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The effect of endodontic sealers on bone
SCIENTIFIC ARTICLES
The effect of endodontic sealers on bone Jeffery Hoover, DMD, MS; George W. Thoma, MD; and Richard M. Madden, DDS, MS
Zinc o x i d e a n d e u g e n o l (ZOE) a n d Z O E w i t h 6% p a r a f o r m a l d e h y d e w e r e i m p l a n t e d in vivo in the m a n d i b l e s of dogs. Z O E p r o d u c e d a z o n e of c h a n g e s in the b o n e w h i c h a p p e a r e d to i n c r e a s e in size t h r o u g h the f o u r - w e e k t e r m of the s t u d y . Z O E with p a r a f o r m a l d e h y d e p r o d u c e d a z o n e w h i c h was g e n e r a l l y s m a l l e r t h a n that of the Z O E s p e c i m e n s , with p o s s i b l e fixation o f the tissue. T h e g r e a t e s t e x t e n t of b o n e d a m a g e w i t h t h e p a r a f o r m a l d e h y d e c e m e n t a p p e a r e d to o c c u r w i t h i n the first w e e k .
The use of zinc oxide and eugenol (ZOE) root canal sealers has been common since Rickert's sealer was introduced in 1927.' Modifications of the basic ZOE formula have been produced by varying several agents which affect color, consistency, radiopacity, and setting time. As observed by Guttuso, -~such modifications may alter tissue response; nevertheless, most published investigations have studied the commercially available products that contain these agents. The techniques used in such studies have included implantation of hardened sealers into bone a.4 and subcutaneous tissues 5-,~ of experimental animals; injection of freshly mixed, unset sealers into subcutaneous tissues-~.'~ simulated clinical endodontic treatment in teeth of animals, ''15 including deliberate overinstrumentation and overfilllL'2"6; implantation of plastic tubes containing freshly mixed sealer into subcutaneous tissues 17''s and bone ~o-2a;
586
and tissue culture studies using sealers in the freshly mixed ~-23 and hardened ~4 states as well as their soluble diffusion products 2'~ and filtrates of the emulsified materialsY Clinically, endodontic sealer is introduced into the root canal in the freshly mixed, unset state; as the setting reaction is incomplete, it is reasonable to expect that individual components may emanate more readily from a semifluid mixture than from the hardened product. Studies by Molnar "~7and Ranly and others 2s have shown that uncombined eugenol is present in fresh ZOE and may diffuse from it, as m a y formaldehyde from a ZOE and paraformaldehyde mixture. The studies of K a w a h a r a and others, -~2 Spangberg and Langeland, 2:' and Curson and Kirk, 29 using both unset and set materials, indicate, not surprisingly, that the former are more irritating. Thus, criticism of investigations which used only hardened materials has some validity.
The use of a plastic vehicle to implant a semifluid mixture in an experimental site inevitably introduces the effect of the vehicle itself and presents difficulties in tissue preparation for microscopic study. Further, whereas studies of soft tissue are useful, those which investigate the biological effect of endodontic materials on bone provide additional information which is needed for application in clinical practice. This study involved a technique that attempts to standardize tissue and material contact area using constant material volume without a vehicle.
MATERIALS A N D METHODS Nine healthy adult mongrel dogs weighing 16.8 to 28.1 kg were anesthetized with 1.5 ml droperidol and fentanyl (Innovar), 200 mg sodium pentobarbital (Nembutal), and 0.08 mg atropine sulfate administered intravenously; and maintained with
JOURNALOF ENDODONTICS I VOL6, NO 6, JUNE 1980
oxygen, nitrous oxide, and methoxyflurane (Metofane) via endotracheal intubation. The surgical sites were shaved; swabbed with iodine followed by a 70% alcohol solution; and draped. Bilateral 6- to 7-era skin incisions were made at the inferior border of the mandible. Three implantation 9sites, approximately 1.5 mm in diameter and 1 rnm in depth, 1.5- to 2-era apart, were made in bone on the inferolateral aspects using a sterile no. 37 inverted cone but at slowspeed with sterile saline coolant and irrigant. The two test mixtures, consisting of 1 gm zinc oxide, USP, with 0.25 ml eugenol, USP, and 0.94 gm zinc oxide, USP, with 0.06 gm paraformaldehyde and 0.25 ml eugenol, USP, were prepared aseptically. On each side of the mandible, one site was filled with the ZOE mixture; another was filled with the ZOE and 6% paraformaldehyde mixture; and the third site was left empty as a control. The incisions were closed with interrupted no. 4-0 silk sutures for one week. Each animal received ampicillin intramuscularly in a dosage of 3 mg/lb postoperatively. Experimental periods were one week, two weeks, and four weeks, with three dogs in each group. At the end of the experimental period the animals were killed by intravenous injection of 10 ml sodium pentobarbital (Sleepaway). The mandibles were resected and segments containing the three, implantation sites from each side were removed en bloc and placed in 10% Formalin solution (Fig 1). After one week, the individual implantation sites were separated, decalcified in agitated 10% formic acid solution for five to seven days, embedded in paraffin (Paraplast),
the margin and absent in others within a zone of 0 to 100~. In contrast to the one-week ZOE specimens, shrunken, occluded vessels were seen frequently within a radius of 100 to 200/tm. Further from the site, bone appeared normal.
