JOURNAL OF ENDODONTICS I VOL 5, NO 7, JULY 1979
Evaluation of systemic immunological reactions to AH-26 root canal sealer Mahmoud
T o r a b i n e j a d , DMD, MSD; James D. K e t t e r i n g , PhD;
a n d Leif K. B a k l a n d , DDS, L o m a Linda, C a l i f
T w e n t y t e e t h in each of t h r e e dogs w e r e cleaned, s h a p e d , a n d t h e n o b t u r a t e d with g u t t a - p e r c h a a n d A H - 2 6 root canal sealer. In m o s t of the teeth, s o m e sealer was i n t e n t i o n a l l y f o r c e d into the periapical tissues. S e v e n d a y s after the r o o t canal p r o c e d u r e s w e r e c o m p l e t e d , passive h e m a g g l u t i n a t i o n , l y m p h o c y t e t r a n s f o r m a t i o n , a n d skin tests were p e r f o r m e d to assess the p r e s e n c e or a b s e n c e of s y s t e m i c a n t i b o d y f o r m a t i o n and d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n to the sealer. T h e results indicate that u n d e r clinical c o n d i t i o n s , A H - 2 6 root canal sealer d o e s not p r o d u c e a n y s y s t e m i c a n t i b o d y f o r m a t i o n or d e l a y e d h y p e r s e n s i t i v i t y reaction.
Several investigators have shown that injecting potential antigens such as bacteria, bovine serum albumin, and horse serum into the root canals of teeth of experimental animals produces antibody formation. ~-G Other experiments in immunized animals also indicate that interactions between antigens and antibodies can occur within the pulp or periapical tissues. T'~ These studies and others ~'3 show that the root canal system is an effective route for host sensitization. Block and associates'"~'~ injected pulp tissue incubated with dental materials intramuscularly and then filled the root canals with the same experimental materials. The incubated pulp tissue was mixed with Freund's incomplete adjuvant and 196
injected intramuscularly in the regions of the cervical and popliteal lymph nodes. Materials tested ineluded N2-type paste, eugenol, formocresol, paraformaldehyde, and root canal sealers. The tanned red blood cell hemagglutination technique, the lymphocyte transformation test, and a skin test were used to determine the presence of antibody formation and cell-mediated reaction to the test materials. The investigators concluded that the foregoing materials, when combined with pulp protein, can cause both systemic humoral and cell-mediated responses, and that the root canal system can be a route for immunization. Recently, Campbell and associates-"' tested hardened or set RG2B,
ProcoSol, calcium hydroxide, and zinc oxide-eugenol cements to assess their ability to induce cell-mediated immune responses in guinea pigs. Skin tests and assays of migration inhibitory factor showed that the test material in the hardened state caused inflammatory changes but no delayed hypersensitivity reactions. It is important to observe that in the studies just described, 14-'-'~protocols were used that differ from routine endodontic procedures. The purpose of the current investigation was to simulate the clinical situation and assess the presence or absence of systemic immunologic reaction to AH-26 root canal sealer, which is suggested to be a hapten, TM by using the passive hemagglutination, lymphocyte transformation, and skin tests.
JOURNAL OF ENDODONTICS I VOL 5, N O 7, JULY 1979
MATERIALS AND METHODS
percha and the sealer, and the access cavities were filled with Cavit.:~
Test animals Three healthy dogs, each weighing 30 to 35 lb, were housed in individua l cages and fed laboratory dog food a d libitum. Two other dogs were used as control animals. Five teeth in each experimental dog were filled at seven-day intervals during a 28-day period, for a total of 20 teeth in each animal. The experimental procedure -.onsisted of the following steps. Each dog was given premedication, a subcutaneous injection of 1/150 grain (0.4 rag) of atropine sulfate* and a half-grain (32.0 mg) of morphine and then intravenously injected with the anesthetic sodium pentobarbitalJ" (35.0 mg/lb). Preoperative radiographs were made of the mandibular incisors, premolars, and maxillary incisors. The teeth were isolated with a rubber dam, and the field of operation was scrubbed with 95% isopropyl alcohol. The pulps were exposed by grinding the occlusal or incisal part of each tooth with a no. 701 fissure bur and then extirpated with barbed broaches and frozen at - 1 0 C in sterile saline solution. After the root canals were irrigated with 2.5% sodium hypochlorite, they were cleaned and shaped at 0.5 to 1.0 mm from the apex. In most instances in which the apical foramenas were not readily negotiable, intentional apical perforations were made with endodontic reamers so that the root canal sealer could extrude into the periapical tissues. After the root canals were cleaned and shaped, they were irrigated with 70% isopropyl alcohol and dried with sterile paper points. AH-26 root canal sealer was mixed to a creamy consistency. The root canals were obturated with gutta-
Incubation of pulp with experimental material Forty-eight hours before the immunologic assays were performed, 25 mg of the frozen pulp from each dog was incubated with AH-26 root canal sealer and placed in the CO. incubator at 37 C for 48 hours. The pulp then was removed from the sealer, rinsed twice with saline solution, and placed in 2 ml of R P M I medium. The treated pulp was cut into small pieces with scalpel blades and then homogenized by crushing with a sterile blunt instrument. This tissue served as the experimental antigenic material and was used for the following tests. Pulp in sterile saline solution was the control. Seven to 14 days after 20 teeth were filled in each dog, the following tests were performed to detect antibody formation and delayed hypersensitivity to AH-26 root canal sealer.
