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Biocompatibility of two calcium hydroxide-based endodontic sealers: A quantitative study in the subcutaneous connective tissue of the rat
0099-2399/88/1405-0229/$02.00/0 JOURNALOF ENDODONTICS Copyright 9 1988 by The American Associationof Endodontists
Printed in U.S.A. VOL. 14, NO. 5, MAY 1988
Biocompatibility of Two Calcium Hydroxide-based Endodontic Sealers: A Quantitative Study in the Subcutaneous Connective Tissue of the Rat Osvaldo Zmener, DDS, Dr. Odont., Maria B. Guglielmotti, DDS, Dr. Odont., and Romulo L. Cabrini, MD
In this study, the biocompatibility of two calcium hydroxide-based endodontic sealers was investigated. Silicone tubes containing freshly mixed Sealapex or CRCS were implanted in the dorsal subcutaneous connective tissue of the rat. Equal size solid silicone rods were also implanted and used as controis. The tissue reaction to test and control materials was histometrically and quantitatively analyzed under light microscopy. After 7, 30, and 90 days of implantation, different grades of tissue reaction to the tested materials were recorded at the end of the tubes. A granulomatous tissue containing foreign body giant cells and macrophages with engulfed material in their cytoplasm as well as many fibroblasts and vessels was initially observed in contact with Sealapex. This reaction increased progressively at the 30- and 90-day observation period. An acute inflammation was detected in tissues in contact with CRCS. However, the severity of this reaction decreased with time and it seemed to be resolved at the 90-day observation period. Taking into account the limitations of the experimental model used in this study, we consider that more extensive experiences will be necessary prior to extrapolating these findings to the actual clinical situation.
able calcium hydroxide-based root canal sealers, i.e. Sealapex (Kerr, Romulus, MI) and CRCS (Hygenic Co., Akron, OH), were analyzed from the point of view of some of their physical characteristics (16, 17). Information about their biological properties, however, is scarce (18, 19). Consequently, we feel that their implantation in small animals will constitute a suitable source of initial information on the biocompatibility of these materials. The purpose of this study was to compare quantitatively the tissue response to both Sealapex and CRCS when they were implanted in the subcutaneous connective tissue of the rat.
MATERIALS AND METHODS In this study, autoclaved silicone tubes (Raholin SRL; V. Madero, Buenos Aires, Argentina), 10-mm long and 1 m m in internal diameter, were filled flush with freshly mixed Sealapex or CRCS. They were immediately implanted into the subcutaneous connective tissue of 30 white male Wistar rats that weighed 90 g each. According to the manufacturer, Sealapex contains calcium hydroxide, barium sulfate, zinc oxide, titanium dioxide, and zinc stearate in a matrix blend of poly(methylene methyl salicylate) resin, isobutyl salicylate, methyl salicylate, ethyl toluene sulfonamide isomers, and pigments. The formulation of CRCS is not totally available; however, it is believed to contain a calcium hydroxide and zinc oxide-eugenol mixture in which approximately 40 to 50% of the eugenol content is replaced by eucaliptol. In addition, solid silicone rods, equal in size, were also implanted and used as inert controls. The implantation procedures were as follows: The animals were anesthetized by intraperitoneal administration of sodium pentobarbital (0.025 g per 1,000g wt); and the dorsal skin was shaved and disinfected with 5% iodine in alcohol. Under aseptic conditions an incision approximately 15-mm long was made through the skin using a scalpel and three separate subcutaneous pockets were prepared by blunt dissection to a depth of 20 mm. Then, three implants (one of each experimental material and one control) were carefully placed into the pockets prepared in each rat. Care was taken to prevent smearing of the test materials on the outside areas of the tubes. After implantation, the wounds were sutured.