Fig 1-Fixed gross specimen. sectioned, and stained with hematoxylin and eosin for microscopic viewing. RESULTS
Two-week control specimens The experimental sites were empty, without either organized clot or immature connective tissue. Bone, however, appeared viable up to the margins with osteocytes in the lacunas (Fig 2).
One-week control specimens The six experimental surgical defects were filled with blood cells or fibrin clot, and surrounded by viable bone with osteocyte-containing lacunas and patent blood vessels within haversian canals. In two cases there was a 0 to 100/~ zone of necrotic bone in which osteocytes were not evident in lacunas.
Zinc oxide and eugenol These specimens disclosed osteocytes present in lacunas beyond a 100 to 300/x radius from the surgical defects. Similarly, beyond this radius blood vessels appeared patent. In contrast to the one-week ZOE specimens, thrombosed blood vessels as well as vacant lacunas were seen within this zone (Fig 3).
Zinc oxide and eugenol ZOE and 6% paraformaldehyde Although cement was still present in the defects at the end of the experimental period, it was removed during preparation of the specimen; the defects, therefore, appeared empty. Whereas patent blood vessels were seen up to the margin of the sites, lacunas were devoid of osteocytes within a marginal zone of 100 to 300~. At greater distances from the experimental lesions, however, bone appeared normal.
ZOE and 6% paraformaldehyde As in the specimens with ZOE alone, the surgical defects appeared empty. Surrounding the experimental sites, osteocytes were present irregularly in some specimens up to
Osteocytes were present irregularly in lacunas for a radius of 0 to 100/L surrounding the empty experimental defects, and were uniformly present at greater distances. Within a similar radius, blood vessels appeared thrombosed.
Four-week control specimens The surgical defects were filled with newly formed bone and cellular fibrous connective tissue. The new bone at the walls of the defects was largely lined with basophilic osteoblasts. Osteocytes in lacunas adjacent to the defects and patent blood vessels characterized these specimens.
587
JOURNAL OF ENDODONTICS I VOL 6, NO 6, JUNE 1980
Ftg 2--Control, two weeks (II&E, orig mag X 125).
Fig 4 - Z O E and 6% paraformaldehyde, four weeks (tlG_.4E, or(g mag • 125).
were seen frequently within a 100/~ radius (Fig 4). DISCUSSION
Fig 3--ZOE, two weeks (II&E, orig raag • 125).
Zinc oxide and eugenol Osteocytes were absent from lacunas in bone surrounding the defects for a radius of 200 to 400/x. Within this radius, haversian canals also appeared empty or contained shrunken, occluded vessels. Beyond this zone of necrotic bone, lacunas contained osteocytes, and blood vessels were patent.