Antibody formation test. The procedure used to determine antibody formation was the passive hemagglutination technique described by Tan and Peebles.'-':' Controls consisted of a human serum with antibodies to nuclear ribonucleoprotein (nRNP) and a negative human serum. Tanned sheep erythrocytes were coated either with an extract of rabbit thymus containing n R N P antigen, with homogenized AH-26-incubated pulp, or with saline-treated pulp. The passive hemagglutination test was performed in a microtiter system. Sera from the dogs were collected before and after treatment and were tested. Different
concentrations (1:2 to 1:1026) of the sera were tested against erythrocytes containing AH-26-incubated pulp or saline-treated pulps. Each dog was tested only against its own pulp. As a positive control, various concentrations of human sera with antibodies to nRNP were added to tanned sheep erythrocytes coated with nRNP antigen. As an additional control, the final sera also were tested against tanned sheep erythrocytes with no antigen attached. Positive hemagglutination consisted of' a smooth mat of cells covering the bottom of the well ( + ) , and a negative result was a clearly defined button in the bottom of the well (0). Titers are expressed as the highest dilution giving positive hemagglutination.
Delayed hypersensitivity test The lymphocyte transformation test was used to detect possible cellmediated immunity in this experiment and was performed according to methods described by Shifrine and associates5 '~ Ten milliliters of blood was drawn from a vein in the leg and placed in a tube containing heparin. The 10.0 ml of heparinized blood was diluted with an equal volume of R P M I 1640 medium. Three ml of Ficoll-Paque (specific gravity, 1.067) was placed into each of four 15.0-ml conical centrifuge tubes. Five milliliters of the diluted blood was carefully layered over the Ficoll-Paque solution with a sterile pipette. These tubes were centrifuged at 1,500 rpm for 40 minutes at room temperature (500 g at the interface). The lymphocyte layer was aspirated from each tube and pooled in a 50-ml sterile conical tube for washing. Cells were washed three times with R P M I 1640
197
T a b l e 1 9 R e s u l t s o f p a s s i v e h e m a g g l u t i n a t i o n test in o n e d o g . *
Dilutions Positive serum controlt Negative serum control Preoperative dog's serum Preoperanve dog's serum Preoperanve dog's serum Postoperauve dog's serum~ Postoperative dog's serum~ Postoperauve dog's serum~ medium. Cells were collected by spinning ten minutes at 1,500 rpm at room temperature. T h e lymphocytes finally were resuspended in 1.0 ml of the R P M I m e d i u m which was supplemented with 10% pooled dog serum. T h e cells were counted with a haemocytometer and diluted to a concentration of 1 to 2 • 10 '~ cells per ml. Staining with Erythrocin B showed 90% to 100% viability of the lymphocytes. Volumes of 0.1 ml then were placed in the wells of a 96-well tissue culture plate. Mitogens tested were phytohemagglutinin ( P H A ) as a control, AH-26 incubated pulp, and saline-treated pulp. Control mitogen was used at optimal dilutions for the system, and AH-26-incubated pulp and saline-treated pulp were tested at dilutions ranging from 1:2 to 1:50. Mitogens were added to the wells in 0.1 ml volumes. Plates were allowed to incubate in a CO2 incubator at 36 C for four days. O n e microcurie of aH-thymidine (specific activity, 6.7 c i / m M ) was added to each well and incubated for approximately 18 to 20 hours. Cells were collected using a M A S H II harvester. Filters containing radioactivity were placed in liquid scintillation vials and counted on a Beckman LSC 250 counter. Stinmlation indexes (SI) were calculated by dividing cell counts stimulated by mitogen or test material with cell counts from untreated controls. A n y SI greater than 2.00 with test material was considered significant and indicated the presence of delayed hypersensitivity reaction.
Skin test Five milligrams of each dog's own AH-26-incubated pulp was homogenized in 0.I ml of R P M I m e d i u m 198
1:2 1:4 1:8 1:16 1:64 + + + + + 0 0 0 0 0 0 0 0 0 0 0
0
0
0
0
0 + + +
0 _+ + 0
0 0 0 0
0 0 0 0
0 0 0 0
1:256 1:1024 + + 0 0 0 0 AH-26 pulp 0 0 No antigen 0 0 Saline pulp 0 0 AH-26 pulp 0 0 No antigen 0 0 Saline pulp
*The results were similar for all three experimental animals. t + indicates positive hemagglutlnation: 0, negative hemagglutination: and -+, incomplete hemagglufination, ~l'he positive hemagglutinations obtained in dilutions 1:2 and 1:4 with postoperative dog's sera are nonspecific because the same results were obtained with untanned erythrocytes as well as AH-26 incubated pulp and saline-incubated pulp.
T a b l e 2 9 R e s u l t s o f l y m p h o c y t e t r a n s f o r m a t i o n test in o n e dog. *
Dilutions
AH-26incubated pulp
Salinetreated pulp
1:2
1.5t
...
1:5 1:10 1:25 1:50 PHA
1.5 1.2 1.7 1.7 14.9
0.9 1.0 1.1 1.1 ...
t *The results were similar for all three experimental animals. *-~Stimulation Index.
and injected intradermally into a cleaned, shaved site on the back of each experimental animal. For control, 5 mg of saline-treated pulp also was homogenized in 0.1 ml of R P M I and injected intradermally into a similarly prepared site on the back of each dog. In addition, 0.1 ml of R P M I was intradermally injected next to these sites. Each site was examined for inflammatory signs such as redness, edema, swelling, induration, or ulceration hourly during the first six hours and then every six to eight hours until 48 hours after the injections.
