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Biological evaluation of root canal sealers using in vitro and in vivo methods
JOURNAL OF ENDODONTICS ] VOL 6, NO 10, OCTOBER 1980
Biological evaluation of root canal sealers using in vitro and in vivo methods All Wennberg,
DDS, PhD, M a l m o , S w e d e n
AH26, U of P sealer, Kloroperka N-O, and Riebler's paste were evaluated with regard to cytotoxicity and initial tissue irritation. The cytotoxicity was assessed in vitro by a cell culture technique by which cellmaterial contact was established through a millipore filter. The initial tissue irritation was assessed in vivo using a nonepithelialized tissue surface. A strong correlation was found between the results obtained in vitro and in vivo. The most severe cell and tissue responses were caused by Riebler's paste; U of P sealer and Kloroperka N-O caused a moderate reaction; and the mildest reaction was seen in the tests with AH26. The toxicity of root canal sealers has been studied by several investigators. '-~ In these studies, the materials were tested either freshly mixed or in the set state, usually a f t e r setting periods of 24 or 48 hours. In clinical use, however, the setting of a sealer takes place within the root canal, in contact with living tissue; therefore, the biological characteristics of a sealer while setting are also of interest. A recently reported in vitro method of assessing the cytotoxicity of a setting material, at various phases of the setting process, 7 was used in this study to evaluate the cytotoxicity pattern of root canal sealers during the setting process. In vitro studies of root canal seal-
784
ers have indicated that the high toxic effect some materials show in the freshly mixed state decreases after the setting of the material has taken place? '8 Because findings on in vivo implication of initial toxicity are invalidated by the surgical trauma resulting from conventional implantation methods, initial tissue response has not been fully assessed. However, a recently developed test method overcomes this difficulty. 9 A further aim of this investigation was to study the initial tissue-irritating effects of root canal sealers and make a comparison with the results obtained in the in vitro experiments. MATERIALS AND METHODS AH26, U of P sealer, Kloroperka N-O, and Riebler's paste were mixed according to the manufacturers' directions. In the in vitro experiments, the materials were loaded into glass rings (diameter 7 ram) and evaluated freshly mixed, or after setting periods of 1, 6, 12, or 24 hours at 37 C and 95% relative humidity. In the in vivo experiments, the materials were evaluated in the freshly mixed state. In the controls, isotonic saline solution was substituted for the test materials. Ten tests were run in each experimental group in the in vitro series, and six in the in vivo series.
In vitro experiments. Mouse fibroblast cells (L 929) were used to establish a cell monolayer on millipore filter disks. The filters were placed on an agar medium bed, cell side down, and the test specimens were placed on top of the filters for two hours. The test specimens were then removed, and the cells, still attached to the millipore filters, were incubated for cytochemical demonstration of succinate dehydrogenase (SDH) activity. After an incubation period of three hours at 37 C, the filters were washed in distilled water and air dried. (For details of the technique, see Wennberg, Hasselgren, and TronstadZ.) The filters were examined macroscopically, and any changes in the staining intensity of the cell monolayer at the test specimen contact area were registered. If an unstained zone was present, the diameter was measured. In vivo experiments. Specially designed teflon bodies were implanted in the thigh muscle of three rabbits. Four implants were inserted into each animal. After six weeks the implants were removed, and an intact, nonepithelialized tissue surface was exposed. The shape of the teflon bodies made three indentations in the tissue at each implantation site. The test materials were placed in the indentations and allowed to influence the tissue for 15
JOURNAL OF ENDODONTICS I VOL 6, NO 10, OCTOBER 1980
Table 1 9 Effect of root canal sealers on cell m o n o l a y e r evaluated by staining for e n z y m e activity.