Z O E a n d 6% p a r a f o r m a l d e h y d e In one case there was a relatively large (100/x) radius of nonviable bone as evidenced by lacunas devoid of osteocytes and empty vascular channels. In other cases, normalappearing osteocytes were present irregularly in lacunas up to the margins. Small, occluded blood vessels
588
As a primary goal of endodontics is the maintenance or regeneration of the bony tissue supporting teeth in a healthy state, the effect of endodontic sealers on bone has clinical significance. Such effect is presumably chemical, being exerted by diffusion of components of the sealer into the periapical region, but it may also occur through the apical foramen when sealer is confined to the pulp canal. Cortical bone pathosis is evaluated by the state of the cells which lie within the bone or which line it, periosteally or endosteally. Changes in the vasculature of the bone also may provide evidence of disease. The bone damage in this study was assessed by the absence of osteocytes from lacunas and haversian canals that contained shrunken, occluded blood vessels, representing thrombosis. The bone necrosis may have occurred by two mechanisms. It is possible that mechanical or thermal trauma during site preparation produced endothelial damage with subsequent vascular thrombosis. Ensuing ischemia could result in
infarction of bone. The chemical effects of ZOE or paraformaldehyde, or both, represent another possible explanation, wherein death of osteocytes and thromboses are directly attributable to the drugs. The small zone of necrosis or its absence in the one-week controls, and its total absence in all two- and four-week controls, indicates that preparation of the surgical defects with rotating bur under irrigation of saline solution causes minimal or reversible damage, or both. As all sites were prepared identically, more extensive changes may be attributed to the respective endodontic sealer. When the prepared sites were filled with ZOE, the zones of damage were uniformly greater than those seen in any of the controls, and were larger in the four-week specimens than in the one- or two-week specimens. In addition, thrombosed blood vessels, not evident at one week, were observed in the two- and four-week specimens. The ZOE appears to exert deleterious effects on bone which, in the quantity used in this study, continue for four weeks. This finding differs from that of Friend and Browne '~ who found that the greatest extent of bone death occurred within two days with zinc oxide and oil of cloves. Although ZOE is known to be an irritant, it might be expected that, in a study of longer term, the zone of necrosis would cease to expand and that some degree of recovery with bone regeneration would follow. The ZOE cement with 6% paraformaldehyde produced seemingly conflicting results, because for all trial periods the adjacent zone of apparent damage contained occluded, shrunken blood vessels within haversian canals, whereas osteocytes were seen in some lacunas up to
JOURNAL OF E N D O D O N T I C S I VOL 6, N O 6, JUNE 1980
the m a r g i n of the e x p e r i m e n t a l site. This result differs from the Z O E specimens in two respects. W i t h ZOE, vascular t h r o m b o s i s seemed to occur subsequent to or c o n c o m i t a n t with loss of osteocytes from lacunas; whereas, with the p a r a f o r m a l d e h y d e sealer, some osteocytes could be seen in lacunas even in the presence of occluded vessels. In a d d i t i o n , the zone of a p p a r e n t d a m a g e with the p a r a f o r m a l d e h y d e c e m e n t was generally smaller t h a n with Z O E alone, for all e x p e r i m e n t a l periods. As 6% p a r a f o r m a l d e h y d e represents the only variable from the Z O E cases, its effects theoretically should account for the difference. It is possible t h a t f o r m a l d e h y d e , a b r e a k d o w n p r o d u c t of p a r a f o r m a l d e hyde, diffusing from the Z O E a n d 6% p a r a f o r m a l d e h y d e sealer, could p r o d u c e e n d o t h e l i a l changes a n d tissue fixation of some osteocytes near the e x p e r i m e n t a l site. D e n a t u r e d proteins, as foreign substances, m i g h t cause a local vasculitis with subsequent thrombosis a n d local ischemia. M e a n w h i l e , fixed osteocytes m a y be preserved in the presence of a local ischemia a n d bone infarction. G u t t u so'-' has suggested t h a t tissue m a y be fixed in the vicinity o f N2. Protein d e n a t u r a t i o n m a y alter diffusion characteristics of the bone such t h a t the Z O E effect m a y occur at smaller distances from the experimental site than for Z O E w i t h o u t paraformaldehyde. This explanation also m a y account for the observation that the zone of d a m a g e did not increase after the first week, in contrast to the Z O E cases. These results differ greatly from those of Friend and Browne, ~9 who f o u n d more e m p ty lacunas a d j a c e n t to N2 t h a n with zinc oxide a n d oil of cloves. Possibly, the fixation p r o p e r t y of p a r a f o r m a l d e h y d e causes a n i n h i b i t i o n of diffu-
sion of o t h e r sealer c o m p o n e n t s with a net decrease in h a r m f u l effect for the short term of four weeks. T h e smaller zone of d a m a g e with the p a r a f o r m a l d e h y d e cement is consistent with such an i n t e r p r e t a t i o n . T h e r e was a noticeable lack of i n f l a m m a t i o n in any of the specimens. As p a r a f o r m a l d e h y d e tissue fixation m a y occur, Guttuso'-' believes t h a t the lack of an acute infiltrate does not preclude the possibility of a severe injury. I n f l a m m a t i o n was observed s u r r o u n d i n g the N2 imp l a n t s of F e l d m a n a n d others :' a n d S p a n g b e r g , 2~ b u t was not a p r o m i nent finding of F r i e n d a n d Browne. ~ T h e absence of such a response confirms that there was m i n i m a l bacterial c o n t a m i n a t i o n , a n d it is conc l u d e d , therefore, that the results of this s t u d y are not due to c o n t a m i n a tion or operative procedures, b u t to the effects of the sealers. A l t h o u g h both sealers induce injury to a d j a c e n t tissue, the relative recovery rates from the effects of Z O E , a n d Z O E and 6% p a r a f o r m a l d e h y d e require study for a longer term. However, as p a r a f o r m a l d e hyde m a y fix a d j a c e n t tissue, it is possible that changes i n d u c e d by the latter are longer lasting a n d that recovery from its effects m a y be slower.