RESULTS W h e n various concentrations of h u m a n sera with antibodies to n R N P were added to red blood cells from tanned sheep with attached n R N P antigen, the results were positive hemagglutination. In contrast, when h u m a n sera without antibodies to n R N P were added to sheep red blood cells containing n R N P antigen, the results were negative hemagglutination. Adding various concentrations of sera of dogs to tanned
or u n t a n n e d sheep red blood cells that had been treated with AH26-incubated pulp or saline-treated pulp produced no positive hemagglutination either in the beginning or at the termination of the experiment. Table 1 shows the results of the passive hemagglutination test in one of the dogs. T h e lymphocyte transformation stimulation indexes for the same dog are shown in Table 2. The results indicate that the positive control substance (PHA) caused Significant lymphocyte stimulation. However, no lymphocyte stimulation occurred with various concentrations of AH-26-incubated pulp or salinetreated pulp. Intradermal injection of AH-26-incubated pulp or salinetreated pulp caused no clinical signs of inflammatory reaction within 48 hours. The same results were obtained where R P M I medium alone had been injected. Since all results were negative with all the tests, no control dogs were used as negative controls.
DISCUSSION In endodontic treatment, various
JOURNAL OF ENDODONTICS I VOL 5, N O 7, JULY 1979
substances are used, such as irrigating solutions, chelating agents, medications for disinfecting the root canal system, filling materials, and sealers. Because the root canal system can act as a pathway for systemic reactions, '-1:3 materials that are used during root canal treatment and which egress into the periapical tissues can potentially act as antigens. As AH-26 root canal sealer is commonly used, this material was chosen for investigation. When boundaries of a host are violated by intruders iantigens), the immune system protects the host's borders by antibody formation and delayed hypersensitivity reactions. Plasma cells synthesize and secrete various classes of antibodies; T-lymphocytes participate in delaved hypersensitivity reactions. If, during antibody formation the host encounters the same antigen to which it is producin~ antibody, an antigen-antibody complex (Arthus) reaction occurs. Antigen-antibody complexes can fix, complement, and attract polymorphonuclear (PMN) leukocytes. -': When immune complexes are phagocytosed by P M N leukocytes, the result is degranulation and elaboration of lysosomal enzymes. ~ The lysosomal enzymes released by PMN leukocytes often cause injury to tissue which leads to erythema, swelling, and even necrosis.-'" Intradermal injection of AH-26incubated pulp was used to detect the presence or absence of antibodies to this sealer. Presumably, the pulpal proteins can act as a carrier for AH-26 -'~ or its components and can combine with antibodies against it in the vessel walls and cause an Arthus reaction. Because the skin test produced a negative Arthus reaction,
it is unlikely that the experimental animals had developed antibodies to AH-26 root canal sealer. A negative hemagglutination test with AH-26incubated pulp and a positive response with the positive control (anti-nRNP + nRNP antigen) confirms the results of the skin test. When a lymphocyte comes into contact with the antigen to which it is sensitized, it starts to proliferate. This phenomenon is called lymphocyte transformation or stimulation and is widely used as an in vitro correlate of cell-mediated or delayed hypersensitivity reactions. :~~ In this test, P H A is used as a positive control to determine the capacity of lymphocytes to proliferate. As shown in Table 2, PHA caused a signifcant lymphocyte transformation (SI = 14.9). In contrast, various concentrations of AH-26-incubated pulp did not produce stimulation indexes greater than 1.7. The stimulation indexes with saline-treated pulp also were insignificant. The results from the passive hemagglutination, the lymphocyte transformation, and the skin test show that in the dog model and under clinical conditions AH-26 root canal sealer does not cause detectable systemic immunologic reactions. The results from the current study confirm our previous findings, a~ In that study, Grossman's sealer was used as the test material and no antibody formation or delayed hypersensitivity reaction was detected. The protocol of that study differed from the present one in that Grossman's sealer was used without first being incubated with pulp tissue for the immunologic assays. The similarity in results obtained with two such dissimilar materials is interesting to observe.
The difference between our results and those of Block and associates ~-'-'~ may be due to the following reasons. First, their experimental animals were preimmunized. Pulp tissue from the dogs' teeth was incubated with various experimental materials, mixed with incomplete Freund's adjuvant, and injected intramuscularly into the animals. In our study, no preimmunization was performed because we tried to simulate the clinical situation. The second reason might be differences in the methods of antigen preparation. According to the procedure reported in their studies, the pulp was incubated with experimental materials for 48 hours, washed, centrifuged, sterilized, sonicated, and concentrated with an Amicon filter. The concentration and nature of the experimental material and the conditions of sterilization, sonication, and concentration by ultrafiltration were not described. These procedures are not performed in the clinical situation and may be responsible for alteration of the pulp tissue. Other investigators have shown that depositing antigens such as bacteria, bovine serum albumin, and horse serum into the root canals of experimental animals produces antibody formation.'-" The difference between these results and ours may be due to the nature of substances tested. Bacteria, bovine serum albumin, and horse serum are potent antigens, whereas neither AH-26 nor any other root canal sealer has been shown conclusively to be antigenic. Our results agree with a clinical human study reported by Longwill and associates? ~ To evaluate the potential of formocresol pulpotomies in causing systemic immunologic reactions, the investigators selected 199
JOURNAL OF ENDODONTICS I VOL S, NO 7, JULY 1979
two groups of patients with and without a history of two or more formocresol pulp0tomies performed five to 18 m o n t h s b e f o r e t h e s t u d y . T h e l y m p h o c y t e t r a n s f o r m a t i o n ass a y w a s u s e d to d e t e c t t h e p r e s e n c e o r absence of delayed hypersensitivity r e a c t i o n to f o r m o c r e s o l . T h e r e s u l t s indicated that formocresol pulpotomies did not cause delayed hypers e n s i t i v i t y r e a c t i o n s in p a t i e n t s w i t h a history of this treatment. The results of the current study suggest that AH-26 root canal sealer used under clinical conditions does not produce systemic immunologic reactions. *Eli Lilly Co. Indianapolis. tNembutal, Abbot Laboratories, North Chicago, Ill. ~Premier Dental Products Co., Philadelphia. Dr. Torabinejad is associate professor of endodontics, School of Dentistry; Dr. Kettering is assistant professor of microbiology, School of Medicine; and Dr. Bakland ks associate professor and chairman, department of endodontic.s, School of Dentistry, Loma Linda University, Loma Linda, Calif 92350. Requests for reprints should be dii'ected to Dr. Mahmoud Torabinejad.