Diameter of unstained zones (mm) minutes. T h e n , a block of tissue surrounding the i d e n t a t i o n s was removed a n d i m m e d i a t e l y frozen in hexane cooled to - 7 5 C with solid CO~. Sectioning was done at - 2 0 C, according to the m e t h o d described by Ullberg, TM a n d after freeze-drying for 48 hours the sections were incubated for histochemical d e m o n s t r a tion of S D H activity. (For details of the technique, see W e n n b e r g , Hasselgren a n d Tronstad~'.) T h e sections were e x a m i n e d microscopically a n d , as an indicator of the degree of tissue reaction, the inhibition of enzyme activity was registered. In the tissue subjacent to each identation, the width of the inhibition zone was measured at a m a g n i f i c a t i o n of 40 times. M e a s u r e m e n t s were carried out on five sections from each identation, and the m e a n value calculated. To evaluate a n y n o n e n z y m a t i c staining reactions, a d d i t i o n a l sections were i n c u b a t e d w i t h o u t substrate. RESULTS bz ~,itro e.~permtents. T h e results are s u m m a r i z e d in T a b l e 1. Outside the contact areas between the test specimens and the filters, the cell monolayer was uniformly stained. Freshlymixed A H 2 6 caused a zone of reduced staining intensity in the cells in all instances. After setting for one hour before a p p l i c a t i o n to the filters, the material caused a circular unstained zone ( d i a m e t e r 10.3 + 1.2 ram). W h e n the setting periods were 6, 12, or 24 hours, no changes in the staining intensity of the cell monolayer were observed (Fig 1). U of P sealer caused a circular unstained zone in the cell m o n o l a y e r in all tests. No difference in width was found between the zone caused by freshly mixed sealer ( d i a m e t e r 9.1 + 0.9 ram), a n d the zones caused
Setting time
Att26"i"
U of P sealer]"
Kloroperka N-6]"
0 hour 1 hour 6 hours 12 hours 24 hours
X~ 10.3 + 1.2 0 0 0
9.1 _+ 0.9 10.2 _+ 1.1 8.4 +_ 0.8 8.5 -+ 0.8 8.6 + 0.7
9.2 + 0.9 xw 0 0 0
Riebler's pastel 20.4 15.1 13.8 11.3 11.3
+ 0.5 _+ 1.8 _+ 2.2 _+ 1.2 + 1.2
9 M e a n ~ standard deviation of 10 tests. t I ) i a m e t e r of test specimen, 7.0 ram. ~Reduced staining inlensit? of cells '.,,as observed in all 10 tesls. w staining intensity of cells was obser'.,ed in four lests; in six tests there was no effect on staining intensity of ceils.
by the m a t e r i a l after setting periods of 1, 6, 12, or 24 hours (P 0.05, S t u d e n t ' s t test) (Fig 1). Freshly mixed K l o r o p e r k a N - O caused a circular unstained zone in the cell monolayer (diameter 9.2 + 0.9 mm). W h e n the m a t e r i a l had set for one hour before a p p l i c a tion to the filters, there was no change in the staining intensity of the cells in six tests; in the r e m a i n i n g four, the staining intensity was reduced. After setting periods of 6, 12, or 24 hours, K l o r o p e r k a N - O h a d no influence on the staining of the cell m o n o l a y e r (Fig 1). Riebler's paste caused a circular u n s t a i n e d zone in the cell m o n o l a y e r in all instances. T h e w i d t h of the
zone r a n g e d from 20.4 _+ 0.5 m m with freshly mixed paste, to 11.3-4- 1.2 m m when the m a t e r i a l had set for 12 or 24 hours before a p p l i c a t i o n to the filters (Fig 1). In vivo experiments. T h e results are s u m m a r i z e d in T a b l e 2. A uniform staining for S D H activity was noticed in the sections from the i n d e n t a t i o n s filled with isotonic saline solution (Fig 2). T h e test materials caused an inhibition of the enzyme activity in the c o n t a c t i n g tissue in all instances. T h e narrowest inhibition zone was caused by A H 2 6 (0.06 _+ 0.03 m m ) (Fig 3). A wider zone was noted in the tests with K l o r o p e r k a N - O (0.15 _+ 0.03 mm), a n d U of P sealer (0.20 _+ 0.06 m m )
Ftg l-Milh[)ore.filters ze,ith cell monolqrer stained for S D H en.:rme activity after being ilyluencd by A1t26 (A ); Riebler5 paste ( R); U of P sealer (U); and Kloroperka N-O ( K ). Left. freshll, mired, all materials reduced or inhibited S D H activi(l, at contact areas. Right, when materials had set for six hours, A t t 2 6 and Kloroperka N-O shozv no e[J'ect on cells. (orzie mag X 1.5). 785
JOURNAL OF ENDODONTICS I VOL 6, NO 10, OCTOBER 1980 Table 2 ~ Effect of root canal sealers on nonepithelialized tissue after 15 m i n contact time evaluated by staining for e n z y m e activity.