Calcium hydroxide has been widely used in endodontics and the evidence shows that it does stimulate or promote hard tissue repair in many instances (1-6). Interestingly, it appears that a direct contact between calcium hydroxide and the tissues is necessary for an adequate inductive action (7) but the actual mechanisms by which calcium hydroxide may act on the surrounding tissues still remain unclear (8-I 1). Despite apical closure by hard tissue deposition found under different experimental conditions when calcium hydroxide was used as a root canal filling (4-6, 12), some complications have been observed. This occurred in some cases in which this material was in direct contact with periapical tissues ( 13-15). Recently, two new commercially avail-
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The animals were killed in groups of 10 each after 7, 30, and 90 days by ether suffocation. The implants along with the surrounding tissues were removed in rectangular blocks and then fixed in 10% formalin solution. After fixation the tissues were processed for paraffin embedding and then longitudinally sectioned through the implants. Serial sections approximately 7-#m thick were obtained from each specimen and then stained with hematoxylin and eosin. In order to obtain a quantitative estimation of the tissue reaction around both ends of the tubes, three sections belonging to the central areas of each specimen were examined. Histometric measurements based on standard stereological methods (20-24) were made on tracings obtained from projections of the histological sections. These projections were made using a projection head mounted on a standard light microscope (Carl Zeiss, Oberkochen, West Germany) at a magnification of x25. The areas of tissue reaction to the implanted materials were measured over the image of each tracing using a transparent celluloid grid marked with equidistant dots every 7 mm. The dots within the outlines of reactive areas and those of the total field were counted and then the areas of reaction were calculated. These values represent the volume proportion of the areas measured on the tracings (24). The values obtained from both ends of each tube were similar in each individual case for all tested materials and for all of the observation periods. For this reason the area of reaction which was finally evaluated in each of the three sections from each individual specimen was randomly selected. Then, a mean value from these sections was calculated. Final values were obtained by transferring the results of the measurements performed on the tracings to the actual size of tissue reaction observed on the histological slides, which were expressed in square millimeters. For cell quantitation, the total number of inflammatory cells, i.e. lymphocytes, polymorphonuclear leukocytes, macrophages, multinucleated giant cells, plasmocytes, and fibroblasts, which were observed in the selected areas of tissue reaction were recorded by counting cells in three separate fields of vision (7.5 mm 2 each) at x400. They were located and spaced at regular intervals within the outlines of these areas. The total number of cells observed in the same sections used for the histometric measurement of the reactive areas of each specimen was counted twice by two different examiners. Since no substantial differences between the two readings were observed, a mean value for these records was obtained. Final results and their standard deviations were derived from the mean values obtained from the total number of specimens of each material examined in each observation period. The data obtained from the measurements of the areas of tissue reaction and from the cell counts were subjected to an analysis of variance and to Duncan's multiple range test. Because of problems inherent to laboratory procedures, some specimens from each observation period and for each material had to be excluded from the study. In these instances, final values were obtained from fewer readings. Definitive data from the number of specimens studied in each observation period are shown in Table 1.
