JOURNAL OF ENDODONTICS I VOL 2, NO 4, APRIL 1976

E v a l u a t i o n of t h e c y t o t o x i c i t y of root c a n a l s e a l i n g a g e n t s on t i s s u e c u l t u r e c e l l s in vitro: Grossman's sealer, N2 (permanent), R i c k e r t ' s s e a l e r , a n d Cavit

Donald D. Antrim, DDS, MS, Loma Linda, Calif

A n in vitro s t u d y with r a d i o a c t i v e l y l a b e l e d tissue culture cells w a s c o n d u c t e d o v e r a s e v e n m o n t h period on G r o s s m a n ' s sealer, N2 (permanent), Rickert's sealer, a n d Cavit to d e t e r m i n e the l a s t i n g a n d relative tissue toxicity of these materials. In this s t u d y it w a s o b s e r v e d that all the m a t e r i a l s s t u d i e d p o s s e s s lasting tissue toxicity. W h e n relative toxicity is c o n s i d e r e d , Grossman.'s is the most toxic, f o l l o w e d b y N2, Rickert's, a n d Cavit. Both G r o s s m a n ' s a n d N2 s h o u l d b e c o n s i d e r e d h i g h l y toxic; Rickert's d i s p l a y s m o d e r a t e toxicity. Cavit s h o u l d b e c o n s i d e r e d mild to m o d e r a t e in toxicity. The u s e of r a d i o a c t i v e - l a b e l e d tissue cells a s a m e t h o d of testing root c a n a l filling m a t e r i a l s is a rapid, h i g h l y sensitive m e t h o d that a l l o w s a d e q u a t e cell-material c o n t a c t a n d permits objective q u a n t i t a t i o n of cell d a m a g e with a c c u r a c y .

The objective of endodontic therapy is restoration of the treated tooth to its proper form and function in the masticatory apparatus. In doing so, the least amount of irritation produced by filling substances is of primary importance. Filling materials inadvertently extruded beyond the confines of the root canal into the periapical tissue can cause inflammation and resultant pain. The degree of discomfort experienced by the patient after a root canal filling should be of

concern to all dentists who perform endodontic therapy. The culmination of careful endodontic treatment involves the ~illing or obliteration of the canal to obtain an adequate seal as close as possible to the cementodentinal junction, with an inert material. To accomplish this the dentist has at his disposal many cements, pastes, plastics, and solids. Sealers and filling materials used in endodontic treatment should be compatible with living connective

tissue to preclude any possible harmful responses. One of the requirements of an ideal root canal sealer or filling material is that it be nonirritating to the periapical tissues. Studies of the biologic response to endodontic materials have been conducted with various species and methods. These pre,cious studies have involved primarily tissue culture experiments, implant studies, or usage tests in animal or human teeth. The biologic effect of various root filling materials can be tested by the examination of animal and human biopsy preparations at different periods of time after implantation or usage. Such examinations are subject to great biologic variation. For successive examinations it is scarcely possible for sufficient human biopsy preparations to be obtained. Preliminary examination of the biologic effect of alien substances can be carried out conveniently in tissue or cell culture studies. The toxicity of root canal filling materials has been studied extensively by in vitro testing. 1-z The results of such experiments, however, have varied not only with 111

JOURNAL OF ENDODONTICS I VOL 2, NO 4, APRIL 1976

the root canal filling material in question, but also with the testing method. Therefore, it became important to develop a more objective method for such evaluation. Such a method was developed by Spangberg. 4 It involved (1) complete material-cell contact; (2) the possibility of varying exposure time related to the stage of setting of the material; and (3) quantitation of the results. The advantages of this in vitro method lie in its simplicity, in the possibility of control, and in the standardizing of experimental conditions. The objective of this study was to compare, by using Spangberg's in vitro method, 4 the lasting and relative tissue toxicity of four root canal sealing materials: Grossman's sealer*; Rickert's sealert; N2 (permanent)z~; and Cavit.w

Test

I I I J

,i

,?be,

sup.... tant

~L I Centrifugation~ ~ I

I

(t-Samples)'-~ 2 ==

--

=~oRelease

0 r-.~mples

I } I f

Fig 1--Spangberg method /or testing cytotoxicity o/ a solid material in vitro. Cells in culture flask are labeled with sodium chromate. Test material is placed in slide culture chamber. Labeled cells are added on top o/ material, followed by incubation at 37 C. A t conclusion of experiment, fluid in culture chamber is transferred to test t u b e s / o r / i n a l centri/ugation, placed in a planchet, dried, and counted in gamma counter.