References 1. Kennedy, D.R.; Hamilton, T.R.; and Syverton, J.T. Effects on monkeys of introduction of hemolytic streptococci into root canals. J Dent Res 36:496-506, 1957. 2. Rosengren, L. The antibody response to experimental streptococci infection ($84) of the dental pulp of the cat. Odontol Tidskr 70:261-360, 1962. 3. Rosengren, L. Inoculation of Streptococcus mutans and other bacteria into dental pulps of rats. Isr J Dent Med 19:1-6, 1970. 4. Rosengren, L., and Winblad, B. Periapieal destructions caused by experimental pulpal, inoculation of Streptococcus mutans in rats. Oral Surg 39(3):479-487, 1975. 5. Barnes, G.W., and Langeland, K. Antibody formation in primates following introduction of antigens into the root canal. J Dent Res 45:t111-1114, 1966.
200
6. Okada, H., and others. Experimental study on focal infection in rabbits by prolonged sensitization through dental pulp canals. Arch Oral Biol 12:1017-1034, 1967. 7. Bergenh0hz, G.; Ahlstedt, S.; and Lindhe, J. Experimental pulpitis in immunized monkeys. Scand J Dent Res 85:396-406, 1977. 8. Torabinejad, M., and Kiger, R.D. Experimentally induced antigen-antibody reactions in periapical tissues of cats. J Dent Res, to be published. 9. Oswald, R.J., and Cohn, S.A. Systemic distribution of lead from root canal fillings. J Endod 1:59-63, 1975. 10. Shapiro, I.M., and others. Blood-lead levels of monkeys treated with a lead-containing (N2) root canal cement', a preliminary report. J Endod 1:294-297, 1975. 11. Chong, R., and Senzer, J. Systemic distribution of 2:~ from root canal fillings. J Endod 2:381-384, 1976. 12. Walton, R.E., and Langeland, K. Migration of materials in the dental pulp of monkeys. J Endod 4:167-177, 1978. 13. Feig!in, B., and Reade, P.C. The distribution of ~'C leucine and a~Sr labeled microspheres from rat incisor root canals. Oral Surg 47:277-281, 1979. 14. Block, R.M., and others. Antibody formation to dog pulp tissue altered by N2type paste within the root canal. J Endod 3:309-315, 1977. 15. Block, R.M., and others. Cell-mediated immune response to dog pulp tissue altered by N2 paste within the root canal. Oral Surg 45(1):131-142, 1978. 16. Block, R.M., and others. Antibody formation to dog pulp tissue altered by eugenol within the root canal. J Endod 4:53-59, 1978. 17. Block, R.M., and others. Cell-mediated immune response to dog pulp tissue altered by formocresol within the root canal. J Endod 3:424-430, 1977. 18. Block, R.M., and others. Cell-mediated immune response to dog pulp tissue altered by 6.5% paraformaldehyde via the root canal. J Endod 4:346-352, 1978. 19. Block, R.M.; Lewis, R.D.; and Sheats, J.B. Immunologic skin test responses to dog pulp tissue altered by endodontic medicaments. J Dent Res 56 (Special Issue B) abstract no. 759, 1977.
20. Block, R.M.; Sheats, J.B.; and Lewis, R.D. Immunologic skin testing of dog pulpal tissue altered by root canal filling materials. J Dent Res 56(Special Issue A) abstract no. 293, 1977. 21. Block, R.M., and others. Cell-mediated immune response to dog pulp tissue altered by Kerr (Rickert's) sealer via the root canal. J Endod 4:110-116, 1978. 22. Block, R.M: Antibody formation to dog pulp tissue altered by procoSol sealer via the root canal. J Endod 4:178-183, 1978. 23. Block, R.M., and others. Antibody formation to dog pulp tissue altered by Grossman's formula sealer via the root canal. Oral Surg 47:179-184, 1979. 24. Campbell, A.D., and others. Cellmediated response to endodontic cements: research report. J Endod 4:147-150, 1978. 25. Tan, E.M., and Peebles, C. Quantitation of antibodies to Smantigen and nuclear ribonucleo protein by bemagglutination. In R .ose, N.R., and Friedman, H. (eds.). Manual of clinical immunology. American Society of Microbiology, 1976, pp 660-665. 26. Shlfrine, M., and others. Comparison of whole blood and purified canine lymphocytes in a lymphocyte-stimulation microassay. Am J Vet Res 39:687-690, 1978. 27. Ward, P.A.; Cochrane, C.G.; and Muller-Eberhard, H.J. Further studies on the chemotactic factors of complement and its formation in vivo. Immunology 11:141-153, 1966. 28. Henson, P.M. The immunologic release of constituents from the neutrophil leukocytes. J Immunol 107:1547-1557, 1971. 29. Henson, P.M. Pathologic mechanisms in neutrophil-mediated injury. Am J Pathol 68:593-610, 1972. 30. Openheim, J.J., and Scbechter, B. Lymphocyte transformation. In Rose, N.R., and Friedman, H., (eds.). Manual of clinical immunology. American Society for Microbiology, 1976, p 81. 31. Torabinejad, M.; Bakland, L.K.; and Kettering, J.D. Detection of systemic immunological reactions to Grossman's sealer. J Dent Res 58 (Special Issue A) abstract no. 149, 1979. 32. Longwill, D.; Creamer, H.R.; and Marshall, J.F. Lymphocyte transformation responses to pulp antigens in formocresol pulpotomy patients. Read before the American Association of Endodontists, Annual Session, Atlanta, 1979.