Width* of unstained zones (mm)
Material
(Fig 3). The widest inhibition zone was caused by Riebler's paste (1.01 + 0.18 mm). The control sections incubated without substrate showed no sign of staining reactions of nonenzymatic origin. DISCUSSION The root canal sealers studied in this paper were selected to represent four different types of sealers. Riebler's paste contains formaldehyde; AH26 is an epoxy resin; U of P sealer is based on zinc oxide-eugenol; and Kloroperka N-O is based on gutta--percha and natural resins, and mixed with chloroform. The first three materials set by a chemical process; Kloroperka N-O sets by an evaporation process. T h e in vitro study showed differences in the toxicity patterns of the various materials during setting. At all setting times, the formaldehydecontaining Riebler's paste showed the widest zone of SDH inhibition, indicating the greatest degree of cytotoxicity of the sealers tested. Freshly mixed, U of P sealer and
AH26 Kloroperka N-5 U of P sealer Riebler's paste
0.06 0.15 0.20 1.01
+ 0.03 _+ 0.03 -+ 0.06 + 0.18
* M e a n -+ standard deviation of six tests.
Kloroperka N-O caused the same reaction--a zone of inhibited S D H activity in the cell monolayer, slightly wider than the diameter of the test specimens. However, while the zinc oxide-eugenol based U of P sealer caused virtually the same cell response at the various phase s of the setting process, the toxic effect of Kloroperka N-O diminished rather quickly, and after setting for an hour it had practically no effect o n the cells. An interesting toxicity pattern was shown by AH26. Freshly mixed, the material had only slight effect on the cells. After setting for an hour, its cytotoxicity was comparable to that of U of P sealer; and after six hours, AH26 had no effect on the cells. The reason for the increase in cytotoxicity of AH26 one hour after mixing is not clear, but m a y be attributable to the effect of polymerisation products, for instance formaldehyde, apparently released during the setting. ~'~
Fig 2--Top, frozen section stained for SDH enzyme activity. Tissue indentation (I) filled with saline solution for 15 minutes. Staining for SDH activity is seen throughout the tissue (orig mag • 20). Bottom, higher magnification of left photomicrograph (orig mag X 60).
I 9
9
w
9
Fig 3--Frozen sections stained for S D H enzyme activity. Tissue indentations (I) filled with AH26 (left) and U of P sealer (right) for 15 minutes. A zone without staining for SDH activity is seen at tissue-material contact area (orig mag • 90). 786
JOURNAL OF ENDODONTICS ] VOL 6, NO 10, OCTOBER 1980
T h e i n vivo s t u d y was l i m i t e d to the testing o f freshly m i x e d m a t e r i als. T h e widest e n z y m e i n h i b i t i o n zone was caused b y R i e b l e r ' s paste. U o f P sealer a n d K l o r o p e r k a N - O c a u s e d a similar, b u t clearly less severe, r e a c t i o n ; a n d t h e mildest response was o b s e r v e d w h e n A H 2 6 was tested. As c a n be seen, there was a s t r o n g c o r r e l a t i o n b e t w e e n the initial tissue response to the materials, a n d their cytotoxicity i n t h e freshly m i x e d state. P r e v i o u s c o m p a r a t i v e studies have s e l d o m s h o w n a clear r e l a t i o n s h i p b e t w e e n in vitro test methods and conventional implantation t e c h n i q u e s , 12-1" a l t h o u g h a s t r o n g c o r r e l a t i o n has b e e n rep o r t e d ? 7"1s H o w e v e r , in previous i m p l a n t a t i o n studies it has n o t b e e n possible to e v a l u a t e t h e i n i t i t a l tissue response to a m a t e r i a l b e c a u s e of t h e i n f l a m m a t o r y r e a c t i o n c a u s e d by the surgery. 9 Because this s t u d y c o m p a r e d the i n i t i a l tissue r e a c t i o n to a m a t e r i a l w i t h the i n i t i a l cell r e a c t i o n to t h e s a m e m a t e r i a l , we d i d n o t expect to find a s t r o n g c o r r e l a t i o n b e t w e e n the results o f t h e in vitro a n d the i n vivo tests.
