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Rat macrophage response to implanted sealer cements
0099-2399/85/1101-0030/$02.00/0 JOURNAL OF ENDODONTICS Copyright 9 1985 by The American Associationof Endodontists
Printed in U.S.A. VOL. 11, NO. 1,J.~NUARY1985
Rat Macrophage Response to Implanted Sealer Cements Respuesta Macrofagica en Rata a Cementos Selladores Implantados John T. Biggs, DDS, MEd, MS, E. J. Kaminski, PhD, and E. M. Osetek, DOS, MEd Un patron satisfactorio para probar la biocompatibilidad de cementos selladores debe incluir la colocacion de los materiales in vivo, donde los efectos sobre una poblacion de macrofagos y otros tipos de c(Hulas pueden ser cuantitativamente evaluados. La cavidad peritoneal de la rata ofrece ese sistema in vivo y el examen del contenido de esta cavidad por irrigacion es un m(~todo aceptado para estudiar la respuesta celular (33 a 35). El proposito de este estudio fue evaluar la cavidad peritoneal de la rata como patron o modelo para testificar la influencia de cementos selladores en la poblacion de macrofagos in vivo.
sistent with the findings reported for the usage studies in which periapical tissues were evaluated. It has been suggested that studies which employ the root canal system (16), as well as those which involve implantation in connective tissues (17), are of limited value for the evaluation of the biocompatibility of dental materials because of the variables which are introduced by the procedures as well as the complex cellular milieu at the test site. Cell culture techniques have been used in an attempt to overcome some of the complexities of implant and usage studies. Sealer cements are known to be cytotoxic to cell lines and tissues in culture (18-21). These techniques, however, involve cells with no capacity for recovery and interaction. It is difficult to compare the effects of dental materials on cultured cells with their actual effects on the periapical region. Root canal sealer cements are known to influence the inflammatory process. Zinc oxide-eugenol cements have been demonstrated to increase neutrophil migration (22) and have been implicated in nonspecific inflammation (23), humoral immunity (24-26), and cell-mediated immunity (27). Although the immunological consequences of placing sealer cements in contadt with periapical tissues are not clear, it is generally agreed that there is an effect on the inflammatory cells. A cell line is available for study which is a wellestablished constituent of the periapical lesion (27-37). It is known to be the primary phagocyte of the body (28) and is known to participate as an antigen processor in both cell-mediated and humoral immune response (29-31). The macrophage is recognized to be a versatile secretory effector cell, whose products influence every stage of the inflammatory process (32). A satisfactory model for testing the biocompatibility of sealer cements should involve the placement of materials into an in vivo system, where the effects on a population of macrophages and other cell types could be quantitatively evaluated. The peritoneal cavity of the rat provides such an in vivo system and examination of the content of this cavity by lavage is an accepted method for studying cellular response (33-35).
The irregularity of root canals which have been prepared for obturation does not allow for an exact fit of the solid or semisolid core which makes up the bulk of root canal fillings (1). Sealer cements are necessary to fill the resultant voids (2, 3), and there is the potential for these materials to come into contact with periapical tissues. The biological effects of these sealer cements are of interest to endodontic practitioners and have been the subject of a number of investigations. Several investigators have studied the effects of overfills (4-8) and extruded sealer cements (9) on the success of root canal treatments. The consensus is that overfills have an adverse effect on healing. Storms (9) has reported that sealer cement which has been extruded into the periapical tissues results in an 11.2% reduction in the success rate of endodontically treated teeth. In studies of factors which influence prognosis, no attempts have been made to correlate failure to heal with specific cellular events in the periapical region. The effects of the sealer cements on the periapical tissues of animals have been evaluated in usage studies by Erasquin and Muruzabal (10, 11), Muruzabal et al. (12), and Davis et al. (13). These investigators have reported that sealer cements cause inflammation and delay healing in the periapical region. Implantation studies in connective tissues by Guttierez et al. (14) and in osseous tissues by Deemer and Tsaknis (15) are con30
Vol. 11, No. 1, January 1985
The purpose of this study was to evaluate the rat peritoneal cavity as a model for assessing the influence of sealer cements on macrophage populations in vivo. MATERIALS AND METHODS
Kerr's antiseptic sealer (Kerr Mfg. Co., Romulus, MI) and Roth's 801 (Roth International, Ltd., Chicago, IL) root canal sealer cement (a Grossman's formulation) were used in this study. Silastic 500-9 (Dow Coming Corp., Midland, MI) was used as the implant control. The Kerr sealer cement was mixed according to manufacturer's instructions, and the Roth's 801 was mixed according to the method described by Fragola et al. (36). The major constituents of Kerr's sealer cement are zinc oxide, elemental silver, thymol ioxide, oleo resins, oil of cloves, and Canada balsam and the constituents of Roth's 801 sealer cement are zinc oxide, bismuth subnitrate, barium sulfate, anhydrous sodium borate, staybelite resin, and eugenol. Immediately after mixing, the sealer cements were placed in implant disk molds which were made by punching circular holes in a 1.60-mm thick piece of 5009 silastic sheeting. The diameter of the holes was 10.5 mm. The sealer cement-filled disk molds were pressed firmly between two 3-inch square glass slabs and immersed in deionized water at 37~ for 5 days. After immersion, the disks were removed from their molds and air dried at room temperature and humidity for 2 days in order to obtain a consistent preimplant weight. The disks were then weighed to an accuracy of 0.001 g. The silastic implants were geometrically identical to the sealer cement implants. Each silastic implant was thoroughly washed in deionized water and allowed to bench dry for 48 h before implantation. Thirty-two CD strain female Charles River rats (Charles River Breeding Laboratory, Portage, MI) were used as implant recipients. The rats were fed a standard laboratory diet ad libitum and weighed 95 to 150 g at the beginning of the study. The animals were divided into four groups. All animals within each group were treated identically. Eleven animals were assigned to a surgical control group and seven animals to each of the implant groups: Kerr, Grossman's formula, and silastic. The control animals were subjected to surgical and anesthetic procedures identical to those experienced by the experimental animals excepting the placement of implants. Research employing the peritoneal lavage technique has demonstrated that the surgical procedure used does not result in significant alterations in the macrophage population of the peritoneal cavity (33, 38). Lavage solutions obtained from the control animals were treated identically to those obtained from the experimental animals.