resected, fixed in a 10% F o r m a l i n solution, decalcified, sectioned, a n d stained for light microscopic s t u d y of the response o f the bone to the sealers. T h e Z O E sealer p r o d u c e d a zone of bone d a m a g e s u r r o u n d i n g the i m p l a n t a t i o n site in which blood vessels were t h r o m b o s e d a n d osteocytes were a b s e n t from lacunas, a n d its effect c o n t i n u e d through the fourweek-term of this study. T h e Z O E a n d 6% p a r a f o r m a l d e h y d e sealer p r o d u c e d a zone of bone d a m a g e that was generally smaller in r a d i u s than t h a t of the Z O E specimens a n d which showed vascular thrombosis and osteocytes irregularly present in lacunas. T h e greatest extent of bone d a m a g e with the p a r a f o r m a l d e h y d e cement a p p e a r e d to occur within the first week. This study was supported by the Donald C. Ruthven, Sr., Research Fund. The authors thank Dr. D. M. Ranly, Dr. A. O. Broome, and Ms. Barbara Hellmers for their assistance. Dr. Hoover is assistant professor, department of endodontics; Dr. Thuma is professor and chairman, department of patbology; and Dr. Madden is professor and chairman, department ofendodontics, University of Texas Health Science Center, Dental Branch, Houston, 77025. Requests for reprints should be directed to Dr. Hoover.
References
SUMMARY A n in vivo study was c o n d u c t e d in which two freshly p r e p a r e d endodontic sealers were i m p l a n t e d surgically in the m a n d i b l e s of nine dogs to observe their effects on the h e a l i n g of the bone. T h e materials s t u d i e d were zinc oxide and eugenol ( Z O E ) a n d Z O E with 6% p a r a f o r m a l d e h y d e . After periods of one week, two weeks, a n d four weeks, three dogs were killed, a n d the m a n d i b l e s were
1. Rickert, U.G. My present conceptions for the control of dental foci of infection. Dent Cosmos 69:451-462, 19279 2. Guttusn, J. Histopathok)gic study of rat connective tissue responses to endodontic materials. Oral Surg 16:713-727, 1963. 3. Feldmann, G.; Nyborg, H.; and Conrado, C.A. Tissue reactions to root filling materials. A comparison between implants of the root filling materials N2 and silver in the jaws of rabbits. Odontol Rev 18:387 393, 1967. 4. Hunter, H.A. The elrect of gutta-percha, silver points, and Rickert's root sealer on bone healing. J Can Dent Assoc 23:385-388, 1957. 589
JOURNAL OF ENDODONTICS I VOL 6, NO 6, JUNE 1980
5. Keresztesi, K.; and Kellner, G. The biological effect of root filling materials. Int Dent J 16:222-231, 1966. 6. Mitchell, D.F. Irritational qualities of dental materials. J A D A 59:954-966, 1959. 7. Rappaport, H.M.; I,illy, G.E.; and Kapsimalis, P. Toxicity of endodontic filling materials. Oral Surg 18:785-802, 1964. 8. Rickert, U.G., and Dixon, C.M. The controlling of root surgery. Transactions eighth international dental congress, Paris. section IIIa. 1931, p 15. 9. Stewart, G.G. A comparative study of three root canal sealing agents. Oral Surg 11:1029-1041, 1174-1178, 1958. 10. Roydhouse, R.H., and Weiss, M.E. Tissue reactions to restorative materials. J Dent Res 43:807, 1964. 11. Barker, B.(~., and l_x~kett, B.C. Reaction of dog tissue to immediate root filling with zinc oxide cement and gutta-percha. Aust Dent J 17:1-8, 1~72. 12. Barker, B.C., and Lockett, B.C. Periapical response to N2 and other paraformaldehyde compounds confined within or extruded beyond the apices of dog root canals. Dent Pratt 22:370-379, 1972. 13. Erausquin,J., and Muruzabal, M. Root canal fillings with zinc oxide-eugenol cement in the rat molar. Oral Surg 24(4):547-558, 1967.