c h a n g e s in o x i d o - r e d u c t a s e e n z y m e activity. T h e most severe c y t o t o x i c effect was c a u s e d b y freshly m i x e d R i e b l e r ' s paste. A less severe effect was c a u s e d b y freshly m i x e d U o f P sealer a n d K l o r o p e r k a N - O ; w h e r e a s a m i l d cell response was c a u s e d b y freshly m i x e d A H 2 6 . R i e b l e r ' s p a s t e also h a d t h e strongest cytotoxic effect after the v a r i o u s s e t t i n g periods. U o f P sealer s h o w e d the s a m e toxic effect t h r o u g h o u t the setting process. A f t e r s e t t i n g for o n e hour, the c y t o t o x i c effect o f K l o r o p e r k a N - O h a d d i m i n ished, w h e r e a s the toxicity of A H 2 6 h a d increased. After six h o u r s , n e i t h er o f the m a t e r i a l s h a d a n y effect o n the cells. I n the in vivo e x p e r i m e n t s , the m o s t severe t i s s u e - i r r i t a t i n g effect was c a u s e d b y R i e b l e r ' s paste. U o f P sealer a n d K l o r o p e r k a N - O c a u s e d a similar, b u t clearly less severe, tissue response; a n d the least tissue-irritati n g effect was seen in the tests w i t h AH26. Dr. Wennberg is research associate, department of endodontics, School of Dentistry, University of Lund, Malmo, Sweden. Requests for reprints should be directed to Dr. Wennberg, School of Dentistry, S-21421 Malmo, Sweden.
SUMMARY F o u r root c a n a l sealers ( A H 2 6 , U of P sealer, K l o r o p e r k a N - O , a n d R i e b l e r ' s paste) were tested w i t h r e g a r d to cytotoxicity a n d i n i t i a l tissue i r r i t a t i o n . T h e c y t o t o x i c i t y was assessed b y a cell c u l t u r e t e c h n i q u e by w h i c h c e l l - m a t e r i a l c o n t a c t was established t h r o u g h a m i l l i p o r e filter. T h e m a t e r i a l s were e v a l u a t e d freshly m i x e d a n d after s e t t i n g p e r i o d s of 1, 6, 12, or 24 hours. T h e i n i t i a l tissue i r r i t a t i o n was e v a l u a t e d u s i n g a n o n e p i t h e l i a l i z e d tissue surface. T h e m a t e r i a l s were tested i n the freshly m i x e d state. Both cell a n d tissue responses were assessed b y r e g i s t e r i n g
References l. Maizumi, H., and Sauerwein, E. Die Wirkung verschiedener Vitalerhaltungs- und Wurzelfullmittel auf Gewebekulturen. Dtsch Zahnaerztl Z 17:1628-1635, 1962. 2. Novak, L.; Nemecek, S.; and Puza, V. Untersuchungen uber die kurz- und langfristige Gewebsreaktion auf die Wurzelfullpaste AH 26. Dtsch Zahnaerztl Z 21:584-591, 1966. 3. Kawahara, H.; Yamagami, A.; and Nakamura, M., Jr. Biological testing of dental materials by means of tissue culture. Int Dent J 18:443-467, 1968. 4. Spangberg, L. Biological effects of root canal filling materials. Experimental investigation of the toxicity of root canal filling materials in vitro and in vivo. Odontol Revy 20(suppl 16):1-32, 1969. 5. Spangberg, L., and Langeland, K. Bio-
logic effects of dental materials. 1. Toxicity of root canal filling materials on HeLa cells in vitro. Oral Surg 35:402-414, 1973. 6. Mohammad, A.R., and others. Cytotoxicity evaluation of root canal sealers by the tissue culture--agar overlay technique. Oral Surg 45(5):768-773, 1978. 7. Wennberg, A.; Hasselgren, G.; and Tronstad, L. A method for toxicity screening of biomaterials using cells cultured on millipore filters. J Biomed Mater Res 13(1):109120, 1979. 8. Kataoka, T. Studies on the tissue irritable action of various canal filling materials by means of tissue culture. J Osaka Dent U 6:158-159, 1972. 9. Wennberg, A.; Hasselgren, G.; and Tronstad, L. A method for evaluation of initial tissue response to biomaterials. Acta Odontol Scand 36(2):67-73, 1978. 10. Ullberg, S. Studies on the distribution and fate of S35labeled benzyl-penicillinin the body. Acta Radiol (suppl 118):1-110, 1954. 11. Keresztesi, K., and Kellner, G. Wurzelfullung mit AH 16. Experimentelle und klinische Untersuchungsergebnisse. Osterr Z Stomatol 69:354-368, 1972. 