MATERIALS AND METHODS

The root canal filling materials had the following compositions: Grossman's root canal sealer: zinc oxide, 42%; Staybelite Resin, 27%, bismuth subcarbonate, 15%; barium sulfate, 15% ; borax, t % ; and eugenol as the liquid component. Rickert's root canal sealer: molecular silver, 25%; zinc oxide, 34%; dithymol diiodide, 11% ; oleo resins, 30%; and clove oil and Canada balsam as the liquid component. N2 (permanent) (RC-2B formula) : hydrocortisone, 1.5%; titanium dioxide, 2.0%; trioxymethylene, 7.0%; lead oxide, 16.5%; zinc oxide, 73.0%; and eugenol ~as the liquid component. Cavit: zinc oxide, calcium sulfate, zinc sulfate, glycol acetate, polyvinyl acetate, polyvinyl chloride-acetate, triethanalamine, and red pigment. All materials were mixed according to the manufacturers' instructions.

The synthetic medium was McCoy's 5A modified medium with glutamine. The medium was supplemented with fetal calf serum (1- to 6-month-old calves) to a final concentration of 1.0%. Added to the medium was 500 international units (IU) benzylpenicillin, 300/xg streptomycin, and 0.25/zg Fungizone per milliliter. The culture medium was buffered with 2.2 mg sodium bicarbonate per milliliter.

Cells

L a b e l i n q of C e l l s

Three- to five-day-old cultures of 112

KB cells derived from a carcinoma of the pharynx were used. During growth the medium was changed every other day and the day the cells were labeled (the day before the experiment). The cells were harvested with ATV, a balanced saline solution of ethylenediaminetetraacetic acid with trypsin.

supplied as sodium chromate in isotonic saline solution. The isotope was added to the target cells in about 1.5 to 2 microcuries per 106 ceils 20 hours before the experiment. After the labeling period, the cells were harvested with A T V and suspended in the experimental medium. The cell suspension was washed and centrifuged (500 g) in medium five times.

Medium

Radioactive chromium (51Cr) was

Experimental Procedures

The procedure is shown schematically in Figure 1. A sample of material (a cylinder 2 mm with a 4.76 mm diameter) to be tested was placed into the center of the culture chamber. Then, 1.0 ml of the labeled cell suspension (2.5 x 10 ~ cells per milliliter) was added to cover the material sample. The materials had hardened for periods of 24 hours, 72 hours, one week, two weeks, one month, two months, three months, five months, six months, and seven months. The chambers were then incubated for

JOURNAL OF ENDODONTICS ] VOL 2, NO 4, APRIL 1976

Table .e Percent release of 51Cr (mean 1 •

Material

72 hr

1 week

2 weok

4-hour cell-material contact SRC 6.3+_0.9 6.8+_0.9 G 66.0_+5.0 19.0+_2.2 N2 22.4-+2.2 13.9+_2.7 R 20.9_+2.8 9.3_+2.6 C 5.3_+1.3 15.8+-4.0

6.9+_1.6 12.6+_1.3 13.7_+2.0 8.2+_1.1 6,4___1.6

6.6+_0.3 14.9_+2.6 14.6__.2.3 8.5• 7.8_+1.3

24-hour SRC G N2 R C

24 hr

1 mo 7.4_+0.7 19.6_+1.8 16.2-+1.9 10.2+1.7 19.0+-3.0

ceil-material contact 21.5+_.0.4 20.5+-0.4 18.8+-0.6 22.5_+0.9 23.0+_1.3 70.0_+1.8 66.2+_2.6 56.7_+4.4 76.4__.2.3 74.4_+2.1 77.5_+1.7 56.7_+5.1 58.5+-1.2 64.0__.2.5 49.9_+1.6 59.0+1.5 31.9_+1.6 31.6_+0.9 38.5_+2.6 38.2_.+2.6 16.8_.+1.7 22.0_+1.9 16.5_+1.8 20.5_+2.0 46.0_+1.8

error).

2 mo

3 mo

5 mo

6 mo

7 mo

5.4+_0.9 12.3• 12.4+_3.2 6.3_+0.7 5.2_+1.0

6.8• 16.1-+2.3 15.6-+2.0 9.9_+0.5 10.9•

9.5--.0.8 11.4-+1.7 29.0-+1.4 11.3_+1.0 19.2+_2.7

8.5_+0.8 11.2+_1.3 31.2_+1.9 13.8_+1.9 10.8+_0.9

7.8+_0.3 18.2-+1.8 21.0• 14.0_+1.9 14.1_+1.0

18.8+_1.4 67.9_+0.8 35.0_+1.3 31.3_+1.8 30.6_+1.3

21.8+-1.7 23.6• 67.2-+1.4 49.8+_2.5 34.1_+2.6 45.2_+3.4 27.3+-0.8 33.4+-1.8 37.2+_2.8 36.0_+4.2

23.2+_1.3 61.6-+2.7 59.3_+2.2 30.9_+1.6 35.7_+_1.9

22.2-+0.9 67.2_+2.2 42.0_+1.9 47.0_+2.9 45.4_+0.6

Note: Six experiments were made for each material and control in each time concentration group. SRC, spontaneous release controls; G, Grossman's sealer; R, Rickert's sealer; C, Cavit.