Response to Implanted Sealer Cements
31
Peritoneal lavages were performed on each animal 1 wk before implantation, on the day of implantation, and at 2, 4, and 6 wk after implantation according to the procedure described by Torraason and Kaminski (33). The abdominal area of each animal was shaved and disinfected with reagent grade ethyl alcohol. Ten cubic centimeters of 37~ Ringer's solution were then slowly injected through a 16-gauge needle into the peritoneal cavity. The needle was removed and the Ringer's solution was mixed with the indigenous peritoneal fluid by rolling the animal. The mix of lavage fluid and peritoneal fluid was collected by allowing it to flow passively through the 16-gauge needle that had been reinserted into the injection site. Ether and subcutaneously injected nembutal (Abbott Laboratories, N. Chicago, IL) were used for anesthesia during lavage, implantation, and suture removal procedures. Implants were inserted by making a midline incision slightly larger than the implant and placing the implant disk into the peritoneal cavity with a cotton forceps. Incisions were closed with continuous 4-0 silk sutures which were removed after 1 wk. At the end of the experimental period (7 wk), the animals were sacrificed with chloroform anesthesia. Implants were recovered and the sealer cement implants were washed in deionized water, air dried for 2 days, and reweighed. The peritoneal lavage fluid from each animal was divided into two portions. One or 2 cm 3 of each solution, depending upon the quantity recovered in the lavage procedure, were placed in Procil-28 (PCR Research Chemicals, Inc., Gainsville, FL) -treated disposable centrifuge tubes and were diluted with equal volumes of 5% acetic acid. The cells contained in the diluted lavage fluid were then vitally stained with 0.005% toluidine blue and counted with the aid of a Spencer hemocytometer (American Optical Corp., Buffalo, NY). Of the cells found in the peritoneal cavity, only mast cells were stained with toluidine blue; therefore, the resulting count differentiated the mast cells present in each sample of fluid. The portion of the lavage solution that was not used for cell counts was diluted to 6 ml with Ringer's solution and centrifuged supernatant was decanted and stored in a separate centrifuge tube. Six cubic centimeters of Ringer's solution were added to the packed cells. Then the mixture was agitated and recentrifuged. The supernatant was again decanted and added to the previously collected volume. Washed packed cells and the supernatant were sealed in their respective containers with Parafilm M (American Can Co., Greenwich, CT) and frozen. At the end of the experiment, the samples were pooled into groups of preoperative and postoperative eluates and macrophages. The pooled samples were analyzed by Trace Elements Inc. (Park Ridge, IL) for silver and for zinc present in either the eluates or the cells.
32
Journal of Endodontics
Biggs et al.
At the time of peritoneal lavage, one drop of each lavage solution was placed on a glass slide and allowed to dry. Subsequently, the slides were fixed in methyl alcohol and stained with Cameco B4130 (Scientific Products, Division of American Hospital Supply Corp., McGraw Park, IL) blood stain (Wright's stain). These slides were decolorized with deionized water and methyl alcohol. Three hundred cells on each slide were counted using an oil immersion lens and the percentages of macrophages, mast cells, lymphocytes, eosinophils, and neutrophils were determined. Counts obtained using the hemocytometer were subjected to two statistical normalization procedures to facilitate comparisons. Macrophage and mast cell counts were adjusted to number of cells/100 g body wt to compensate for differences attributable to using a constant volume of lavage fluid in animals of different sizes. A secod normalization was performed by subtracting the number of cells from each group necessary to bring the first mean count to the mean value for the surgical control animals. This normalization ensured that the relative numbers of cells would not obscure the changes which resulted from the effects of the experimental procedures. All data were subjected to a one-way analysis of variance. Where indicated, a t test for differences between groups was performed.
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FiG 1, All animals demonstrated a consistent gain in weight over the 7-wk period.
RESULTS 1"511
All animals gained weight consistently during the experimental period (Fig. 1). An analysis of variance indicates that the regressions of the mean animal weights by week are not significantly different (p > 0.05). Vitally stained mast cells and macrophages aspirated from the peritoneal cavities of implanted and unimplanted animals and counted with the aid of a hemocytometer demonstrated a wide variation in numbers both within and between groups (Figs. 2 and 3). In the control animals, macrophages gradually increased in number throughout the experiment. This increase was attributed to inflammation resulting from the initial surgical intervention and subsequent lavages. Macrophage numbers in experimental animals increased similarly, except for the first postimplant count (wk 3). In both sealer-implanted groups, the mean number of macrophages decreased during the first 2 wk after implantation (Fig. 3). The number of macrophages in the two sealer cement test groups began to increase thereafter, until the 6th week after implantation, when they achieved the same cell population as the control groups. An analysis of variance indicates that the regressions of the group values are different and that this difference cannot be attributed to random variation (p = 0.0028, F = 5.68). A t test indicates that the numbers of
Surglcol Control
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Surgical Control Grossmon tormula $11ast|c Kerr
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1.03 -1.00--
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FtG 2. Implantation procedures were performed at wk 1. The mean vital macrophage counts of sealer cement-implanted animals dropped at wk 3, but returned to normal levels by wk 7.
macrophages in the surgical control and the silastic groups are not significantly different (p > 0.05). The Kerr-implanted and the Grossman formula-implanted groups were also statistically similar (p > 0.05). The sealer cement groups, however, were found to be statistically different from both control groups (p < 0.05).
Vol. 11, No. 1, January 1985
Response to Implanted Sealer Cements
Vitally stained mast cell counts exhibited even more variation than the macrophage counts (Fig. 3). The vital mast cell counts decreased significantly following the first injections of Ringer's solution and did not return to the initial levels during the experimental period. An analysis of variance indicates that the group regressions are different (p < 0.05). The t test indicates that the sealer cement-implanted groups are not significantly different from each other (p > 0.05). At the same level of significance, however, the sealer cement-implanted groups are different from the control groups and the control groups are different from each other. The differential counts of the lavage fluid were reasonably consistent with respect to all cell types within each group for any given week (Table 1). The drop in mast cell numbers reported for vitally stained specimens was also observed in the differential count specimens, but to a lesser extent. The number of eosinophils increased dramatically after implantation of the sealer cement disks and did not return to preimplantation values during the experimental time period. The percentage of eosinophils remained within normal limits for the silastic-implanted animals and for the surgical control animals (Fig. 4). An analysis of variance indicates that the regressions of the eosinophil values of the four groups are significantly different (p < 0.05). A t test indicates that each of the four groups is significantly different from the others (p < 0.05) with respect to percentages of eosinophils. Although variations in neutrophil and lymphocyte numbers were proportionately greater than for other cell types, .both of these cell types were found in very
TABLE 1. Mean differential cell counts in percentages by week Wk 0
A
v
t~
Wk 1
Wk 3
Wk 5
Wk 7
Surgical Control Macrophage Mast cell Eosinophil PMN leukocyte Symphocyte
89.6 5.3 4.5 0.5 0.3
(2.8)* 89.5 (3.0) 89.3 (3.0) 89.1 (1.6) 89.4 (1.5) (0.9) 5.1 (1.3) 4.9 (1.6) 5.4 (0.8) 4.9 (0.83) (2.3) 4.1 (1.6) 4.6 (1.6) 4.6 (0.9) 4.4 (0.9) (0.5) 0.5 (.5) 0.8 (0.3) 0.8 (0.3) 0.6 (0.5) (.4) 0.4 (.4) 0.5 (0.4) 0.5 (0.4) 0.7 (0.6)
87.6 5.6 5.4 0.9 0.5
(2.8) (1.2) (4.4) (0.8) (1.1)
90.1 4.8 3.3 0.8 1.1
(1.1) 90.8(1.0) 86.8(1.3) 87.3(1.2) 85.3(1.2) (0.7) 4.1 (0.8) 2.4 (0.5) 2.6 (0.7) 2.6 (0.7) (0.5) 3.9 (0.8) 10.0 (1.4) 9.4 (0.7) 11.6 (2.0) (0.7) 1.0 (0.5) 0.3 (0.5) 0.4 (0.4) 0.2 (0.3) (0.7) 1+0 (0.5) 0.6 (0.5) 0.4 (0.5) 0.3 (3.8)
90.9 4.5 3.3 0.5 0.6
(2.7) 91.4 (1.5) 96.9 (1.5) 85.2 (1.7) 86.2 (1.3) (1.5) 3.5 (1.1) 2.5 (0.5) 2.5 (0.5) 2.8 (0.7) (0.5) 3.4 (0.7) 9.5 (1.2) 10.5 (1.4) 9.6 (0.5) (0.4) 0.8(0.3) 0.7 (0.6) 0.6 (0.4) 0.5 (0.5) (0.4) 0.8(0.3) 0.5 (0.5) 0.7 (0.2) 0.8 (1.0)
Silastic Control Macrophage Mast cell Eosinophil PMN leukocyte Lymphocyte
86.3 6.2 6.2 2.7 0.6
(2.8) 87.8 (6.6) 88.4 (1.6) 88.4 (3.8) (1.8) 5.8 (3.4) 5.4 (1.6) 5.4 (2.0) (1.8) 5.2 (2.6) 5.2 (1.2) 5.2 (2.8) (4.9) 0.9 (0.4) 0.4 (0.4) 0.4 (1.2) (1.2) 0.3 (0.6) 0.4 (1.2) 0.6 (1.2)
Kerr Macrophage Mast cell Eosinophil PMN leukocyte Lymphocyte Grossman Macrophage Mast cell Eosinophil PMN leukocyte Lymphocyte
* SD's in parentheses.