12. Rappaport, H.M.; Lilly, G.E.; and Kapsimalis, P. Toxicity of endodontic filling materials. Oral Surg 18:785-802, 1964. 13. Rosenbluth, S.A., and others. Tissue culture method for screening toxicity of plastic materials to be used in medical practice. J Pharm Sci 54:156-159, 1965. 14. Grasso, P.; Gaydon, J.; and Hendy, R.J. The safety testing of medical plastics. II. An assessment of lysosomal changes as an index of toxicity in cell cultures. Food Cosmet Toxicol 11:255-263, 1973. 15. Wilsnack, R.E.; Meyer, F.J.; and Smith, J.G. Human cell culture toxicity testing of medical devices and correlation to animal tests. Biomater Med Devices Artif Organs 1(3):543-562, 1973. 16. Mjor, I.A.; Hensten-Pettersen, A.; and Skogedal, O. Biologic evaluation of filling materials. A comparison of results using cell culture techniques, implantation tests and pulp studies. Int Dent J 27(2):124-129, 1977. 17. Lawrence, W.H.; Turner, J.E.; and Autian, J. Reevaluation of plastic tubings currently used in medical and paramedical applications. J Biomed Mater Res 3:291-303, 1969. 18. Pelling, D.; Sharratt, M.; and Hardy, J. The safety testing of medical plastics.'I. An assessment of methods. Food Cosmet Toxicol 11:69-83, 1973. 787
Biological evaluation of root canal sealers using in vitro and in vivo methods All Wennberg,
DDS, PhD, M a l m o , S w e d e n
AH26, U of P sealer, Kloroperka N-O, and Riebler's paste were evaluated with regard to cytotoxicity and initial tissue irritation. The cytotoxicity was assessed in vitro by a cell culture technique by which cellmaterial contact was established through a millipore filter. The initial tissue irritation was assessed in vivo using a nonepithelialized tissue surface. A strong correlation was found between the results obtained in vitro and in vivo. The most severe cell and tissue responses were caused by Riebler's paste; U of P sealer and Kloroperka N-O caused a moderate reaction; and the mildest reaction was seen in the tests with AH26. The toxicity of root canal sealers has been studied by several investigators. '-~ In these studies, the materials were tested either freshly mixed or in the set state, usually a f t e r setting periods of 24 or 48 hours. In clinical use, however, the setting of a sealer takes place within the root canal, in contact with living tissue; therefore, the biological characteristics of a sealer while setting are also of interest. A recently reported in vitro method of assessing the cytotoxicity of a setting material, at various phases of the setting process, 7 was used in this study to evaluate the cytotoxicity pattern of root canal sealers during the setting process. In vitro studies of root canal seal-
784
ers have indicated that the high toxic effect some materials show in the freshly mixed state decreases after the setting of the material has taken place? '8 Because findings on in vivo implication of initial toxicity are invalidated by the surgical trauma resulting from conventional implantation methods, initial tissue response has not been fully assessed. However, a recently developed test method overcomes this difficulty. 9 A further aim of this investigation was to study the initial tissue-irritating effects of root canal sealers and make a comparison with the results obtained in the in vitro experiments. MATERIALS AND METHODS AH26, U of P sealer, Kloroperka N-O, and Riebler's paste were mixed according to the manufacturers' directions. In the in vitro experiments, the materials were loaded into glass rings (diameter 7 ram) and evaluated freshly mixed, or after setting periods of 1, 6, 12, or 24 hours at 37 C and 95% relative humidity. In the in vivo experiments, the materials were evaluated in the freshly mixed state. In the controls, isotonic saline solution was substituted for the test materials. Ten tests were run in each experimental group in the in vitro series, and six in the in vivo series.