Evaluation of systemic immunological reactions to AH-26 root canal sealer Mahmoud
T o r a b i n e j a d , DMD, MSD; James D. K e t t e r i n g , PhD;
a n d Leif K. B a k l a n d , DDS, L o m a Linda, C a l i f
T w e n t y t e e t h in each of t h r e e dogs w e r e cleaned, s h a p e d , a n d t h e n o b t u r a t e d with g u t t a - p e r c h a a n d A H - 2 6 root canal sealer. In m o s t of the teeth, s o m e sealer was i n t e n t i o n a l l y f o r c e d into the periapical tissues. S e v e n d a y s after the r o o t canal p r o c e d u r e s w e r e c o m p l e t e d , passive h e m a g g l u t i n a t i o n , l y m p h o c y t e t r a n s f o r m a t i o n , a n d skin tests were p e r f o r m e d to assess the p r e s e n c e or a b s e n c e of s y s t e m i c a n t i b o d y f o r m a t i o n and d e l a y e d h y p e r s e n s i t i v i t y r e a c t i o n to the sealer. T h e results indicate that u n d e r clinical c o n d i t i o n s , A H - 2 6 root canal sealer d o e s not p r o d u c e a n y s y s t e m i c a n t i b o d y f o r m a t i o n or d e l a y e d h y p e r s e n s i t i v i t y reaction.
Several investigators have shown that injecting potential antigens such as bacteria, bovine serum albumin, and horse serum into the root canals of teeth of experimental animals produces antibody formation. ~-G Other experiments in immunized animals also indicate that interactions between antigens and antibodies can occur within the pulp or periapical tissues. T'~ These studies and others ~'3 show that the root canal system is an effective route for host sensitization. Block and associates'"~'~ injected pulp tissue incubated with dental materials intramuscularly and then filled the root canals with the same experimental materials. The incubated pulp tissue was mixed with Freund's incomplete adjuvant and 196
injected intramuscularly in the regions of the cervical and popliteal lymph nodes. Materials tested ineluded N2-type paste, eugenol, formocresol, paraformaldehyde, and root canal sealers. The tanned red blood cell hemagglutination technique, the lymphocyte transformation test, and a skin test were used to determine the presence of antibody formation and cell-mediated reaction to the test materials. The investigators concluded that the foregoing materials, when combined with pulp protein, can cause both systemic humoral and cell-mediated responses, and that the root canal system can be a route for immunization. Recently, Campbell and associates-"' tested hardened or set RG2B,
ProcoSol, calcium hydroxide, and zinc oxide-eugenol cements to assess their ability to induce cell-mediated immune responses in guinea pigs. Skin tests and assays of migration inhibitory factor showed that the test material in the hardened state caused inflammatory changes but no delayed hypersensitivity reactions. It is important to observe that in the studies just described, 14-'-'~protocols were used that differ from routine endodontic procedures. The purpose of the current investigation was to simulate the clinical situation and assess the presence or absence of systemic immunologic reaction to AH-26 root canal sealer, which is suggested to be a hapten, TM by using the passive hemagglutination, lymphocyte transformation, and skin tests.
JOURNAL OF ENDODONTICS I VOL 5, N O 7, JULY 1979
MATERIALS AND METHODS
percha and the sealer, and the access cavities were filled with Cavit.:~
Test animals Three healthy dogs, each weighing 30 to 35 lb, were housed in individua l cages and fed laboratory dog food a d libitum. Two other dogs were used as control animals. Five teeth in each experimental dog were filled at seven-day intervals during a 28-day period, for a total of 20 teeth in each animal. The experimental procedure -.onsisted of the following steps. Each dog was given premedication, a subcutaneous injection of 1/150 grain (0.4 rag) of atropine sulfate* and a half-grain (32.0 mg) of morphine and then intravenously injected with the anesthetic sodium pentobarbitalJ" (35.0 mg/lb). Preoperative radiographs were made of the mandibular incisors, premolars, and maxillary incisors. The teeth were isolated with a rubber dam, and the field of operation was scrubbed with 95% isopropyl alcohol. The pulps were exposed by grinding the occlusal or incisal part of each tooth with a no. 701 fissure bur and then extirpated with barbed broaches and frozen at - 1 0 C in sterile saline solution. After the root canals were irrigated with 2.5% sodium hypochlorite, they were cleaned and shaped at 0.5 to 1.0 mm from the apex. In most instances in which the apical foramenas were not readily negotiable, intentional apical perforations were made with endodontic reamers so that the root canal sealer could extrude into the periapical tissues. After the root canals were cleaned and shaped, they were irrigated with 70% isopropyl alcohol and dried with sterile paper points. AH-26 root canal sealer was mixed to a creamy consistency. The root canals were obturated with gutta-
Incubation of pulp with experimental material Forty-eight hours before the immunologic assays were performed, 25 mg of the frozen pulp from each dog was incubated with AH-26 root canal sealer and placed in the CO. incubator at 37 C for 48 hours. The pulp then was removed from the sealer, rinsed twice with saline solution, and placed in 2 ml of R P M I medium. The treated pulp was cut into small pieces with scalpel blades and then homogenized by crushing with a sterile blunt instrument. This tissue served as the experimental antigenic material and was used for the following tests. Pulp in sterile saline solution was the control. Seven to 14 days after 20 teeth were filled in each dog, the following tests were performed to detect antibody formation and delayed hypersensitivity to AH-26 root canal sealer.