Kerr Eoslnophlls GrossmanEoslnophlls I l~r StlasticEoslnophils 20-~ ~ SurgicalControl Eosinophlls 1
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Kerr Mast Cells GrossmanMast Cells SilasUcMast Cells SurgicalControl MastCells
11.010+09,0r.j
2.8 2.7 26 2,5- A 2.4-2.3-2.2-2,12.0-1.9-1.s1.7-
33
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FIG 4. After implantation(wk 1), the percentage of eosinophilsin the sealer cement groups increaseddramatically.The mast cell percentage in these groups dropped below preimplantationvalues,
.8.7.6.5-
I 1
i 3
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WEEKS
FIG 3. After the initiallavage (wk 0), the vital mast cell counts declined and did not return to p r e l a v a g e l e v e l s during the e x p e r i m e n t a l period.
small numbers in all animals. Neither the mean percentage of lymphocytes nor the mean percentage of neutrophils exceeded 1.1% in any of the four groups (Table 1). An analysis of variance for both neutrophil and lymphocyte counts indicates that there is no significant difference between the groups (p > 0.1).
34
Biggs et
Journal of Endodontics
al.
The content of silver and zinc in macrophages and pooled eluate samples taken by peritoneal lavage
TABLE 2.
Group
Metal Content (g/macrophages) Zinc
Surgical control preoperative Surgical control postoperative Silastic control preoperative Silastic control postoperative Kerr implant preoperative Kerr implant postoperative Grossman implant preoperative Grossman implant postoperative
7 . 2 5 x 1 0 -15
Silver 3 . 7 x 1 0 -15
7.25 x 10 -15
in
Metal Concentration (g/liter of etuate) Zinc
Silver
8 x 1 0 -5 0 . 3 x 1 0 -~ 8 x 10 -5
7 . 2 5 x 1 0 -15 3 . 7 5 x 1 0 -15 3 . 3 x 1 0 -s 0 . 3 x 1 0 -5 7.22 x 10 -15
8 x 10 -5
7.25 x 10 -15 3.75 x 10 -is 2 0 x 1 0 -15
7 . 5 x 1 0 -15
0.3 x 10 -s 6 x 1 0 -5 0 . 3 x 1 0 -5 0 . 3 x 1 0 -5 0 . 3 x 1 0 -5
11.04 x 10 -15
11 • 10 -5
The trace metal analysis of the pooled samples of macrophages and eluates indicates that the metal ions from both Kerr and Grossman formula root canal sealer cements were associated with the macrophage fraction of the peritoneal fluid (Table 2). Furthermore, the zinc concentration in the peritoneal lavage fluid of the cement-implanted animals indicates that the materials are soluble in peritoneal fluid. DISCUSSION
In this investigation, the cell populations of the rat peritoneal cavity were used as a model system for the evaluation of the biocompatibility of endodontic sealer cements. The in vivo method used offers the simplicity of cell culture techniques while retaining the basic concepts of connective tissue implant methods. The continuous monitoring of macrophages, mast cells, and other cells in control and experimental animals was accomplished using the methods of Toraason and Kaminski (33), who reported that differential cell counts of macrophages and mast cells could be determined accurately using vitally stained samples. They also reported that vitally stained mast cell counts could be expected to be consistently 2 to 2.5% lower than counts obtained on samples treated with Wright's stain. The findings of these investigators are confirmed by this study. Both vitally stained samples and those processed with Wright's stain must be used to determine both the relative numbers of cells and the total numbers of macrophages and mast cells collected. In the absence of inflammation, the peritoneal exudate should consist of approximately 83 to 90% macrophages, 5 to 7% mast cells, 3 to 6% eosinophils, and 2 to 4% lymphocytes and neutrophils (38, 39). The differential counts reported in this study confirm that acute inflammation does not result from implantation procedures or from the presence of set sealer cements
in the peritoneal cavity. The sealer cements did, however, result in an eosinophilia which persisted throughout the postimplantation period. This eosinophilia did not occur in silastic-implanted or surgical control animals. Since eosinophils were not stained by the vital staining procedures used in this study, they were counted as macrophages in the vitally stained counts. This eosinophilia would tend to obscure the effect which sealer cement implants exert on vitally counted macrophages. Young animals were selected for this investigation so that general observations could be made concerning the effects of implants on growth and development. Weight gain was monitored to assess this effect. No significant difference was found between groups. Since growth, as reflected by weight gain, was not affected by the experimental procedures, systemic toxicity was not considered to be a factor in the decreased numbers of macrophages observed in those animals receiving the sealer cement implants. The decrease in the number of macrophages may result from a local toxicity to that cell line or alternatively to an inhibition of chemotaxis or a suppression of proliferation. This is noteworthy since macrophages are necessary in all inflammatory modalities (29, 30) as well as in the debridement of the periapical region following endodontic treatment (40). A compromise in the periapical population may have a significant influence upon the rate and quality of periapical healing (40). The slow return to nearly normal levels of macrophages following the implantation of sealer cements may indicate that the degradative products exert their influence on macrophages for only a short period of time. The mechanism involved in the decline of the macrophage population or in the return to normal numbers of macrophages was not determined in this study. Both Grossman's formula and Kerr sealer cements contain materials which are potentially toxic. Eugenol has been established as a protoplasmic poison and has been implicated in sealer cement toxicity by Antrim (21), Mohammed et al. (20), Spangberg et al. (18), and Munaco et al. (19). Zinc oxide, silver, barium sulfate, bismuth, and sodium borate have also been determined to be toxic at the appropriate dose levels (41-45). The analysis for trace amounts of zinc and silver in macrophages and in the diluted peritoneal fluid indicates that the degradative products of the materials are associated with the macrophage fraction of the peritoneal cavity. Macrophages apparently engulf and transport minute fragments of the materials to the liver, spleen, and regional lymph nodes. These may ultimately become excreted in the feces or urine (46). An unanticipated finding of this study was the postimplantation eosinophilia of the sealer cement-implanted animals. The presence of eosinophils as a major constituent of the peritoneal cavity is well established (38, 47, 48). Their biological role, however, has not been