4-hour and 24-hour periods at 37 C. In all experiments, 0.5-ml samples were withdrawn randomly during dispersion of the labeled cells into the chambers with the material. These samples (r samples) were used as reference points for calculating the nlCr release in the experiments. In each experiment, empty culture chambers were prepared with cell suspension and were used as spontaneous release controls. At the conclusion of the experiment, the medium in the culture chambers was withdrawn, transferred to test tubes, and centrifuged (500 g) for ten minutes. Then 0.5 ml of supernatant (t samples) was transferred to planchets, dried under a heat lamp, and counted for ten minutes in a gamma particle counter. The percentage of 5~Cr release was calculated on .the basis of the total amount of label incorporated in the target cells; that is: release % =51Cr in t samples X 100. 5~Cr in r samples

Approximately 585 experiments were performed in this way. The amount of toxicity displayed by the materials is determined by the amount of cell lysis and subsequent release of radioactive material. Sanderson 5 showed that 100% lysed cells resulted in about 60% to 70% 51Cr release. It is, therefore, reasonable to ,assume that, independent of any changes or variations produced by experimental conditions, any release of 51Cr exceeding 70% would indicate that cytolysis has occurred in aU cells. Consequently, an approximation of degree of toxicity can be determined based on the percent release the materials have displayed. A n y material near the 70% release could be considered highly toxic. Materials near the percent release of the spontaneous release control would be considered mild in toxicity. Materials falling in between these extremes would range from mild to moderate to highly toxic, depending on the percent of 5xCr released.

RESULTS

The results of the cell-material interactions .and subsequent percent release of radioactive material are listed in the Table. The same information is shown graphically in Figures 2 and 3. The four-hour cell-material contact showed a relatively stable picfure. The initial 24-hour responses of Grossman's sealer, N 2 (permanent), and Rickert's sealer were significantly more toxic than the spontaneous release control ( P < 0 . 0 0 1 ) . As the experiments progressed in time the three-, five-, six-, and seven-month samples of N 2 (permanent) proved to be significantly more toxic than the spontaneous release control. This would indicate that N2 (permanent) is slightly more toxic, with some significance at the older ages, than the other materials. Cavit showed somewhat erratic results. The 24-hour cell-material contact showed significant toxicity for all materials when compared to the spontaneous release control ( P < 0 . 0 0 1 ) . 113

JOURNAL OF ENDODONTICS [ VOI. 2, NO 4, APRIL 1976

Fig 2 - - P e r c e n t release over speci/ied time intervals, /our-hour contact.

Cavit initially showed a low toxicity, but this increased at the one-month time period and maintained a high level of toxicity thereafter. Although some fluctuation existed with each material, the toxic effect was present throughout all age levels and thus displayed lasting toxicity. A n overall analysis of the data was done using a general linear hypothesis. A comparison of all materials with the spontaneous release control showed a significant difference in all materials ( P ( 0 . 0 0 1 ) . A n overall ranking of relative toxicity showed Grossman's sealer to be the most toxic, followed by N2 (permanent), Rickert's sealer, and Cavit. Grossman's sealer was significantly more toxic when compared directly with N2 (permanent) ( P ( 0 . 0 0 1 ) , and Rickert's sealer was more toxic than Cavit. An individual comparison of the relative toxicity of all the materials was made by use of the q test of multiple confidence intervals using a comparison of means. These comparisons showed, as can be seen in Figure 2, that the results of the four-hour contact were virtually nonsignificant, 114

Fig 3---Percent release over speci/ied time intervals, 24-hour contact.

with a few exceptions which have been mentioned. A n analysis of the 24-hour cellmaterial contact seemed to correlate with the results seen in the general linear hypothesis analysis. Grossman's sealer was the most toxic, followed by N2 (permanent), Rickert's sealer, and Cavit. There was some overlapping of the various materials, as can be seen in Figure 3. When compared with the spontaneous release controls at the 4-hour and 24-hour levels, the materials show a greater degree of toxicity at the 24hour level. DISCUSSION

