealers. J Endod 17(11):537-40, 1991.

24. Gerosa R, Borin M, et al, In vitro evaluation of the cytotoxicity of pure eugenol. J Endod 22(10):532-4, 1996.

25. Catanzaro Guimares SA and Percinoto C, Effect of some endodontic materials on the influx of macrophages and multinucleated giant cell development in experimental granulomas. J Endod 10(3):101-4, 1984.

26. Becker RM, Hume WR, Wolinsky LE, Release of eugenol from mixtures of ZOE in vitro. J Pedod 8(1):71-7, 1983.

27. Maseki T, Nakata K, et al, Lack of correlation between the amount of eugenol release from zinc oxide eugenol sealer and cytotoxicity of the sealer. J Endod 17(2):76-9, 1991.

To request a printed copy of this article, please contact / Ahmad Sadeghein, DDS, MS, 29 West Nahid Av./Valiasr St., Tehran, Iran or Sadegha@farabi.hbi.dmr.or.ir.

Figure 1. The calculation formula for the adherence index.

Figure 2. The adherence index results of the AH sealer immediately after mixing.

Figure 3. The adherence index results of the AH sealer 12 hours after mixing.

Figure 4. The adherence index results of the AH sealer 24 hours after mixing.

Figure 5. The adherence index results of the AH sealer 48 hours after mixing.