Vol. 11, No. 1, January 1985
completely described (45). It has been demonstrated that materials which cause mast cell degranulation will cause a subsequent increase in the number of eosinophils (47-49). Mast cells are also known to release active eosinophil chemotactic factor of anaphylaxis (50) along with histamine and heparin upon degranulation (49). This mechanism cannot be considered to be completely responsible for the eosinophilia of the sealer cement-implanted animals as the proportion of eosinophils was increased in these animals well beyond the decrease in mast cells noted in the same groups. We would therefore conclude that other interactions may also be involved in this observed increase of eosinophils. CONCLUSIONS The following may be concluded from the observations and analysis of this study: 1. Kerr and Grossman formula root canal sealer implants result in a decline of rat peritoneal macrophages in vivo. 2. Kerr and Grossman formula root canal sealer cements cause a significant increase in the numbers of eosinophils in the rat peritoneal cavity. This peritoneal eosinophilia makes vital cell counts inadequate to determine material influences on macrophages unless Wright-stained specimens are also evaluated. 3. None of the procedures used in this study result in an acute inflammatory response. 4. The macrophage fraction is associated with accumulated degradative products of Kerr and Grossman formula sealer cements. These products are either phagocytosed or they adhere to the macrophage cell surface. 5. The rat peritoneal cavity offers an effective model for the determination of the potential influence of endodontic materials upon macrophages. 6. The lavage dilution and vital staining procedures resulted in a consistent decrease in vital mast cell counts. This variation is not reflected in the percentage of mast cells observed during differential counts of cells using a Wright stain preparation.
References 1. Brayton S, Davis S, Goldman M. Gutta-percha root canal fillings, an in vitro analysis. Part I. Oral Surg. 1973;35:226-31. 2. Langeland K. The histopathologic basis in endodontic treatment. Dent Clin North Am 1967;11:491-520. 3. Langeland K Root canal sealants and pastes. Dent Clin North Am 1974:18:309_27. 4. Ingle JL Endodontics. Philadelphia: Lea 8, Febiger, 1974:64. 5. Seltzer S, Soltanoff W, Sinai I, Smith J. Biologic aspects of endodontics. IV. Periapical tissue reaction to root filled teeth whose canals had been instrumented short of their apices. Oral Surg 1969;28:724-38. 6. Seltzer S, Bender IB, Turkenkoph S. Factors affecting successful repair after root canal therapy. J Am Dent Assoc 1963;67:651-62. 7. Strindberg LZ. The dependence of the results of pulp therapy on certain factors. Acta Odontol Scand 1956;14:1-175. 8. Frostell G. Factors influencing the prognosis of endodontie therapy. In:
Response to Implanted Sealer Cements
35
Grossman L, ed. Transactions of the third international conference on endodontics. Philadelphia: University of Pennsylvania Printing Office, 1963:161-73. 9. Storms JL. Factors that influence the success of endodontic treatment. J Can Dent Assoc 1969;35:83-97. 10. Erasquin J, Muruzabal M. Tissue reaction to root canal cements in the rat molar. Oral Surg 1968;26:360-73. 11. Erasquin J, Muruzabal M Root canal filling with zinc oxide and eugenol cement in the rat molar. Oral Surg 1967;24:547-58. 12. Muruzabal M, Erasquin J, Devote F. A study of periapical overfilling in root canal treatment in the molar of the rat. Arch Oral Bio11966;11:373-83. 13. Davis M, Joseph S, Bucher J. Periapical and intracanal healing following incomplete root canal fillings in dogs. Oral Surg 1971 ;31:662-75. 14. Gutierrez HH, Gigoux C, Escobar F. Histotogic reactions to root canal fillings. Oral Surg 1969;28:557-66. 15. Deemer JP, Tsaknis PJ. The effects of overfilled polyethylene tube intraosseous implants in rats. Oral Surg 1979;48:358-73. 16. Friend LA, Browne RM Tissue reactions to some fillling materials. Br Dent J 1968;125:291-8. 17. Langeland K, Guttuso J, Langeland L, Tobor G. Methods in the study of biologic responses to endodontic materials, tissue response to N2. Oral Surg 1969;27:522-42. 18. Spangberg L, Langeland K. Biologic effect of dental materials. I. Toxicity of root canal filling materials on He-La cells in vitro. Oral Surg 1978;35:40244. 19. Munaco FS, Miller WA, Everett MM A study of long term toxicity of endodontic materials with use of an in vitro model. J Endodon 1978;4:151-7. 20. Mohammed AR, Mincer HH, Younis D, Dillingham E, Siskin M. Cytotoxicity evaluation of root canal sealers by tissue culture-agar overlay technique. Oral Surg 1978;45:766-73. 21. Antrim DD. Evaluation of the cytotoxicity of root canal sealing agents on tissue culture cells in vitro: Grossman's sealer, N2 permanent, Rickert's sealer, Cavit. J Endodon 1976;2:111-2. 22. Doblecki W, Turner DW, Osetek EM, Pelleu GB. Leukocyte migration response to dental materials using Boyden chambers. J Endodon 1980;6:63640. 23. Campbell AD, Gear RD, Turner DW, Cunningham CJ, Bell WC. Cell mediated response to endodontic cements: research report. J Endodon 1978;4:147-50. 24. Langeland K, Block RM, Grossman LI. A histopathologic and histobacteriologic study of 35 periapical endodontic surgical specimens. J Endodon 1977;3:8-23. 25. Morse DR, Lasater DR, White D. Presence of immunoglobin producing cells in periapical lesions. J Endodon 1975;1:338-43. 26. Barnes GW, Langeland K. Antibody formation in primates following introduction of antigens into the root canal. J Dent Res 1966;45:111-4. 27. Farber PA. Scanning electron microscopy of cells from periapical lesions. J Endodon 1975;1:291-3. 28. Spector WG. The granulomatous inflammatory exudate. Int Rev Exp Patho11969;8:1-55. 