RESULTS Macroscopic examination showed that wound healing was satisfactory in all observation periods. Histological evaluation
of the implants revealed that they were surrounded by fibrous connective encapsulation of irregular thickness. It could be easily distinguished from the reaction of the tissues in contact with the experimental materials at both ends of the tubes. The quantitative measurements of the areas of necrosis or inflammatory responses are summarized in Table 2. The results of cell quantitation are shown in Tables 3 to 5. At the 7-day observation the extent of tissue reaction to Sealapex and CRCS was similar. In this respect no significant differences were found. However, these results strongly differed from those obtained from the controls which were much less. At the ends of the tubes filled with Sealapex and CRCS, some amounts of necrotic tissue as well as the presence of macrophages were observed in direct contact with these materials. Whereas a concentration of lymphocytes and polymorphonuclear leukocytes were present only in contact with CRCS (Table 3). Some foreign body giant cells with engulfed particles of the test materials were also seen in the areas surrounding the ends of the tubes filled with Sealapex and CRCS (Fig. l, A to E). In addition, a fibroblastic proliferation was also observed around both of these materials. In general, a thin fibrous connective tissue composed of young fibroblasts and few inflammatory cells was observed in contact with the controls (Fig. 1F). At the 30-day observation, significant differences between the areas of tissue reaction to Sealapex and CRCS as well as between these two materials and the controls were observed. As is noted in Table 2, the values obtained from the measurements made on CRCS and the control specimen projections were less than those obtained from Sealapex. In the areas in contact with Sealapex, some fragments of this material were surrounded by granulomatous tissue that appeared to be invaginated into the lumen of the tubes (Fig. 2A). Macrophages and foreign body giant cells containing many dark particles in their cytoplasm were seen in contact or at some distance from the end of the tubes (Fig. 2B). In contact with CRCS, the inflammatory reaction subsisted (Fig. 3) but it was much less than those observed at the 7-day period (Table 4). A consistent fibroblastic proliferation at the ends of the specimens was also detected. In contact with the
TABLE 1. Observation periods and number of analyzed
specimens Days
Sealapex CRCS Control
7
30
90
9 8 10
9 9 9
10 10 8
TABLE 2. Areas of tissue reaction to the implanted materials*
Days 7 30 90
Sealapex
CRCS
Control
(mm2)
p
0.66 ___0.311" 0.82 -+ 0.36 1.23 _ 0.54
0.68 - 0.281" 0.63 _ 0.25 0.30 _+ 0.12
0.31 _ 0.08 0.33 _ 0.16 0.00 - 0.00
<0,05 <0.05 <0.01
(mm2)
(mm2)
*The results of the histometric measurements were expressedas the actual size in square millimeters of tissue reaction obeervedon the histological slides. 1"No significant differences were observed between Sealapex and CRCS speomens (+SD).
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TABLE 3. Differential cell counts, 7 days after implantation* Cell Type
Lymphocytes Polymorphonuclear leukocytes Macrophages Giant cells Piasmocytes Fibroblasts
Sealapex
3.0 2.0 22.0 3.5 0.0 51.0
-+ 0.7 _+ 1.5 _+ 7.0 -+ 1.3 + 0.0 -+ 25.5
CRCS
Control
p
19.0 _+ 8.0 24.0 _+ 20.0 30.0 +- 14.0 1.0 _+ 2.0 0.4 _+ 0.5 45.0 + 18.0
9.0 -+ 6.0 6.0 _+ 5.0 9.0 -+ 6.0 0.0 -+ 0.0 0.0 -+ 0.0 58.0 -+ 23.0
<0.01 <0.01 <0.01 >0.05 >0.05
9The resultsof the cellcountswereexpressedas the meanvalueobtained from the total numberof cells whichwere countedin all specimensof eachmaterial(+ SD).
controls, a thin fibrous capsule containing some scattered inflammatory cells could be observed. At the 90-day observation, the specimens of Sealapex showed the greatest areas of tissue reaction whereas the values obtained from the measurements of CRCS and control specimens were markedly reduced (Table 2). The results of cell quantitation (Table 5) showed that macrophages and foreign body giant cells with important amounts of engulfed material persist at the ends of the tubes filled with Sealapex. The majority of these specimens showed some extent of granulomatous tissue invagination into the lumen of the tubes (Fig. 4A). In addition, fragments of the filling material that appeared to be surrounded by macrophages and multinucleated giant cells were detected in the invaginated tissues, especially in the areas of the specimen end (Fig. 4B). At these sites the granulomatous tissues were generally walled off by fibrous encapsulation. Interestingly, the accumulation of acute inflammatory cells that was noted in the areas of contact with CRCS decreased as the observation period increased. In spite of these findings, some lymphocyte concentration persisted (Fig. 5,4). On the other hand, a consistent fibrous connective tissue capsule with the presence of only occasional lymphocytes was detected in contact with the controls (Fig. 5B).