Antibody formation test. The procedure used to determine antibody formation was the passive hemagglutination technique described by Tan and Peebles.'-':' Controls consisted of a human serum with antibodies to nuclear ribonucleoprotein (nRNP) and a negative human serum. Tanned sheep erythrocytes were coated either with an extract of rabbit thymus containing n R N P antigen, with homogenized AH-26-incubated pulp, or with saline-treated pulp. The passive hemagglutination test was performed in a microtiter system. Sera from the dogs were collected before and after treatment and were tested. Different
concentrations (1:2 to 1:1026) of the sera were tested against erythrocytes containing AH-26-incubated pulp or saline-treated pulps. Each dog was tested only against its own pulp. As a positive control, various concentrations of human sera with antibodies to nRNP were added to tanned sheep erythrocytes coated with nRNP antigen. As an additional control, the final sera also were tested against tanned sheep erythrocytes with no antigen attached. Positive hemagglutination consisted of' a smooth mat of cells covering the bottom of the well ( + ) , and a negative result was a clearly defined button in the bottom of the well (0). Titers are expressed as the highest dilution giving positive hemagglutination.
Delayed hypersensitivity test The lymphocyte transformation test was used to detect possible cellmediated immunity in this experiment and was performed according to methods described by Shifrine and associates5 '~ Ten milliliters of blood was drawn from a vein in the leg and placed in a tube containing heparin. The 10.0 ml of heparinized blood was diluted with an equal volume of R P M I 1640 medium. Three ml of Ficoll-Paque (specific gravity, 1.067) was placed into each of four 15.0-ml conical centrifuge tubes. Five milliliters of the diluted blood was carefully layered over the Ficoll-Paque solution with a sterile pipette. These tubes were centrifuged at 1,500 rpm for 40 minutes at room temperature (500 g at the interface). The lymphocyte layer was aspirated from each tube and pooled in a 50-ml sterile conical tube for washing. Cells were washed three times with R P M I 1640
197
T a b l e 1 9 R e s u l t s o f p a s s i v e h e m a g g l u t i n a t i o n test in o n e d o g . *
Dilutions Positive serum controlt Negative serum control Preoperative dog's serum Preoperanve dog's serum Preoperanve dog's serum Postoperauve dog's serum~ Postoperative dog's serum~ Postoperauve dog's serum~ medium. Cells were collected by spinning ten minutes at 1,500 rpm at room temperature. T h e lymphocytes finally were resuspended in 1.0 ml of the R P M I m e d i u m which was supplemented with 10% pooled dog serum. T h e cells were counted with a haemocytometer and diluted to a concentration of 1 to 2 • 10 '~ cells per ml. Staining with Erythrocin B showed 90% to 100% viability of the lymphocytes. Volumes of 0.1 ml then were placed in the wells of a 96-well tissue culture plate. Mitogens tested were phytohemagglutinin ( P H A ) as a control, AH-26 incubated pulp, and saline-treated pulp. Control mitogen was used at optimal dilutions for the system, and AH-26-incubated pulp and saline-treated pulp were tested at dilutions ranging from 1:2 to 1:50. Mitogens were added to the wells in 0.1 ml volumes. Plates were allowed to incubate in a CO2 incubator at 36 C for four days. O n e microcurie of aH-thymidine (specific activity, 6.7 c i / m M ) was added to each well and incubated for approximately 18 to 20 hours. Cells were collected using a M A S H II harvester. Filters containing radioactivity were placed in liquid scintillation vials and counted on a Beckman LSC 250 counter. Stinmlation indexes (SI) were calculated by dividing cell counts stimulated by mitogen or test material with cell counts from untreated controls. A n y SI greater than 2.00 with test material was considered significant and indicated the presence of delayed hypersensitivity reaction.
Skin test Five milligrams of each dog's own AH-26-incubated pulp was homogenized in 0.I ml of R P M I m e d i u m 198
1:2 1:4 1:8 1:16 1:64 + + + + + 0 0 0 0 0 0 0 0 0 0 0
0
0
0
0
0 + + +
0 _+ + 0
0 0 0 0
0 0 0 0
0 0 0 0
1:256 1:1024 + + 0 0 0 0 AH-26 pulp 0 0 No antigen 0 0 Saline pulp 0 0 AH-26 pulp 0 0 No antigen 0 0 Saline pulp
*The results were similar for all three experimental animals. t + indicates positive hemagglutlnation: 0, negative hemagglutination: and -+, incomplete hemagglufination, ~l'he positive hemagglutinations obtained in dilutions 1:2 and 1:4 with postoperative dog's sera are nonspecific because the same results were obtained with untanned erythrocytes as well as AH-26 incubated pulp and saline-incubated pulp.
T a b l e 2 9 R e s u l t s o f l y m p h o c y t e t r a n s f o r m a t i o n test in o n e dog. *
Dilutions
AH-26incubated pulp
Salinetreated pulp
1:2
1.5t
...
1:5 1:10 1:25 1:50 PHA
1.5 1.2 1.7 1.7 14.9
0.9 1.0 1.1 1.1 ...
t *The results were similar for all three experimental animals. *-~Stimulation Index.
and injected intradermally into a cleaned, shaved site on the back of each experimental animal. For control, 5 mg of saline-treated pulp also was homogenized in 0.1 ml of R P M I and injected intradermally into a similarly prepared site on the back of each dog. In addition, 0.1 ml of R P M I was intradermally injected next to these sites. Each site was examined for inflammatory signs such as redness, edema, swelling, induration, or ulceration hourly during the first six hours and then every six to eight hours until 48 hours after the injections.