29. Basten A, Mitchell J. Role of macrophages in 1- cell and B cell collaboration in antibody production. In: Nelson DS, ed. Immunobiology of the macrophage. New York: Academic Press, 1976:45-90. 30. Dannenburg AM. Macrophages in inflammation and infection. N Engl J Med 1975;293:489-93. 31. Gordon S, Unkeless JC, Cohn ZA. Induction of macrophage plasminogen activator by endotoxin stimulation and phagoeytosis, evidence for a two stage process. J Exp Med 1974;140:995-1010. 32. Nathan GF, Murray HW, Cohn ZA. The macrophages as an effector cell. N Engl J Med 1980;298:622-6. 33. Toraason J, Kaminski EK. Accuracy of mast cell counts with different staining procedures. Med Lab Techno11979;32:17-28. 34. Toh K, Sato N, Kikuchi K. Effect of concanavalin A on the cytotoxicity of rat peritoneal macrophages. J Reticuloendothel Soc 1979;25:17-28. 35. Pugh-Humphreys RGP, Thomson AW. An ultrastructural study of peritoneal mononuclear phagocytes from cornybacteriumparvum injected mice. Br J Exp Patho1197;60:259-68. 36. Fragola A, Pascal S, Rosengarten M, Smith A, Blechman H. The effect of varying particle size on the components of Grossman's cement. J Endodon 1981 ;5:336-9. 37. Stern MH, Driezens S, Mackler BF, Selbst AG, Levy BM. Quantitative analysis of cellular composition of human periapical granuloma. J Endodon 1981 ;7:117-22. 38. Bosworth N, Archer GT. The eosinophil content of the peritoneal cavity of the rat. Aust J Exp Biol 1961 ;39:165-70. 39. Johnson GR, Nicholas WL, Metcalf D, McKenzie IF, Mitchel GF. Peritoneal cell population of mice infected with mesocestoides corti as a source of eosinophits, fnt Arch Allergy Appl lmmunol 1979;59:315-22. 40. Spector WG. Chronic inflammation. J Endodon 1917;3:218-23. 41. Subcommittee on zinc. Committee on Medical and Biological Effects of Environment Pollutants, Division of Medical Sciences, Assembly of Life Sciences, National Research Council. Zinc. Baltimore; University Park Press, 1979;341. 42. Venugopal B, Luckey TD. Medical toxicity in mammals 2. New York: Plenum Press, 1977:32-7. 43. Browning EC. Toxicity of industrial metals. London: Butterworth,
36
Biggs et al.
1961:262-7. 44. Robinson FR, Johnson MT. Histopathological studies of tissue reactions to various metals implanted in cat brains. Aeronautical systems division technical report 61-397, t96. Wrigf~t Patterson Air Force Base, OH: US Government Press, 2:1-13. 45. Arena J M Poisoning: toxicology, symptoms, treatments. American lecture series no 903, American lectures in living chemistry. 3rd ed. Springfield, IL: Charles C Thomas, 1974. 46. Luckey TD, Venugopal B, Metal toxicity in mammals 1 Physiologic and Chemical Basis for Metal Toxicity. New York: Plenum Press, 1977:88.
Journal of Endodontics 47. Pincus SJ. Production of eosinophil-fich guinea pig peritoneal exudates. Blood 1978;52:127-34. 48. Gleioh GL, Loegering D. Selective stimulation and purification of eosinophils and neutrop~ils from ~uinea pig peritoneaI fluids. J Lab Clin Med 1973;82:522-8 49 Ward PA, Chemotaxis of human eosinophils. Am J Pathol 1969;54: 121-8. 50. Kownatzki E, Till G, Gagelmann M, Terwort G, Gemsa D. Histamine induces release of an eosinophil immobilizing factor from mononuclear cells. Nature 1977;270:67-9.
Printed in U.S.A. VOL. 11, NO. 1,J.~NUARY1985
Rat Macrophage Response to Implanted Sealer Cements Respuesta Macrofagica en Rata a Cementos Selladores Implantados John T. Biggs, DDS, MEd, MS, E. J. Kaminski, PhD, and E. M. Osetek, DOS, MEd Un patron satisfactorio para probar la biocompatibilidad de cementos selladores debe incluir la colocacion de los materiales in vivo, donde los efectos sobre una poblacion de macrofagos y otros tipos de c(Hulas pueden ser cuantitativamente evaluados. La cavidad peritoneal de la rata ofrece ese sistema in vivo y el examen del contenido de esta cavidad por irrigacion es un m(~todo aceptado para estudiar la respuesta celular (33 a 35). El proposito de este estudio fue evaluar la cavidad peritoneal de la rata como patron o modelo para testificar la influencia de cementos selladores en la poblacion de macrofagos in vivo.
sistent with the findings reported for the usage studies in which periapical tissues were evaluated. It has been suggested that studies which employ the root canal system (16), as well as those which involve implantation in connective tissues (17), are of limited value for the evaluation of the biocompatibility of dental materials because of the variables which are introduced by the procedures as well as the complex cellular milieu at the test site. Cell culture techniques have been used in an attempt to overcome some of the complexities of implant and usage studies. Sealer cements are known to be cytotoxic to cell lines and tissues in culture (18-21). These techniques, however, involve cells with no capacity for recovery and interaction. It is difficult to compare the effects of dental materials on cultured cells with their actual effects on the periapical region. Root canal sealer cements are known to influence the inflammatory process. Zinc oxide-eugenol cements have been demonstrated to increase neutrophil migration (22) and have been implicated in nonspecific inflammation (23), humoral immunity (24-26), and cell-mediated immunity (27). Although the immunological consequences of placing sealer cements in contadt with periapical tissues are not clear, it is generally agreed that there is an effect on the inflammatory cells. A cell line is available for study which is a wellestablished constituent of the periapical lesion (27-37). It is known to be the primary phagocyte of the body (28) and is known to participate as an antigen processor in both cell-mediated and humoral immune response (29-31). The macrophage is recognized to be a versatile secretory effector cell, whose products influence every stage of the inflammatory process (32). A satisfactory model for testing the biocompatibility of sealer cements should involve the placement of materials into an in vivo system, where the effects on a population of macrophages and other cell types could be quantitatively evaluated. The peritoneal cavity of the rat provides such an in vivo system and examination of the content of this cavity by lavage is an accepted method for studying cellular response (33-35).