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14. Rowe, A.H. Effect of root filling materials on the periapical tissues. Br Dent J 122:98-102, 1967. 15. Snyder, D.E.; Seltzer. S.; and Moodnik, R. Effects of N2 in experimental endodontic therapy. Oral Surg 21:635-656, 1966. 16. Erausquin, J., and Muruzabal, M. Tissue reaction to root canal cements in the rat molar. Oral Surg 26:360-373, 1968. 17. Friend, L.A., and Browne, R.M. Tissue reactions to some root filling materials. Br Dent J 125:291-298, 1968. 18. Langeland, K., and others. Methods in the study of biologic responses to endodontic materials. Tissue response to N2. Oral Surg 27(4):522-542, 1969. 19. Friend, L.A., and Browne, R.M. Tissue reactions to some root filling materials implanted in the bone of rabbits. Arch Oral Biol 14:629-638, 1969. 20. Spangberg, L. Biological effects of root canal filling materials. Reaction of bony tissue to implanted root canal filling materials in guinea pigs. Odontol Tidsk 77:133-159, 1969. 21. Spangherg, I.. Biologic effects of root canal filling materials. The effect on bone tissue of two formaldehyde-containing root canal pastes: N2 and Riebler's paste. Oral Surg 38(6):934-944, 1974.
22. Kawahara, H.; Yamagami, A.; and Nakamura, M. Biological testing of dental materials by m e a n s of tissue culture. Int Dent J 18:443-467, 1968. 23. Spangberg, L., and Langeland, K. Biologic effects of dental materials. Toxicity of root canal filling materials on HeLa cells in vitro. Oral Surg 35:402-414, 1973. 24. Antrim, D.D. Evaluation of the cytotoxicity of root canal sealing agents on tissue culture cells in vitro: Grossman's sealer, N2 (permanent), Rickert's sealer, and Cavit. J Endod 2(4):11 !-116, 1976. 25. Spangberg, L. Biological effects of root canal filling materials. Effect in vitro of water-soluble components of root canal filling material on HeLa ceils. Odontol Rev 20:133145, 1969. 26. Spangberg, L. Biological effects of root canal filling materials. Toxic effect in vitro of root canal filling materials on HeLa cells and h u m a n skin fibroblasts. Odontol Rev 20:427436, 1969. 27. Molnar, E.J. Residual eugenol from zinc oxide eugenol compounds. J Dent Res 46:645-649, 1967. 28. Ranly, D.M.; Montgomery, E.H.; and Pope, H.O. The loss of 3H-formaldehyde from zinc oxide-eugenol c e m e n t - a n in vitro study. J Dent Child 42(2):128-132, 1975. 29. Curson, I., and Kirk, E.E. An assessment of root canal-sealing cements. Oral Surg 26:229-236, 1968.
The effect of endodontic sealers on bone Jeffery Hoover, DMD, MS; George W. Thoma, MD; and Richard M. Madden, DDS, MS
Zinc o x i d e a n d e u g e n o l (ZOE) a n d Z O E w i t h 6% p a r a f o r m a l d e h y d e w e r e i m p l a n t e d in vivo in the m a n d i b l e s of dogs. Z O E p r o d u c e d a z o n e of c h a n g e s in the b o n e w h i c h a p p e a r e d to i n c r e a s e in size t h r o u g h the f o u r - w e e k t e r m of the s t u d y . Z O E with p a r a f o r m a l d e h y d e p r o d u c e d a z o n e w h i c h was g e n e r a l l y s m a l l e r t h a n that of the Z O E s p e c i m e n s , with p o s s i b l e fixation o f the tissue. T h e g r e a t e s t e x t e n t of b o n e d a m a g e w i t h t h e p a r a f o r m a l d e h y d e c e m e n t a p p e a r e d to o c c u r w i t h i n the first w e e k .
The use of zinc oxide and eugenol (ZOE) root canal sealers has been common since Rickert's sealer was introduced in 1927.' Modifications of the basic ZOE formula have been produced by varying several agents which affect color, consistency, radiopacity, and setting time. As observed by Guttuso, -~such modifications may alter tissue response; nevertheless, most published investigations have studied the commercially available products that contain these agents. The techniques used in such studies have included implantation of hardened sealers into bone a.4 and subcutaneous tissues 5-,~ of experimental animals; injection of freshly mixed, unset sealers into subcutaneous tissues-~.'~ simulated clinical endodontic treatment in teeth of animals, ''15 including deliberate overinstrumentation and overfilllL'2"6; implantation of plastic tubes containing freshly mixed sealer into subcutaneous tissues 17''s and bone ~o-2a;
586
and tissue culture studies using sealers in the freshly mixed ~-23 and hardened ~4 states as well as their soluble diffusion products 2'~ and filtrates of the emulsified materialsY Clinically, endodontic sealer is introduced into the root canal in the freshly mixed, unset state; as the setting reaction is incomplete, it is reasonable to expect that individual components may emanate more readily from a semifluid mixture than from the hardened product. Studies by Molnar "~7and Ranly and others 2s have shown that uncombined eugenol is present in fresh ZOE and may diffuse from it, as m a y formaldehyde from a ZOE and paraformaldehyde mixture. The studies of K a w a h a r a and others, -~2 Spangberg and Langeland, 2:' and Curson and Kirk, 29 using both unset and set materials, indicate, not surprisingly, that the former are more irritating. Thus, criticism of investigations which used only hardened materials has some validity.