RESULTS W h e n various concentrations of h u m a n sera with antibodies to n R N P were added to red blood cells from tanned sheep with attached n R N P antigen, the results were positive hemagglutination. In contrast, when h u m a n sera without antibodies to n R N P were added to sheep red blood cells containing n R N P antigen, the results were negative hemagglutination. Adding various concentrations of sera of dogs to tanned
or u n t a n n e d sheep red blood cells that had been treated with AH26-incubated pulp or saline-treated pulp produced no positive hemagglutination either in the beginning or at the termination of the experiment. Table 1 shows the results of the passive hemagglutination test in one of the dogs. T h e lymphocyte transformation stimulation indexes for the same dog are shown in Table 2. The results indicate that the positive control substance (PHA) caused Significant lymphocyte stimulation. However, no lymphocyte stimulation occurred with various concentrations of AH-26-incubated pulp or salinetreated pulp. Intradermal injection of AH-26-incubated pulp or salinetreated pulp caused no clinical signs of inflammatory reaction within 48 hours. The same results were obtained where R P M I medium alone had been injected. Since all results were negative with all the tests, no control dogs were used as negative controls.
DISCUSSION In endodontic treatment, various
JOURNAL OF ENDODONTICS I VOL 5, N O 7, JULY 1979
substances are used, such as irrigating solutions, chelating agents, medications for disinfecting the root canal system, filling materials, and sealers. Because the root canal system can act as a pathway for systemic reactions, '-1:3 materials that are used during root canal treatment and which egress into the periapical tissues can potentially act as antigens. As AH-26 root canal sealer is commonly used, this material was chosen for investigation. When boundaries of a host are violated by intruders iantigens), the immune system protects the host's borders by antibody formation and delayed hypersensitivity reactions. Plasma cells synthesize and secrete various classes of antibodies; T-lymphocytes participate in delaved hypersensitivity reactions. If, during antibody formation the host encounters the same antigen to which it is producin~ antibody, an antigen-antibody complex (Arthus) reaction occurs. Antigen-antibody complexes can fix, complement, and attract polymorphonuclear (PMN) leukocytes. -': When immune complexes are phagocytosed by P M N leukocytes, the result is degranulation and elaboration of lysosomal enzymes. ~ The lysosomal enzymes released by PMN leukocytes often cause injury to tissue which leads to erythema, swelling, and even necrosis.-'" Intradermal injection of AH-26incubated pulp was used to detect the presence or absence of antibodies to this sealer. Presumably, the pulpal proteins can act as a carrier for AH-26 -'~ or its components and can combine with antibodies against it in the vessel walls and cause an Arthus reaction. Because the skin test produced a negative Arthus reaction,
it is unlikely that the experimental animals had developed antibodies to AH-26 root canal sealer. A negative hemagglutination test with AH-26incubated pulp and a positive response with the positive control (anti-nRNP + nRNP antigen) confirms the results of the skin test. When a lymphocyte comes into contact with the antigen to which it is sensitized, it starts to proliferate. This phenomenon is called lymphocyte transformation or stimulation and is widely used as an in vitro correlate of cell-mediated or delayed hypersensitivity reactions. :~~ In this test, P H A is used as a positive control to determine the capacity of lymphocytes to proliferate. As shown in Table 2, PHA caused a signifcant lymphocyte transformation (SI = 14.9). In contrast, various concentrations of AH-26-incubated pulp did not produce stimulation indexes greater than 1.7. The stimulation indexes with saline-treated pulp also were insignificant. The results from the passive hemagglutination, the lymphocyte transformation, and the skin test show that in the dog model and under clinical conditions AH-26 root canal sealer does not cause detectable systemic immunologic reactions. The results from the current study confirm our previous findings, a~ In that study, Grossman's sealer was used as the test material and no antibody formation or delayed hypersensitivity reaction was detected. The protocol of that study differed from the present one in that Grossman's sealer was used without first being incubated with pulp tissue for the immunologic assays. The similarity in results obtained with two such dissimilar materials is interesting to observe.
The difference between our results and those of Block and associates ~-'-'~ may be due to the following reasons. First, their experimental animals were preimmunized. Pulp tissue from the dogs' teeth was incubated with various experimental materials, mixed with incomplete Freund's adjuvant, and injected intramuscularly into the animals. In our study, no preimmunization was performed because we tried to simulate the clinical situation. The second reason might be differences in the methods of antigen preparation. According to the procedure reported in their studies, the pulp was incubated with experimental materials for 48 hours, washed, centrifuged, sterilized, sonicated, and concentrated with an Amicon filter. The concentration and nature of the experimental material and the conditions of sterilization, sonication, and concentration by ultrafiltration were not described. These procedures are not performed in the clinical situation and may be responsible for alteration of the pulp tissue. Other investigators have shown that depositing antigens such as bacteria, bovine serum albumin, and horse serum into the root canals of experimental animals produces antibody formation.'-" The difference between these results and ours may be due to the nature of substances tested. Bacteria, bovine serum albumin, and horse serum are potent antigens, whereas neither AH-26 nor any other root canal sealer has been shown conclusively to be antigenic. Our results agree with a clinical human study reported by Longwill and associates? ~ To evaluate the potential of formocresol pulpotomies in causing systemic immunologic reactions, the investigators selected 199
JOURNAL OF ENDODONTICS I VOL S, NO 7, JULY 1979
two groups of patients with and without a history of two or more formocresol pulp0tomies performed five to 18 m o n t h s b e f o r e t h e s t u d y . T h e l y m p h o c y t e t r a n s f o r m a t i o n ass a y w a s u s e d to d e t e c t t h e p r e s e n c e o r absence of delayed hypersensitivity r e a c t i o n to f o r m o c r e s o l . T h e r e s u l t s indicated that formocresol pulpotomies did not cause delayed hypers e n s i t i v i t y r e a c t i o n s in p a t i e n t s w i t h a history of this treatment. The results of the current study suggest that AH-26 root canal sealer used under clinical conditions does not produce systemic immunologic reactions. *Eli Lilly Co. Indianapolis. tNembutal, Abbot Laboratories, North Chicago, Ill. ~Premier Dental Products Co., Philadelphia. Dr. Torabinejad is associate professor of endodontics, School of Dentistry; Dr. Kettering is assistant professor of microbiology, School of Medicine; and Dr. Bakland ks associate professor and chairman, department of endodontic.s, School of Dentistry, Loma Linda University, Loma Linda, Calif 92350. Requests for reprints should be dii'ected to Dr. Mahmoud Torabinejad.