The irregularity of root canals which have been prepared for obturation does not allow for an exact fit of the solid or semisolid core which makes up the bulk of root canal fillings (1). Sealer cements are necessary to fill the resultant voids (2, 3), and there is the potential for these materials to come into contact with periapical tissues. The biological effects of these sealer cements are of interest to endodontic practitioners and have been the subject of a number of investigations. Several investigators have studied the effects of overfills (4-8) and extruded sealer cements (9) on the success of root canal treatments. The consensus is that overfills have an adverse effect on healing. Storms (9) has reported that sealer cement which has been extruded into the periapical tissues results in an 11.2% reduction in the success rate of endodontically treated teeth. In studies of factors which influence prognosis, no attempts have been made to correlate failure to heal with specific cellular events in the periapical region. The effects of the sealer cements on the periapical tissues of animals have been evaluated in usage studies by Erasquin and Muruzabal (10, 11), Muruzabal et al. (12), and Davis et al. (13). These investigators have reported that sealer cements cause inflammation and delay healing in the periapical region. Implantation studies in connective tissues by Guttierez et al. (14) and in osseous tissues by Deemer and Tsaknis (15) are con30
Vol. 11, No. 1, January 1985
The purpose of this study was to evaluate the rat peritoneal cavity as a model for assessing the influence of sealer cements on macrophage populations in vivo. MATERIALS AND METHODS
Kerr's antiseptic sealer (Kerr Mfg. Co., Romulus, MI) and Roth's 801 (Roth International, Ltd., Chicago, IL) root canal sealer cement (a Grossman's formulation) were used in this study. Silastic 500-9 (Dow Coming Corp., Midland, MI) was used as the implant control. The Kerr sealer cement was mixed according to manufacturer's instructions, and the Roth's 801 was mixed according to the method described by Fragola et al. (36). The major constituents of Kerr's sealer cement are zinc oxide, elemental silver, thymol ioxide, oleo resins, oil of cloves, and Canada balsam and the constituents of Roth's 801 sealer cement are zinc oxide, bismuth subnitrate, barium sulfate, anhydrous sodium borate, staybelite resin, and eugenol. Immediately after mixing, the sealer cements were placed in implant disk molds which were made by punching circular holes in a 1.60-mm thick piece of 5009 silastic sheeting. The diameter of the holes was 10.5 mm. The sealer cement-filled disk molds were pressed firmly between two 3-inch square glass slabs and immersed in deionized water at 37~ for 5 days. After immersion, the disks were removed from their molds and air dried at room temperature and humidity for 2 days in order to obtain a consistent preimplant weight. The disks were then weighed to an accuracy of 0.001 g. The silastic implants were geometrically identical to the sealer cement implants. Each silastic implant was thoroughly washed in deionized water and allowed to bench dry for 48 h before implantation. Thirty-two CD strain female Charles River rats (Charles River Breeding Laboratory, Portage, MI) were used as implant recipients. The rats were fed a standard laboratory diet ad libitum and weighed 95 to 150 g at the beginning of the study. The animals were divided into four groups. All animals within each group were treated identically. Eleven animals were assigned to a surgical control group and seven animals to each of the implant groups: Kerr, Grossman's formula, and silastic. The control animals were subjected to surgical and anesthetic procedures identical to those experienced by the experimental animals excepting the placement of implants. Research employing the peritoneal lavage technique has demonstrated that the surgical procedure used does not result in significant alterations in the macrophage population of the peritoneal cavity (33, 38). Lavage solutions obtained from the control animals were treated identically to those obtained from the experimental animals.
Response to Implanted Sealer Cements
31
Peritoneal lavages were performed on each animal 1 wk before implantation, on the day of implantation, and at 2, 4, and 6 wk after implantation according to the procedure described by Torraason and Kaminski (33). The abdominal area of each animal was shaved and disinfected with reagent grade ethyl alcohol. Ten cubic centimeters of 37~ Ringer's solution were then slowly injected through a 16-gauge needle into the peritoneal cavity. The needle was removed and the Ringer's solution was mixed with the indigenous peritoneal fluid by rolling the animal. The mix of lavage fluid and peritoneal fluid was collected by allowing it to flow passively through the 16-gauge needle that had been reinserted into the injection site. Ether and subcutaneously injected nembutal (Abbott Laboratories, N. Chicago, IL) were used for anesthesia during lavage, implantation, and suture removal procedures. Implants were inserted by making a midline incision slightly larger than the implant and placing the implant disk into the peritoneal cavity with a cotton forceps. Incisions were closed with continuous 4-0 silk sutures which were removed after 1 wk. At the end of the experimental period (7 wk), the animals were sacrificed with chloroform anesthesia. Implants were recovered and the sealer cement implants were washed in deionized water, air dried for 2 days, and reweighed. The peritoneal lavage fluid from each animal was divided into two portions. One or 2 cm 3 of each solution, depending upon the quantity recovered in the lavage procedure, were placed in Procil-28 (PCR Research Chemicals, Inc., Gainsville, FL) -treated disposable centrifuge tubes and were diluted with equal volumes of 5% acetic acid. The cells contained in the diluted lavage fluid were then vitally stained with 0.005% toluidine blue and counted with the aid of a Spencer hemocytometer (American Optical Corp., Buffalo, NY). Of the cells found in the peritoneal cavity, only mast cells were stained with toluidine blue; therefore, the resulting count differentiated the mast cells present in each sample of fluid. The portion of the lavage solution that was not used for cell counts was diluted to 6 ml with Ringer's solution and centrifuged supernatant was decanted and stored in a separate centrifuge tube. Six cubic centimeters of Ringer's solution were added to the packed cells. Then the mixture was agitated and recentrifuged. The supernatant was again decanted and added to the previously collected volume. Washed packed cells and the supernatant were sealed in their respective containers with Parafilm M (American Can Co., Greenwich, CT) and frozen. At the end of the experiment, the samples were pooled into groups of preoperative and postoperative eluates and macrophages. The pooled samples were analyzed by Trace Elements Inc. (Park Ridge, IL) for silver and for zinc present in either the eluates or the cells.
32
Journal of Endodontics
Biggs et al.
At the time of peritoneal lavage, one drop of each lavage solution was placed on a glass slide and allowed to dry. Subsequently, the slides were fixed in methyl alcohol and stained with Cameco B4130 (Scientific Products, Division of American Hospital Supply Corp., McGraw Park, IL) blood stain (Wright's stain). These slides were decolorized with deionized water and methyl alcohol. Three hundred cells on each slide were counted using an oil immersion lens and the percentages of macrophages, mast cells, lymphocytes, eosinophils, and neutrophils were determined. Counts obtained using the hemocytometer were subjected to two statistical normalization procedures to facilitate comparisons. Macrophage and mast cell counts were adjusted to number of cells/100 g body wt to compensate for differences attributable to using a constant volume of lavage fluid in animals of different sizes. A secod normalization was performed by subtracting the number of cells from each group necessary to bring the first mean count to the mean value for the surgical control animals. This normalization ensured that the relative numbers of cells would not obscure the changes which resulted from the effects of the experimental procedures. All data were subjected to a one-way analysis of variance. Where indicated, a t test for differences between groups was performed.
240I~
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A Kerr ~ GrosDman
~
~. SJ|asilc
190U) 180 -
:S
170 160-
110-
0 '
i WEEKS
FiG 1, All animals demonstrated a consistent gain in weight over the 7-wk period.