DISCUSSION In conjunction with previous reports (25, 26), our results showed that the histological response to endodontic materials implanted in the subcutaneous connective tissue of the rat could be used as a preliminary source of information on their biocompatibility. The introduction of experimental materials into tubes is amply recognized as a suitable method for the implantation of these materials. This methodology allows a precise control of the amount of material that will be in contact with the tissues (27). However, in spite of handling all implants carefully to prevent smearing of the test materials on the outside of the tubes, our results revealed that both of these materials, i.e. Sealapex and CRCS, easily flowed out of the tubes or broke up. In this respect, particles of these materials were seen in the tissues surrounding the end of the tubes and this could be the main cause of the extent of the tissue reaction during the short observation periods. Different materials such as polyethylene or Teflon are normally used for implantation purposes. Since these materials show no irritation properties on the surrounding tissues (28, 29), the reactions at both ends of the implants are normally related to the toxicity of the material that is contained in the lumen of the tubes. In this study, we used silicone tubes in order to eliminate some methodological problems. For example, the tissues in
contact with polyethylene or Teflon implants have the tendency to adhere to the implant surfaces. Therefore, the interface adjacent to these materials is frequently destroyed or removed during the laboratory procedures. In this study, the silicone tubes or the solid silicone rods were found to be surrounded by a thin fibrous connective capsule without signs of adherence to the material surface. In addition, there was no evidence of the presence of foreign body giant cells in contact with the lateral walls of the tubes or at the ends of the solid silicone rods. These cells were occasionally seen in contact with polyethylene or Teflon as reported elsewhere (30-32). The use of quantitative methods for the evaluation of the tissue reaction to freshly prepared materials has been subjected to criticism (33). However, we consider the use of histometric procedures to quantificate some of the histological parameters related to the toxicity of endodontic materials to be an objective method of evaluation. There were no evident differences in simple observation which could be detected. The results of the histometric evaluation showed that the greatest areas of tissue reaction were seen in contact with Sealapex. At the 7-day observation no significant differences between this material and CRCS were observed, but the amount of tissue reaction to Sealapex increased after 30 and 90 days. In the majority of these cases, Sealapex was fragmented or it was lost from the inside of the tubes and the test material was progressively replaced by invagination of the granulomatous tissues which were initially located at the external areas of the end of the implants (Figs. 2A and 4A). Consequently, significant areas of tissue reaction measured in surface units resulted from the analysis of Sealapex specimens at the 30- and 90-day observations. In this respect the statistical analysis of the data showed that the differences observed between both Sealapex and CRCS or between these two materials and the controls were significant (p < 0.01), especially in the late observation period. The results of cell quantitation revealed that a granulomatous tissue composed by macrophages and foreign body giant cells with engulfed material in their cytoplasm as well as a high number of fibroblasts and vessels were observed in contact with Sealapex, and that this reaction increased severely at the 30- and 90-day observation periods. The presence of particles of the material in macrophages and foreign body giant cells or in vessels in areas located at some distance from the end of the tubes revealed transportation of the filling material to sites far away from the implants. This was probably due to some of the components of Sealapex as titanium dioxide which can be easily dispersed into the surrounding tissues, provoking an intense foreign body reaction (34, 35). In contact with CRCS, an initial acute inflammation was observed. In this respect, the presence of polymorphonuclear leukocytes at the 7-day observation or their persistence through other