The use of a plastic vehicle to implant a semifluid mixture in an experimental site inevitably introduces the effect of the vehicle itself and presents difficulties in tissue preparation for microscopic study. Further, whereas studies of soft tissue are useful, those which investigate the biological effect of endodontic materials on bone provide additional information which is needed for application in clinical practice. This study involved a technique that attempts to standardize tissue and material contact area using constant material volume without a vehicle.
MATERIALS A N D METHODS Nine healthy adult mongrel dogs weighing 16.8 to 28.1 kg were anesthetized with 1.5 ml droperidol and fentanyl (Innovar), 200 mg sodium pentobarbital (Nembutal), and 0.08 mg atropine sulfate administered intravenously; and maintained with
JOURNALOF ENDODONTICS I VOL6, NO 6, JUNE 1980
oxygen, nitrous oxide, and methoxyflurane (Metofane) via endotracheal intubation. The surgical sites were shaved; swabbed with iodine followed by a 70% alcohol solution; and draped. Bilateral 6- to 7-era skin incisions were made at the inferior border of the mandible. Three implantation 9sites, approximately 1.5 mm in diameter and 1 rnm in depth, 1.5- to 2-era apart, were made in bone on the inferolateral aspects using a sterile no. 37 inverted cone but at slowspeed with sterile saline coolant and irrigant. The two test mixtures, consisting of 1 gm zinc oxide, USP, with 0.25 ml eugenol, USP, and 0.94 gm zinc oxide, USP, with 0.06 gm paraformaldehyde and 0.25 ml eugenol, USP, were prepared aseptically. On each side of the mandible, one site was filled with the ZOE mixture; another was filled with the ZOE and 6% paraformaldehyde mixture; and the third site was left empty as a control. The incisions were closed with interrupted no. 4-0 silk sutures for one week. Each animal received ampicillin intramuscularly in a dosage of 3 mg/lb postoperatively. Experimental periods were one week, two weeks, and four weeks, with three dogs in each group. At the end of the experimental period the animals were killed by intravenous injection of 10 ml sodium pentobarbital (Sleepaway). The mandibles were resected and segments containing the three, implantation sites from each side were removed en bloc and placed in 10% Formalin solution (Fig 1). After one week, the individual implantation sites were separated, decalcified in agitated 10% formic acid solution for five to seven days, embedded in paraffin (Paraplast),
the margin and absent in others within a zone of 0 to 100~. In contrast to the one-week ZOE specimens, shrunken, occluded vessels were seen frequently within a radius of 100 to 200/tm. Further from the site, bone appeared normal.
Fig 1-Fixed gross specimen. sectioned, and stained with hematoxylin and eosin for microscopic viewing. RESULTS
Two-week control specimens The experimental sites were empty, without either organized clot or immature connective tissue. Bone, however, appeared viable up to the margins with osteocytes in the lacunas (Fig 2).
One-week control specimens The six experimental surgical defects were filled with blood cells or fibrin clot, and surrounded by viable bone with osteocyte-containing lacunas and patent blood vessels within haversian canals. In two cases there was a 0 to 100/~ zone of necrotic bone in which osteocytes were not evident in lacunas.
Zinc oxide and eugenol These specimens disclosed osteocytes present in lacunas beyond a 100 to 300/x radius from the surgical defects. Similarly, beyond this radius blood vessels appeared patent. In contrast to the one-week ZOE specimens, thrombosed blood vessels as well as vacant lacunas were seen within this zone (Fig 3).
Zinc oxide and eugenol ZOE and 6% paraformaldehyde Although cement was still present in the defects at the end of the experimental period, it was removed during preparation of the specimen; the defects, therefore, appeared empty. Whereas patent blood vessels were seen up to the margin of the sites, lacunas were devoid of osteocytes within a marginal zone of 100 to 300~. At greater distances from the experimental lesions, however, bone appeared normal.