A comparison of the toxicity of the sealers used in the current study (except Cavit) with the study of Spangberg a n d . Langeland 6 shows some agreement. Spangberg and Langeland showed that all of these materials were highly toxic, that is, total cell lysis, when the material was set and tested at 1-hour, 4-hour, and 24-hour cell-material contact times. 6 In the current study the 24hour sample (set material) showed total lysis when the cells were in con-

tact with the material for 24 hours. The 4-hour cell-material contact, however, did not display as much toxicity as that reported by Spangberg and Langeland. 6 Only Grossman's sealer displayed total lysis. Rickert's sealer and N2 (permanent) showed approximately 50% lysis. Preliminary examination of the biologic effect of alien substances can be carried out conveniently in tissue or cell culture. The advantages of the in vitro method lies not only in the possibilities of control and observation of reaction, but also in the possibility of standardizing experimental conditions. In establishing a measure of cytotoxicity, Spangbe.rg 4 has used a relatively quantitative in vitro system that has already been proved to be of considerable value in immunologic studies for the determination of cell lysis. His new method for testing dental materials is rapid, highly sensitive, allows adequate cell-material contact, and permits objective quantitation of cell damage with good accuracy. It is apparent, however, that caution must be exercised in attempting to apply the results obtained by .this

JOURNAL OF ENDODONTICS [ VOL 2, NO 4, APRIL 1976

method to the clinical situation. The results of in vitro studies .are quite different from those of in vivo experiments. It is difficult, if not impossible, to extrapolate the results of an in vitro assessment of cytotoxicity involving tissue culture cells to connective ceils in vivo. Rappaport, Lilly, and Kapsimalis 3 showed some degree of correlation between tissue culture testing and in vivo implantation studies. They found that the materials tested were fairly consistent from study to study. Some variables were noted, but for the most part the materials exhibited a fairly uniform response within each test group when all materials were compared. Spangberg 1 tested several materials with the tissue culture test, implantation with polyethylene tubes, and use in dogs' teeth. He said that there is an adequate correlation between in vitro and in vivo tests. Kawahara 2 recognized differences in the results of in vitro and in vivo studies. He further stated that it was, however, necessary to investigate analytically biologic responses to various dental materials by means of in vitro studies before clinical use. He felt with the development of tissue culture techniques in recent years that the in vitro study may become .one of the important methods for the biologic standardization of dental materials. Spangberg's 4 refinements of the technique may well have solidified this prediction. One deviation in the current study from the technique of Spangberg and Langeland 6 involved the material sample size. They spread the material over the entire bottom of the culture chamber. This allowed complete cellmaterial contact. Preliminary experiments showed that the material that was spread over the bottom of the chamber and allowed to set for more than 24 hours produced a cracking,

peeling, dried-out product unsuitable for cell-material contact. To elimina.te this problem, the material was placed in polyethylene tube pieces 2-mm thick with an inside diameter of 4.76 mm. This resulted in material samples with uniform surface area. Kawahara 2 felt that one important problem of the biologic testing of cytotoxicity of dental materials is to control strictly the active surface area of the test piece that is in contact with the culture medium. The use of the tubing satisfies this requirement. The tubing was removed from the test sample before addition of the tissue culture cells. This experiment was conducted over a seven-month period. It was, therefore, necessary to use several batches of cells, since various groups of aged materials were grouped and evaluated at different times. Although the numbers of cells used for each experiment can be approximated, exact numbers cannot be obtained. The cells are quite temperamental and sometimes are difficult to grow. To ensure proper labeling and subsequent satisfactory experimental results, cells that look and act healthy and normal should be used. The strength of the 51Cr is altered by time and great care must be taken in getting the proper amount of labeling into the cell samples. These factors may be responsible for the fluctuation seen in the individual materials in this experiment from the 24-hour to the seven-month time periods. Ideally, the experiment should be conducted at one time on all groups of aged material using a common supply of cells labeled with the same strength of ~lCr. Another variable that must be considered is material degeneration or alteration during aging. All materials were mixed according to the manufacturers' instructions in sterile conditions, placed into the polyethylene tubing, and allowed to set. The ma-