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FiG 1. Tissue reaction to implanted materials at the end of the tubes after 7 days of implantation. A and B, Sealapex. A, Low-power photomicrograph showing the tissue response at the areas of tissue-material contact (hematoxylin and eosin; original magnification x70). B, Higher magnification of tissues in direct contact with Sealapex showing the presence of particles of the filling material in foreign body giant cells and macrophages. Note also the presence of fibroblasts and blood vessels (hematoxylin and eosin; original magnification x650). C, Low-power photomicrograph showing the tissue response at the end of a tube filled with CRCS. There is a dense cell concentration at the areas of tissuematerial contact (hematoxylin and eosin; original magnification • D, Higher magnification of C showing the tissue-material interface with lymphocytes, polymorphonuclear leukocytes, fibroblasts, macrophages, and some foreign body giant cells (hematoxylin and eosin; original magnification • E, Foreign body giant cell (arrow) and macrophages containing particles of CRCS in the cytoplasm (hematoxylin and eosin; original magnification • F, Silicone rod (control). Note the presence of a thin fibrous connective tissue capsule and a mild tissue response in the areas surrounding the implant (hematoxylin and eosin; original magnification •
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233
observation periods may reveal that the material still remains toxic even after 30 days of implantation. This irritative quality may be due to the presence of eugenol in the composition of CRCS. In spite of the manufacturer's claims that eugenol is present in low concentrations in CRCS, our results agreed with those of previous reports in which other sealers containing eugenol showed some irritative action in the surrounding tissues (30, 36-38). In addition, the presence of dark granules in the cytoplasm of macrophages which persists in the lumen of the tubes or into the tissues surrounding the end of the implants may also reveal that there is a permanent transportation of the material. However, the severity of this reaction decreased with time and it seemed to be resolved after 90 days of implantation. The histological evaluation of the controls initially showed a delicate fibrous capsule and a mild inflammatory reaction in contact with the implants, probably as a result of the
FIG 2. Tissue reaction at the end of a silicone tube filled with Sealapex. Time of implantation, 30 days. A, Granulomatous tissue invagination into the lumen of the tube. Within this, note the presence of many dark particles of the filling material (hematoxylin and eosin; original magnification x70). B, Higher magnification of the tissues in direct contact with Sealapex showing multinucleated foreign body giant cells and macrophages containing particles of the filling material in their cytoplasm (hematoxylin and eosin; original magnification x1,000).
F=G 3. Photomicrograph of an inflammatory reaction in the tissues in contact with CRCS after 30 days of implantation showing lymphocytes, polymorphonuclear leukocytes, and macrophages as well as many fibroblasts and newly formed vessels (hematoxylin and eosin; original magnification x400).
TABLE 4. Differential cell counts, 30 days after implantation* Cell Type
Sealapex
Lymphocytes Polymorphonuclear leukocytes Macrophages Giant cells Plasmocytes Fibroblasts
13.0 0.0 35.0 4.0 0.0 58.0
+_ 8.0 _ 0.0 _+ 16.0 _+ 1.5 __.0.0 _+8.0
CRCS
Control
p
12.0 +_ 3.5 8.5 _+ 1.5 27.0 -F 12.0 0.3 -+ 0.5 1.0 -+ 1.0 48.0 +_ 18.0
11.0 +- 2.0 1.0 - 1.5 4.0 _+ 5.0 0.0 +- 0.0 1.0 -+ 0.0 74.0 _ 37.0
>0.05 <0.01 <0.01 <0.01 >0.05 >0.05
9 The results of the cell counts were expressed as the mean value obtained from the total number of ceils which were counted in all specimens of each material (•
TABLE 5. Differential cell counts, 90 days after implantation* Cell Type Lymphocytes Polymorphonuclear leukocytes Macrophages Giant cells Plasmocytes Fibroblasts
Sealapex 16.0 1.3 41.0 3.0 0.0 83.0
_+ 5.0 +_ 1.5 _ 6.5 _+ 2.0 +_ 0.0 _+27.0
CRCS
Control
p
32.0 _+ 6.0 3.5 -+ 1.5 5.0 _ 1.0 0.0 -+ 0.0 0.0 +--0.0 74.0 _+ 35.0
4.3 -+ 3.0 0.0 -+ 0.0 0.0 -+ 0.0 0.0 -+ 0.0 0.0 +--0.0 62.0 -+ 30.0
<0.01 >0.05 <0.01 <0.05 >0.05
* The results of the cell counts were expressed as the mean value obtained from the total number of cells whiCh were counted in all speornens of each material (_+SD).