RESULTS 1"511
All animals gained weight consistently during the experimental period (Fig. 1). An analysis of variance indicates that the regressions of the mean animal weights by week are not significantly different (p > 0.05). Vitally stained mast cells and macrophages aspirated from the peritoneal cavities of implanted and unimplanted animals and counted with the aid of a hemocytometer demonstrated a wide variation in numbers both within and between groups (Figs. 2 and 3). In the control animals, macrophages gradually increased in number throughout the experiment. This increase was attributed to inflammation resulting from the initial surgical intervention and subsequent lavages. Macrophage numbers in experimental animals increased similarly, except for the first postimplant count (wk 3). In both sealer-implanted groups, the mean number of macrophages decreased during the first 2 wk after implantation (Fig. 3). The number of macrophages in the two sealer cement test groups began to increase thereafter, until the 6th week after implantation, when they achieved the same cell population as the control groups. An analysis of variance indicates that the regressions of the group values are different and that this difference cannot be attributed to random variation (p = 0.0028, F = 5.68). A t test indicates that the numbers of
Surglcol Control
A x IAI
0 ~, ,~ A
Surgical Control Grossmon tormula $11ast|c Kerr
1.45"] 1.42--1 1.39-1.36-1.30-1.27-1.24-1,21-1.18-1,151,12-
1.03 -1.00--
0
I 1
I 3
1 5
I 7
WEEKS
FtG 2. Implantation procedures were performed at wk 1. The mean vital macrophage counts of sealer cement-implanted animals dropped at wk 3, but returned to normal levels by wk 7.
macrophages in the surgical control and the silastic groups are not significantly different (p > 0.05). The Kerr-implanted and the Grossman formula-implanted groups were also statistically similar (p > 0.05). The sealer cement groups, however, were found to be statistically different from both control groups (p < 0.05).
Vol. 11, No. 1, January 1985
Response to Implanted Sealer Cements
Vitally stained mast cell counts exhibited even more variation than the macrophage counts (Fig. 3). The vital mast cell counts decreased significantly following the first injections of Ringer's solution and did not return to the initial levels during the experimental period. An analysis of variance indicates that the group regressions are different (p < 0.05). The t test indicates that the sealer cement-implanted groups are not significantly different from each other (p > 0.05). At the same level of significance, however, the sealer cement-implanted groups are different from the control groups and the control groups are different from each other. The differential counts of the lavage fluid were reasonably consistent with respect to all cell types within each group for any given week (Table 1). The drop in mast cell numbers reported for vitally stained specimens was also observed in the differential count specimens, but to a lesser extent. The number of eosinophils increased dramatically after implantation of the sealer cement disks and did not return to preimplantation values during the experimental time period. The percentage of eosinophils remained within normal limits for the silastic-implanted animals and for the surgical control animals (Fig. 4). An analysis of variance indicates that the regressions of the eosinophil values of the four groups are significantly different (p < 0.05). A t test indicates that each of the four groups is significantly different from the others (p < 0.05) with respect to percentages of eosinophils. Although variations in neutrophil and lymphocyte numbers were proportionately greater than for other cell types, .both of these cell types were found in very
TABLE 1. Mean differential cell counts in percentages by week Wk 0
A
v
t~
Wk 1
Wk 3
Wk 5
Wk 7
Surgical Control Macrophage Mast cell Eosinophil PMN leukocyte Symphocyte
89.6 5.3 4.5 0.5 0.3
(2.8)* 89.5 (3.0) 89.3 (3.0) 89.1 (1.6) 89.4 (1.5) (0.9) 5.1 (1.3) 4.9 (1.6) 5.4 (0.8) 4.9 (0.83) (2.3) 4.1 (1.6) 4.6 (1.6) 4.6 (0.9) 4.4 (0.9) (0.5) 0.5 (.5) 0.8 (0.3) 0.8 (0.3) 0.6 (0.5) (.4) 0.4 (.4) 0.5 (0.4) 0.5 (0.4) 0.7 (0.6)
87.6 5.6 5.4 0.9 0.5
(2.8) (1.2) (4.4) (0.8) (1.1)
90.1 4.8 3.3 0.8 1.1
(1.1) 90.8(1.0) 86.8(1.3) 87.3(1.2) 85.3(1.2) (0.7) 4.1 (0.8) 2.4 (0.5) 2.6 (0.7) 2.6 (0.7) (0.5) 3.9 (0.8) 10.0 (1.4) 9.4 (0.7) 11.6 (2.0) (0.7) 1.0 (0.5) 0.3 (0.5) 0.4 (0.4) 0.2 (0.3) (0.7) 1+0 (0.5) 0.6 (0.5) 0.4 (0.5) 0.3 (3.8)
90.9 4.5 3.3 0.5 0.6
(2.7) 91.4 (1.5) 96.9 (1.5) 85.2 (1.7) 86.2 (1.3) (1.5) 3.5 (1.1) 2.5 (0.5) 2.5 (0.5) 2.8 (0.7) (0.5) 3.4 (0.7) 9.5 (1.2) 10.5 (1.4) 9.6 (0.5) (0.4) 0.8(0.3) 0.7 (0.6) 0.6 (0.4) 0.5 (0.5) (0.4) 0.8(0.3) 0.5 (0.5) 0.7 (0.2) 0.8 (1.0)
Silastic Control Macrophage Mast cell Eosinophil PMN leukocyte Lymphocyte
86.3 6.2 6.2 2.7 0.6
(2.8) 87.8 (6.6) 88.4 (1.6) 88.4 (3.8) (1.8) 5.8 (3.4) 5.4 (1.6) 5.4 (2.0) (1.8) 5.2 (2.6) 5.2 (1.2) 5.2 (2.8) (4.9) 0.9 (0.4) 0.4 (0.4) 0.4 (1.2) (1.2) 0.3 (0.6) 0.4 (1.2) 0.6 (1.2)
Kerr Macrophage Mast cell Eosinophil PMN leukocyte Lymphocyte Grossman Macrophage Mast cell Eosinophil PMN leukocyte Lymphocyte
* SD's in parentheses.
Kerr Eoslnophlls GrossmanEoslnophlls I l~r StlasticEoslnophils 20-~ ~ SurgicalControl Eosinophlls 1
---~" ,,.[]., ...~r. "-'~'-
Kerr Mast Cells GrossmanMast Cells SilasUcMast Cells SurgicalControl MastCells
11.010+09,0r.j
2.8 2.7 26 2,5- A 2.4-2.3-2.2-2,12.0-1.9-1.s1.7-
33
8.07.0-
Q SurgicalControl "i~ Slllmtlc O Grossman Kerr
6.0- ..........
"......
.............
4.0.-
~
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20
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1.0
1,51+41.3-
0
1.21.11.0+9-
i
+ .......... .~:...
i 1
! 3
i 5
i 7
WEEKS
FIG 4. After implantation(wk 1), the percentage of eosinophilsin the sealer cement groups increaseddramatically.The mast cell percentage in these groups dropped below preimplantationvalues,
.8.7.6.5-
I 1
i 3
I 5
I 7
WEEKS
FIG 3. After the initiallavage (wk 0), the vital mast cell counts declined and did not return to p r e l a v a g e l e v e l s during the e x p e r i m e n t a l period.
small numbers in all animals. Neither the mean percentage of lymphocytes nor the mean percentage of neutrophils exceeded 1.1% in any of the four groups (Table 1). An analysis of variance for both neutrophil and lymphocyte counts indicates that there is no significant difference between the groups (p > 0.1).