ZOE and 6% paraformaldehyde As in the specimens with ZOE alone, the surgical defects appeared empty. Surrounding the experimental sites, osteocytes were present irregularly in some specimens up to
Osteocytes were present irregularly in lacunas for a radius of 0 to 100/L surrounding the empty experimental defects, and were uniformly present at greater distances. Within a similar radius, blood vessels appeared thrombosed.
Four-week control specimens The surgical defects were filled with newly formed bone and cellular fibrous connective tissue. The new bone at the walls of the defects was largely lined with basophilic osteoblasts. Osteocytes in lacunas adjacent to the defects and patent blood vessels characterized these specimens.
587
JOURNAL OF ENDODONTICS I VOL 6, NO 6, JUNE 1980
Ftg 2--Control, two weeks (II&E, orig mag X 125).
Fig 4 - Z O E and 6% paraformaldehyde, four weeks (tlG_.4E, or(g mag • 125).
were seen frequently within a 100/~ radius (Fig 4). DISCUSSION
Fig 3--ZOE, two weeks (II&E, orig raag • 125).
Zinc oxide and eugenol Osteocytes were absent from lacunas in bone surrounding the defects for a radius of 200 to 400/x. Within this radius, haversian canals also appeared empty or contained shrunken, occluded vessels. Beyond this zone of necrotic bone, lacunas contained osteocytes, and blood vessels were patent.
Z O E a n d 6% p a r a f o r m a l d e h y d e In one case there was a relatively large (100/x) radius of nonviable bone as evidenced by lacunas devoid of osteocytes and empty vascular channels. In other cases, normalappearing osteocytes were present irregularly in lacunas up to the margins. Small, occluded blood vessels
588
As a primary goal of endodontics is the maintenance or regeneration of the bony tissue supporting teeth in a healthy state, the effect of endodontic sealers on bone has clinical significance. Such effect is presumably chemical, being exerted by diffusion of components of the sealer into the periapical region, but it may also occur through the apical foramen when sealer is confined to the pulp canal. Cortical bone pathosis is evaluated by the state of the cells which lie within the bone or which line it, periosteally or endosteally. Changes in the vasculature of the bone also may provide evidence of disease. The bone damage in this study was assessed by the absence of osteocytes from lacunas and haversian canals that contained shrunken, occluded blood vessels, representing thrombosis. The bone necrosis may have occurred by two mechanisms. It is possible that mechanical or thermal trauma during site preparation produced endothelial damage with subsequent vascular thrombosis. Ensuing ischemia could result in
infarction of bone. The chemical effects of ZOE or paraformaldehyde, or both, represent another possible explanation, wherein death of osteocytes and thromboses are directly attributable to the drugs. The small zone of necrosis or its absence in the one-week controls, and its total absence in all two- and four-week controls, indicates that preparation of the surgical defects with rotating bur under irrigation of saline solution causes minimal or reversible damage, or both. As all sites were prepared identically, more extensive changes may be attributed to the respective endodontic sealer. When the prepared sites were filled with ZOE, the zones of damage were uniformly greater than those seen in any of the controls, and were larger in the four-week specimens than in the one- or two-week specimens. In addition, thrombosed blood vessels, not evident at one week, were observed in the two- and four-week specimens. The ZOE appears to exert deleterious effects on bone which, in the quantity used in this study, continue for four weeks. This finding differs from that of Friend and Browne '~ who found that the greatest extent of bone death occurred within two days with zinc oxide and oil of cloves. Although ZOE is known to be an irritant, it might be expected that, in a study of longer term, the zone of necrosis would cease to expand and that some degree of recovery with bone regeneration would follow. The ZOE cement with 6% paraformaldehyde produced seemingly conflicting results, because for all trial periods the adjacent zone of apparent damage contained occluded, shrunken blood vessels within haversian canals, whereas osteocytes were seen in some lacunas up to
JOURNAL OF E N D O D O N T I C S I VOL 6, N O 6, JUNE 1980