In vitro experiments. Mouse fibroblast cells (L 929) were used to establish a cell monolayer on millipore filter disks. The filters were placed on an agar medium bed, cell side down, and the test specimens were placed on top of the filters for two hours. The test specimens were then removed, and the cells, still attached to the millipore filters, were incubated for cytochemical demonstration of succinate dehydrogenase (SDH) activity. After an incubation period of three hours at 37 C, the filters were washed in distilled water and air dried. (For details of the technique, see Wennberg, Hasselgren, and TronstadZ.) The filters were examined macroscopically, and any changes in the staining intensity of the cell monolayer at the test specimen contact area were registered. If an unstained zone was present, the diameter was measured. In vivo experiments. Specially designed teflon bodies were implanted in the thigh muscle of three rabbits. Four implants were inserted into each animal. After six weeks the implants were removed, and an intact, nonepithelialized tissue surface was exposed. The shape of the teflon bodies made three indentations in the tissue at each implantation site. The test materials were placed in the indentations and allowed to influence the tissue for 15
JOURNAL OF ENDODONTICS I VOL 6, NO 10, OCTOBER 1980
Table 1 9 Effect of root canal sealers on cell m o n o l a y e r evaluated by staining for e n z y m e activity.
Diameter of unstained zones (mm) minutes. T h e n , a block of tissue surrounding the i d e n t a t i o n s was removed a n d i m m e d i a t e l y frozen in hexane cooled to - 7 5 C with solid CO~. Sectioning was done at - 2 0 C, according to the m e t h o d described by Ullberg, TM a n d after freeze-drying for 48 hours the sections were incubated for histochemical d e m o n s t r a tion of S D H activity. (For details of the technique, see W e n n b e r g , Hasselgren a n d Tronstad~'.) T h e sections were e x a m i n e d microscopically a n d , as an indicator of the degree of tissue reaction, the inhibition of enzyme activity was registered. In the tissue subjacent to each identation, the width of the inhibition zone was measured at a m a g n i f i c a t i o n of 40 times. M e a s u r e m e n t s were carried out on five sections from each identation, and the m e a n value calculated. To evaluate a n y n o n e n z y m a t i c staining reactions, a d d i t i o n a l sections were i n c u b a t e d w i t h o u t substrate. RESULTS bz ~,itro e.~permtents. T h e results are s u m m a r i z e d in T a b l e 1. Outside the contact areas between the test specimens and the filters, the cell monolayer was uniformly stained. Freshlymixed A H 2 6 caused a zone of reduced staining intensity in the cells in all instances. After setting for one hour before a p p l i c a t i o n to the filters, the material caused a circular unstained zone ( d i a m e t e r 10.3 + 1.2 ram). W h e n the setting periods were 6, 12, or 24 hours, no changes in the staining intensity of the cell monolayer were observed (Fig 1). U of P sealer caused a circular unstained zone in the cell m o n o l a y e r in all tests. No difference in width was found between the zone caused by freshly mixed sealer ( d i a m e t e r 9.1 + 0.9 ram), a n d the zones caused
Setting time
Att26"i"
U of P sealer]"
Kloroperka N-6]"
0 hour 1 hour 6 hours 12 hours 24 hours
X~ 10.3 + 1.2 0 0 0
9.1 _+ 0.9 10.2 _+ 1.1 8.4 +_ 0.8 8.5 -+ 0.8 8.6 + 0.7
9.2 + 0.9 xw 0 0 0
Riebler's pastel 20.4 15.1 13.8 11.3 11.3
+ 0.5 _+ 1.8 _+ 2.2 _+ 1.2 + 1.2
9 M e a n ~ standard deviation of 10 tests. t I ) i a m e t e r of test specimen, 7.0 ram. ~Reduced staining inlensit? of cells '.,,as observed in all 10 tesls. w staining intensity of cells was obser'.,ed in four lests; in six tests there was no effect on staining intensity of ceils.