34
Biggs et
Journal of Endodontics
al.
The content of silver and zinc in macrophages and pooled eluate samples taken by peritoneal lavage
TABLE 2.
Group
Metal Content (g/macrophages) Zinc
Surgical control preoperative Surgical control postoperative Silastic control preoperative Silastic control postoperative Kerr implant preoperative Kerr implant postoperative Grossman implant preoperative Grossman implant postoperative
7 . 2 5 x 1 0 -15
Silver 3 . 7 x 1 0 -15
7.25 x 10 -15
in
Metal Concentration (g/liter of etuate) Zinc
Silver
8 x 1 0 -5 0 . 3 x 1 0 -~ 8 x 10 -5
7 . 2 5 x 1 0 -15 3 . 7 5 x 1 0 -15 3 . 3 x 1 0 -s 0 . 3 x 1 0 -5 7.22 x 10 -15
8 x 10 -5
7.25 x 10 -15 3.75 x 10 -is 2 0 x 1 0 -15
7 . 5 x 1 0 -15
0.3 x 10 -s 6 x 1 0 -5 0 . 3 x 1 0 -5 0 . 3 x 1 0 -5 0 . 3 x 1 0 -5
11.04 x 10 -15
11 • 10 -5
The trace metal analysis of the pooled samples of macrophages and eluates indicates that the metal ions from both Kerr and Grossman formula root canal sealer cements were associated with the macrophage fraction of the peritoneal fluid (Table 2). Furthermore, the zinc concentration in the peritoneal lavage fluid of the cement-implanted animals indicates that the materials are soluble in peritoneal fluid. DISCUSSION
In this investigation, the cell populations of the rat peritoneal cavity were used as a model system for the evaluation of the biocompatibility of endodontic sealer cements. The in vivo method used offers the simplicity of cell culture techniques while retaining the basic concepts of connective tissue implant methods. The continuous monitoring of macrophages, mast cells, and other cells in control and experimental animals was accomplished using the methods of Toraason and Kaminski (33), who reported that differential cell counts of macrophages and mast cells could be determined accurately using vitally stained samples. They also reported that vitally stained mast cell counts could be expected to be consistently 2 to 2.5% lower than counts obtained on samples treated with Wright's stain. The findings of these investigators are confirmed by this study. Both vitally stained samples and those processed with Wright's stain must be used to determine both the relative numbers of cells and the total numbers of macrophages and mast cells collected. In the absence of inflammation, the peritoneal exudate should consist of approximately 83 to 90% macrophages, 5 to 7% mast cells, 3 to 6% eosinophils, and 2 to 4% lymphocytes and neutrophils (38, 39). The differential counts reported in this study confirm that acute inflammation does not result from implantation procedures or from the presence of set sealer cements
in the peritoneal cavity. The sealer cements did, however, result in an eosinophilia which persisted throughout the postimplantation period. This eosinophilia did not occur in silastic-implanted or surgical control animals. Since eosinophils were not stained by the vital staining procedures used in this study, they were counted as macrophages in the vitally stained counts. This eosinophilia would tend to obscure the effect which sealer cement implants exert on vitally counted macrophages. Young animals were selected for this investigation so that general observations could be made concerning the effects of implants on growth and development. Weight gain was monitored to assess this effect. No significant difference was found between groups. Since growth, as reflected by weight gain, was not affected by the experimental procedures, systemic toxicity was not considered to be a factor in the decreased numbers of macrophages observed in those animals receiving the sealer cement implants. The decrease in the number of macrophages may result from a local toxicity to that cell line or alternatively to an inhibition of chemotaxis or a suppression of proliferation. This is noteworthy since macrophages are necessary in all inflammatory modalities (29, 30) as well as in the debridement of the periapical region following endodontic treatment (40). A compromise in the periapical population may have a significant influence upon the rate and quality of periapical healing (40). The slow return to nearly normal levels of macrophages following the implantation of sealer cements may indicate that the degradative products exert their influence on macrophages for only a short period of time. The mechanism involved in the decline of the macrophage population or in the return to normal numbers of macrophages was not determined in this study. Both Grossman's formula and Kerr sealer cements contain materials which are potentially toxic. Eugenol has been established as a protoplasmic poison and has been implicated in sealer cement toxicity by Antrim (21), Mohammed et al. (20), Spangberg et al. (18), and Munaco et al. (19). Zinc oxide, silver, barium sulfate, bismuth, and sodium borate have also been determined to be toxic at the appropriate dose levels (41-45). The analysis for trace amounts of zinc and silver in macrophages and in the diluted peritoneal fluid indicates that the degradative products of the materials are associated with the macrophage fraction of the peritoneal cavity. Macrophages apparently engulf and transport minute fragments of the materials to the liver, spleen, and regional lymph nodes. These may ultimately become excreted in the feces or urine (46). An unanticipated finding of this study was the postimplantation eosinophilia of the sealer cement-implanted animals. The presence of eosinophils as a major constituent of the peritoneal cavity is well established (38, 47, 48). Their biological role, however, has not been
Vol. 11, No. 1, January 1985
completely described (45). It has been demonstrated that materials which cause mast cell degranulation will cause a subsequent increase in the number of eosinophils (47-49). Mast cells are also known to release active eosinophil chemotactic factor of anaphylaxis (50) along with histamine and heparin upon degranulation (49). This mechanism cannot be considered to be completely responsible for the eosinophilia of the sealer cement-implanted animals as the proportion of eosinophils was increased in these animals well beyond the decrease in mast cells noted in the same groups. We would therefore conclude that other interactions may also be involved in this observed increase of eosinophils. CONCLUSIONS The following may be concluded from the observations and analysis of this study: 1. Kerr and Grossman formula root canal sealer implants result in a decline of rat peritoneal macrophages in vivo. 2. Kerr and Grossman formula root canal sealer cements cause a significant increase in the numbers of eosinophils in the rat peritoneal cavity. This peritoneal eosinophilia makes vital cell counts inadequate to determine material influences on macrophages unless Wright-stained specimens are also evaluated. 3. None of the procedures used in this study result in an acute inflammatory response. 4. The macrophage fraction is associated with accumulated degradative products of Kerr and Grossman formula sealer cements. These products are either phagocytosed or they adhere to the macrophage cell surface. 5. The rat peritoneal cavity offers an effective model for the determination of the potential influence of endodontic materials upon macrophages. 6. The lavage dilution and vital staining procedures resulted in a consistent decrease in vital mast cell counts. This variation is not reflected in the percentage of mast cells observed during differential counts of cells using a Wright stain preparation.
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