the m a r g i n of the e x p e r i m e n t a l site. This result differs from the Z O E specimens in two respects. W i t h ZOE, vascular t h r o m b o s i s seemed to occur subsequent to or c o n c o m i t a n t with loss of osteocytes from lacunas; whereas, with the p a r a f o r m a l d e h y d e sealer, some osteocytes could be seen in lacunas even in the presence of occluded vessels. In a d d i t i o n , the zone of a p p a r e n t d a m a g e with the p a r a f o r m a l d e h y d e c e m e n t was generally smaller t h a n with Z O E alone, for all e x p e r i m e n t a l periods. As 6% p a r a f o r m a l d e h y d e represents the only variable from the Z O E cases, its effects theoretically should account for the difference. It is possible t h a t f o r m a l d e h y d e , a b r e a k d o w n p r o d u c t of p a r a f o r m a l d e hyde, diffusing from the Z O E a n d 6% p a r a f o r m a l d e h y d e sealer, could p r o d u c e e n d o t h e l i a l changes a n d tissue fixation of some osteocytes near the e x p e r i m e n t a l site. D e n a t u r e d proteins, as foreign substances, m i g h t cause a local vasculitis with subsequent thrombosis a n d local ischemia. M e a n w h i l e , fixed osteocytes m a y be preserved in the presence of a local ischemia a n d bone infarction. G u t t u so'-' has suggested t h a t tissue m a y be fixed in the vicinity o f N2. Protein d e n a t u r a t i o n m a y alter diffusion characteristics of the bone such t h a t the Z O E effect m a y occur at smaller distances from the experimental site than for Z O E w i t h o u t paraformaldehyde. This explanation also m a y account for the observation that the zone of d a m a g e did not increase after the first week, in contrast to the Z O E cases. These results differ greatly from those of Friend and Browne, ~9 who f o u n d more e m p ty lacunas a d j a c e n t to N2 t h a n with zinc oxide a n d oil of cloves. Possibly, the fixation p r o p e r t y of p a r a f o r m a l d e h y d e causes a n i n h i b i t i o n of diffu-
sion of o t h e r sealer c o m p o n e n t s with a net decrease in h a r m f u l effect for the short term of four weeks. T h e smaller zone of d a m a g e with the p a r a f o r m a l d e h y d e cement is consistent with such an i n t e r p r e t a t i o n . T h e r e was a noticeable lack of i n f l a m m a t i o n in any of the specimens. As p a r a f o r m a l d e h y d e tissue fixation m a y occur, Guttuso'-' believes t h a t the lack of an acute infiltrate does not preclude the possibility of a severe injury. I n f l a m m a t i o n was observed s u r r o u n d i n g the N2 imp l a n t s of F e l d m a n a n d others :' a n d S p a n g b e r g , 2~ b u t was not a p r o m i nent finding of F r i e n d a n d Browne. ~ T h e absence of such a response confirms that there was m i n i m a l bacterial c o n t a m i n a t i o n , a n d it is conc l u d e d , therefore, that the results of this s t u d y are not due to c o n t a m i n a tion or operative procedures, b u t to the effects of the sealers. A l t h o u g h both sealers induce injury to a d j a c e n t tissue, the relative recovery rates from the effects of Z O E , a n d Z O E and 6% p a r a f o r m a l d e h y d e require study for a longer term. However, as p a r a f o r m a l d e hyde m a y fix a d j a c e n t tissue, it is possible that changes i n d u c e d by the latter are longer lasting a n d that recovery from its effects m a y be slower.
resected, fixed in a 10% F o r m a l i n solution, decalcified, sectioned, a n d stained for light microscopic s t u d y of the response o f the bone to the sealers. T h e Z O E sealer p r o d u c e d a zone of bone d a m a g e s u r r o u n d i n g the i m p l a n t a t i o n site in which blood vessels were t h r o m b o s e d a n d osteocytes were a b s e n t from lacunas, a n d its effect c o n t i n u e d through the fourweek-term of this study. T h e Z O E a n d 6% p a r a f o r m a l d e h y d e sealer p r o d u c e d a zone of bone d a m a g e that was generally smaller in r a d i u s than t h a t of the Z O E specimens a n d which showed vascular thrombosis and osteocytes irregularly present in lacunas. T h e greatest extent of bone d a m a g e with the p a r a f o r m a l d e h y d e cement a p p e a r e d to occur within the first week. This study was supported by the Donald C. Ruthven, Sr., Research Fund. The authors thank Dr. D. M. Ranly, Dr. A. O. Broome, and Ms. Barbara Hellmers for their assistance. Dr. Hoover is assistant professor, department of endodontics; Dr. Thuma is professor and chairman, department of patbology; and Dr. Madden is professor and chairman, department ofendodontics, University of Texas Health Science Center, Dental Branch, Houston, 77025. Requests for reprints should be directed to Dr. Hoover.
References
SUMMARY A n in vivo study was c o n d u c t e d in which two freshly p r e p a r e d endodontic sealers were i m p l a n t e d surgically in the m a n d i b l e s of nine dogs to observe their effects on the h e a l i n g of the bone. T h e materials s t u d i e d were zinc oxide and eugenol ( Z O E ) a n d Z O E with 6% p a r a f o r m a l d e h y d e . After periods of one week, two weeks, a n d four weeks, three dogs were killed, a n d the m a n d i b l e s were
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