by the m a t e r i a l after setting periods of 1, 6, 12, or 24 hours (P 0.05, S t u d e n t ' s t test) (Fig 1). Freshly mixed K l o r o p e r k a N - O caused a circular unstained zone in the cell monolayer (diameter 9.2 + 0.9 mm). W h e n the m a t e r i a l had set for one hour before a p p l i c a tion to the filters, there was no change in the staining intensity of the cells in six tests; in the r e m a i n i n g four, the staining intensity was reduced. After setting periods of 6, 12, or 24 hours, K l o r o p e r k a N - O h a d no influence on the staining of the cell m o n o l a y e r (Fig 1). Riebler's paste caused a circular u n s t a i n e d zone in the cell m o n o l a y e r in all instances. T h e w i d t h of the
zone r a n g e d from 20.4 _+ 0.5 m m with freshly mixed paste, to 11.3-4- 1.2 m m when the m a t e r i a l had set for 12 or 24 hours before a p p l i c a t i o n to the filters (Fig 1). In vivo experiments. T h e results are s u m m a r i z e d in T a b l e 2. A uniform staining for S D H activity was noticed in the sections from the i n d e n t a t i o n s filled with isotonic saline solution (Fig 2). T h e test materials caused an inhibition of the enzyme activity in the c o n t a c t i n g tissue in all instances. T h e narrowest inhibition zone was caused by A H 2 6 (0.06 _+ 0.03 m m ) (Fig 3). A wider zone was noted in the tests with K l o r o p e r k a N - O (0.15 _+ 0.03 mm), a n d U of P sealer (0.20 _+ 0.06 m m )
Ftg l-Milh[)ore.filters ze,ith cell monolqrer stained for S D H en.:rme activity after being ilyluencd by A1t26 (A ); Riebler5 paste ( R); U of P sealer (U); and Kloroperka N-O ( K ). Left. freshll, mired, all materials reduced or inhibited S D H activi(l, at contact areas. Right, when materials had set for six hours, A t t 2 6 and Kloroperka N-O shozv no e[J'ect on cells. (orzie mag X 1.5). 785
JOURNAL OF ENDODONTICS I VOL 6, NO 10, OCTOBER 1980 Table 2 ~ Effect of root canal sealers on nonepithelialized tissue after 15 m i n contact time evaluated by staining for e n z y m e activity.
Width* of unstained zones (mm)
Material
(Fig 3). The widest inhibition zone was caused by Riebler's paste (1.01 + 0.18 mm). The control sections incubated without substrate showed no sign of staining reactions of nonenzymatic origin. DISCUSSION The root canal sealers studied in this paper were selected to represent four different types of sealers. Riebler's paste contains formaldehyde; AH26 is an epoxy resin; U of P sealer is based on zinc oxide-eugenol; and Kloroperka N-O is based on gutta--percha and natural resins, and mixed with chloroform. The first three materials set by a chemical process; Kloroperka N-O sets by an evaporation process. T h e in vitro study showed differences in the toxicity patterns of the various materials during setting. At all setting times, the formaldehydecontaining Riebler's paste showed the widest zone of SDH inhibition, indicating the greatest degree of cytotoxicity of the sealers tested. Freshly mixed, U of P sealer and
AH26 Kloroperka N-5 U of P sealer Riebler's paste
0.06 0.15 0.20 1.01
+ 0.03 _+ 0.03 -+ 0.06 + 0.18
* M e a n -+ standard deviation of six tests.
Kloroperka N-O caused the same reaction--a zone of inhibited S D H activity in the cell monolayer, slightly wider than the diameter of the test specimens. However, while the zinc oxide-eugenol based U of P sealer caused virtually the same cell response at the various phase s of the setting process, the toxic effect of Kloroperka N-O diminished rather quickly, and after setting for an hour it had practically no effect o n the cells. An interesting toxicity pattern was shown by AH26. Freshly mixed, the material had only slight effect on the cells. After setting for an hour, its cytotoxicity was comparable to that of U of P sealer; and after six hours, AH26 had no effect on the cells. The reason for the increase in cytotoxicity of AH26 one hour after mixing is not clear, but m a y be attributable to the effect of polymerisation products, for instance formaldehyde, apparently released during the setting. ~'~
Fig 2--Top, frozen section stained for SDH enzyme activity. Tissue indentation (I) filled with saline solution for 15 minutes. Staining for SDH activity is seen throughout the tissue (orig mag • 20). Bottom, higher magnification of left photomicrograph (orig mag X 60).