terials were then placed into sterile test tubes with a damp cotton roll and incubated at 37 C. It was thought that these conditions approximated the atmosphere found in the oral cavity. All materials held up well except Cavit, which showed some deterioration, This seemed to be due to its absorption of the moisture content within the test tube. This could somewhat explain Cavit's fluctuant behavior, although it proved to be the least toxic material. Tissue reaction to a material reflects a combination of the toxic effect of the material per se and the response to some of its physical properties. A material that is toxic in vitro can always be expected to cause tissue irritation. Low toxicity in vitro, however, is not equal to low tissue irritation. This depends on variations in resorbability, solubility in tissue fluids, or fragmentation, by which inflammatory reactions may be caused.r, s Materials that have aged up to seven months may show tissue toxicity in vitro. However, in human tissues these materials may have been affected by physiologic processes to the point where they are being contairred by the body and no longer present a problem. Even though one material may prove to be more toxic in vitro than another, it m a y be removed by the body defenses more readily and thus do less damage than one that displayed less toxicity. Because of the highly toxic nature of Grossman's sealer and N2 (permanent), and to a lesser degree Rickert's sealer, it is not recommended that these materials be used as total root canal filling materials. Use of these materials as sealers in conjunction with a solid or semisolid filling material should be done judiciously to prevent extrusion of gross amounts into periapical tissues during obturation of the canal. 115

JOURNAL OF ENDODONTICS

SUMMARY This study was directed toward determining, by in vitro means, the lasting and relative toxicity of four root canal sealing agents. The method used involved radioactively labeled tissue culture cells. These cells were allowed to contact the material samples. This resulted in cell lysis and release of the radioactive particles. The particles were counted in a gamma counter. Comparisons of the amount of release gave an accurate quantitative comparison between the materials tested and, thus, a relative degree of toxicity. Performing the experiment over periods of from 24 hours to 7 months and comparing the amounts of release gave an indication of lasting toxicity. The four sealing materials were Grossman's sealer, N2 (permanent), Rickert's sealer, and Cavit.

CONCLUSIONS The use of radioactive-labeled tissue culture cells as a method of testing dental materials in vitro is rapid and highly sensitive; it allows adequate cell-material contact and permits objective quantitation of cell

116

damage with accuracy. All four sealing materials--Grossman's sealer, N2 (permanent), Rickert's sealer, and Cavit--possess some degree of lasting tissue toxicity. The relative toxicity of these materials in descending degree of toxicity is Grossman's sealer, N2 (permanent), Rickert's sealer, and Cavit. Both Grossman's sealer and N2 (permanent), should be considered highly toxic, with Rickert's displaying moderate toxicity. Cavit should be considered mild to moderate in toxicity. *Walser Drugs, San Marino, Calif. tPatterson Dental Supply Co., Anaheim, Calif. ~tSteri-Kem, Inc., Whittier, Calif. w Dental Products Co., Philadelphia. The author acknowledges the technical assistance of Ms. Robin Hill. The opinions or assertions contained herein are those of the author and are not to be construed as official or as reflecting the views of the Department of the Navy or of the Department of Defense. Dr. Antrim, formerly a graduate student, division of endodontics, School of Dentistry, Loma Linda University, is

VOL 2, NO 4, APRIL 1976

a Commander, DC, USN. Reprint requests should be directed to Cdr. D. D. Antrim, 1615 E Mission Rd, Fallbrook, Calif 92028. RefeFence

et

1. Spangberg, L. Biological effects of root canal filling materials. Odontol Rev 20:123, 133, 283, 289, 427, 1969. 2. Kawahara, H.; Yamagami, A.; and Nakamura, M., Jr. Biological testing of dental materials by means of tissue culture. Int Dent J 18:443 June 1968. 3. Rappaport, H.M.; Lilly, G.E.; and Kapsimalis, P. Toxicity of endodontic filling materials. Oral Surg 18:785 Dec 1964. 4. Spangberg, L. Kinetic and quantitative evaluation of material cytotoxicity in vitro. Oral Surg 35:389 March 1973. 5. Sanderson, A.T. Applications of isoimmune cytolysis using radiolabeled target ceils. Nature 204:250 Oct 1964. 6. Spangberg, L., and Langeland, K. Biologic effects of dental materials. 1. Toxicity of root canal filling materials on HeLa cells .in vitro. Oral Surg 35:402 March 1973. 7. Langeland, K.; Guttuso, J.; Langeland, L.K.; and Tobon, G. Methods in the study of biologic responses to endodontic materials; tissue response to N2. Oral Surg 27:522 April 1969. 8. Spangberg, L. Biological effects of root canal filling materials. 7. Reaction of bony tissue to implanted root canal filling material in guinea pigs. Odontol Tidsk 77:133 April 15, 1969.

Evaluation of the cytotoxicity of root canal sealing agents on tissue culture cells in vitro: Grossman's sealer, N2 (permanent), Rickerts's sealer, and Cavit.

JOURNAL OF ENDODONTICS I VOL 2, NO 4, APRIL 1976 E v a l u a t i o n of t h e c y t o t o x i c i t y of root c a n a l s e a l i n g a g e n t s on...
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