References 1. Kennedy, D.R.; Hamilton, T.R.; and Syverton, J.T. Effects on monkeys of introduction of hemolytic streptococci into root canals. J Dent Res 36:496-506, 1957. 2. Rosengren, L. The antibody response to experimental streptococci infection ($84) of the dental pulp of the cat. Odontol Tidskr 70:261-360, 1962. 3. Rosengren, L. Inoculation of Streptococcus mutans and other bacteria into dental pulps of rats. Isr J Dent Med 19:1-6, 1970. 4. Rosengren, L., and Winblad, B. Periapieal destructions caused by experimental pulpal, inoculation of Streptococcus mutans in rats. Oral Surg 39(3):479-487, 1975. 5. Barnes, G.W., and Langeland, K. Antibody formation in primates following introduction of antigens into the root canal. J Dent Res 45:t111-1114, 1966.
200
6. Okada, H., and others. Experimental study on focal infection in rabbits by prolonged sensitization through dental pulp canals. Arch Oral Biol 12:1017-1034, 1967. 7. Bergenh0hz, G.; Ahlstedt, S.; and Lindhe, J. Experimental pulpitis in immunized monkeys. Scand J Dent Res 85:396-406, 1977. 8. Torabinejad, M., and Kiger, R.D. Experimentally induced antigen-antibody reactions in periapical tissues of cats. J Dent Res, to be published. 9. Oswald, R.J., and Cohn, S.A. Systemic distribution of lead from root canal fillings. J Endod 1:59-63, 1975. 10. Shapiro, I.M., and others. Blood-lead levels of monkeys treated with a lead-containing (N2) root canal cement', a preliminary report. J Endod 1:294-297, 1975. 11. Chong, R., and Senzer, J. Systemic distribution of 2:~ from root canal fillings. J Endod 2:381-384, 1976. 12. Walton, R.E., and Langeland, K. Migration of materials in the dental pulp of monkeys. J Endod 4:167-177, 1978. 13. Feig!in, B., and Reade, P.C. The distribution of ~'C leucine and a~Sr labeled microspheres from rat incisor root canals. Oral Surg 47:277-281, 1979. 14. Block, R.M., and others. Antibody formation to dog pulp tissue altered by N2type paste within the root canal. J Endod 3:309-315, 1977. 15. Block, R.M., and others. Cell-mediated immune response to dog pulp tissue altered by N2 paste within the root canal. Oral Surg 45(1):131-142, 1978. 16. Block, R.M., and others. Antibody formation to dog pulp tissue altered by eugenol within the root canal. J Endod 4:53-59, 1978. 17. Block, R.M., and others. Cell-mediated immune response to dog pulp tissue altered by formocresol within the root canal. J Endod 3:424-430, 1977. 18. Block, R.M., and others. Cell-mediated immune response to dog pulp tissue altered by 6.5% paraformaldehyde via the root canal. J Endod 4:346-352, 1978. 19. Block, R.M.; Lewis, R.D.; and Sheats, J.B. Immunologic skin test responses to dog pulp tissue altered by endodontic medicaments. J Dent Res 56 (Special Issue B) abstract no. 759, 1977.
20. Block, R.M.; Sheats, J.B.; and Lewis, R.D. Immunologic skin testing of dog pulpal tissue altered by root canal filling materials. J Dent Res 56(Special Issue A) abstract no. 293, 1977. 21. Block, R.M., and others. Cell-mediated immune response to dog pulp tissue altered by Kerr (Rickert's) sealer via the root canal. J Endod 4:110-116, 1978. 22. Block, R.M: Antibody formation to dog pulp tissue altered by procoSol sealer via the root canal. J Endod 4:178-183, 1978. 23. Block, R.M., and others. Antibody formation to dog pulp tissue altered by Grossman's formula sealer via the root canal. Oral Surg 47:179-184, 1979. 24. Campbell, A.D., and others. Cellmediated response to endodontic cements: research report. J Endod 4:147-150, 1978. 25. Tan, E.M., and Peebles, C. Quantitation of antibodies to Smantigen and nuclear ribonucleo protein by bemagglutination. In R .ose, N.R., and Friedman, H. (eds.). Manual of clinical immunology. American Society of Microbiology, 1976, pp 660-665. 26. Shlfrine, M., and others. Comparison of whole blood and purified canine lymphocytes in a lymphocyte-stimulation microassay. Am J Vet Res 39:687-690, 1978. 27. Ward, P.A.; Cochrane, C.G.; and Muller-Eberhard, H.J. Further studies on the chemotactic factors of complement and its formation in vivo. Immunology 11:141-153, 1966. 28. Henson, P.M. The immunologic release of constituents from the neutrophil leukocytes. J Immunol 107:1547-1557, 1971. 29. Henson, P.M. Pathologic mechanisms in neutrophil-mediated injury. Am J Pathol 68:593-610, 1972. 30. Openheim, J.J., and Scbechter, B. Lymphocyte transformation. In Rose, N.R., and Friedman, H., (eds.). Manual of clinical immunology. American Society for Microbiology, 1976, p 81. 31. Torabinejad, M.; Bakland, L.K.; and Kettering, J.D. Detection of systemic immunological reactions to Grossman's sealer. J Dent Res 58 (Special Issue A) abstract no. 149, 1979. 32. Longwill, D.; Creamer, H.R.; and Marshall, J.F. Lymphocyte transformation responses to pulp antigens in formocresol pulpotomy patients. Read before the American Association of Endodontists, Annual Session, Atlanta, 1979.