I 9
9
w
9
Fig 3--Frozen sections stained for S D H enzyme activity. Tissue indentations (I) filled with AH26 (left) and U of P sealer (right) for 15 minutes. A zone without staining for SDH activity is seen at tissue-material contact area (orig mag • 90). 786
JOURNAL OF ENDODONTICS ] VOL 6, NO 10, OCTOBER 1980
T h e i n vivo s t u d y was l i m i t e d to the testing o f freshly m i x e d m a t e r i als. T h e widest e n z y m e i n h i b i t i o n zone was caused b y R i e b l e r ' s paste. U o f P sealer a n d K l o r o p e r k a N - O c a u s e d a similar, b u t clearly less severe, r e a c t i o n ; a n d t h e mildest response was o b s e r v e d w h e n A H 2 6 was tested. As c a n be seen, there was a s t r o n g c o r r e l a t i o n b e t w e e n the initial tissue response to the materials, a n d their cytotoxicity i n t h e freshly m i x e d state. P r e v i o u s c o m p a r a t i v e studies have s e l d o m s h o w n a clear r e l a t i o n s h i p b e t w e e n in vitro test methods and conventional implantation t e c h n i q u e s , 12-1" a l t h o u g h a s t r o n g c o r r e l a t i o n has b e e n rep o r t e d ? 7"1s H o w e v e r , in previous i m p l a n t a t i o n studies it has n o t b e e n possible to e v a l u a t e t h e i n i t i t a l tissue response to a m a t e r i a l b e c a u s e of t h e i n f l a m m a t o r y r e a c t i o n c a u s e d by the surgery. 9 Because this s t u d y c o m p a r e d the i n i t i a l tissue r e a c t i o n to a m a t e r i a l w i t h the i n i t i a l cell r e a c t i o n to t h e s a m e m a t e r i a l , we d i d n o t expect to find a s t r o n g c o r r e l a t i o n b e t w e e n the results o f t h e in vitro a n d the i n vivo tests.
c h a n g e s in o x i d o - r e d u c t a s e e n z y m e activity. T h e most severe c y t o t o x i c effect was c a u s e d b y freshly m i x e d R i e b l e r ' s paste. A less severe effect was c a u s e d b y freshly m i x e d U o f P sealer a n d K l o r o p e r k a N - O ; w h e r e a s a m i l d cell response was c a u s e d b y freshly m i x e d A H 2 6 . R i e b l e r ' s p a s t e also h a d t h e strongest cytotoxic effect after the v a r i o u s s e t t i n g periods. U o f P sealer s h o w e d the s a m e toxic effect t h r o u g h o u t the setting process. A f t e r s e t t i n g for o n e hour, the c y t o t o x i c effect o f K l o r o p e r k a N - O h a d d i m i n ished, w h e r e a s the toxicity of A H 2 6 h a d increased. After six h o u r s , n e i t h er o f the m a t e r i a l s h a d a n y effect o n the cells. I n the in vivo e x p e r i m e n t s , the m o s t severe t i s s u e - i r r i t a t i n g effect was c a u s e d b y R i e b l e r ' s paste. U o f P sealer a n d K l o r o p e r k a N - O c a u s e d a similar, b u t clearly less severe, tissue response; a n d the least tissue-irritati n g effect was seen in the tests w i t h AH26. Dr. Wennberg is research associate, department of endodontics, School of Dentistry, University of Lund, Malmo, Sweden. Requests for reprints should be directed to Dr. Wennberg, School of Dentistry, S-21421 Malmo, Sweden.
SUMMARY F o u r root c a n a l sealers ( A H 2 6 , U of P sealer, K l o r o p e r k a N - O , a n d R i e b l e r ' s paste) were tested w i t h r e g a r d to cytotoxicity a n d i n i t i a l tissue i r r i t a t i o n . T h e c y t o t o x i c i t y was assessed b y a cell c u l t u r e t e c h n i q u e by w h i c h c e l l - m a t e r i a l c o n t a c t was established t h r o u g h a m i l l i p o r e filter. T h e m a t e r i a l s were e v a l u a t e d freshly m i x e d a n d after s e t t i n g p e r i o d s of 1, 6, 12, or 24 hours. T h e i n i t i a l tissue i r r i t a t i o n was e v a l u a t e d u s i n g a n o n e p i t h e l i a l i z e d tissue surface. T h e m a t e r i a l s were tested i n the freshly m i x e d state. Both cell a n d tissue responses were assessed b y r e g i s t e r i n g
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