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FtG 4. Sealapex, 90 days. A, Low-power photomicrograph showing significant tissue invagination into the lumen of the tube and fragments of
the filling material (hematoxylin and eosin; original magnification • B, Higher magnification of A showing particles of the filling material in the granulomatous tissue at the end of the tube. Note fibrous encapsulation of these particles (arrow) and the presence of many foreign body giant cells and macrophages containing particles of engulfed material in their cytoplasm (hematoxylin and eosin; original magnification x400).
Our results indicate that after 90 days of implantation, the tissues adjacent to CRCS will heal progressively whereas in those in contact with Sealapex the granulomatous reaction persists even in the late observation period. However, the limitations of the experimental model used in this study must be seriously considered. In this respect we feel that more extensive investigations on this issue will be necessary prior to extrapolation of the above observations to the actual clinical situation. These experiments are now in progress. SUMMARY
FtG 5. A, CRCS, 90 days. Low-power photomicrograph showing
resolution of acute inflammation at the end of the tube. Inflammatory infiltrate was progressively replaced by fibrous encapsulation (hematoxylin and eosin; original magnification • B, Silicone rod, 90 days. A continuous fibrous connective tissue capsule without signs of inflammation is observed in contact with the implant (hematoxylin and eosin; original magnification •
surgical trauma. With time this picture appeared to be resolved and a well-organized fibrous capsule which was free of inflammatory cells was seen around the implants, especially at the 90-day observation period.
In this study, the biocompatibility of two calcium hydroxide-based endodontic sealers was investigated. Silicone tubes, 10-mm long and l mm in internal diameter, containing freshly mixed Sealapex or CRCS were implanted into the subcutaneous connective tissue of 30 white male Wistar rats. Solid silicone rods equal in size were likewise implanted as controls. The observation periods were 7, 30, and 90 days, after which the areas of tissue reaction to the implanted materials were histometrically analyzed. In addition, the cellular population of these reactions was quantitated. Our results showed that different grades of tissue reaction among the tested materials were recorded at the ends of the tubes. Quantitative results revealed that a granulomatous tissue with many multinucleate foreign body giant cells and macrophages with engulfed material was observed in contact with Sealapex. This reaction increased in severity in the late observation periods. In spite of the occurrence of an initial acute inflammation when in direct contact with CRCS, the severity of this reaction decreased with time and it seemed to be resolved after 90 days of implantation. On the other hand, a wellorganized fibrous tissue capsule was seen around the control implants, especially at the 90-day observation period. We wish to thank Dr. R. L. Mechi for the valuable collaboration in the statistical analysis and Miss O. Chiessa and P. Mandalunis for their technical assistance,
Calcium Hydroxide-based Sealers
Vol. 14, No. 5, May 1988 Dr. Zmener is affiliated with the Department of Oral Pathology, Faculty of Odontology, University of Buenos Aires, Buenos Aires, Argentina, and in pdvate practice limited to endodonUcs. Dr. Guglielmotti is an associate researcher, Department of Radiobiology, National Atomic Commission and Department of Oral Pathology, Faculty of Odontology, University of Buenos Aires. Dr. Cabrini is a head professor, Department of Radiobiology, National Atomic Commission and Department of Oral Pathology, Faculty of Odontology, University of Buenos Aires. Address requests for repdnts to Dr. Osvaldo Zmener, Cfitedra de Anatomia Patolbglca, Facultad de Odontologia, Universidad de Buenos Aires. Marcelo T. de Alvear 2142 (1122), Buenos Aires, Rep=3blicaArgentina.
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1978;7:185-91. 4. Holland R, de Mello W, Nery MJ, Bemab~ PFE, de Souza V. Reaction of human pedapicel tissue to pulp extirpation and immediate root canal filling with calcium hydroxide. J Endodon 1977;3:63-7. 5. Holland R, de Souza V, Nery MJ, Bemal~ PFE, de Mello W, Otoboni JA. Apical hard-tissue deposition in adult teeth of monkeys with use of calcium hydroxide. Aust Dent J 1980;25:189-92. 6. Holland R, de Souza V, Nery MJ, de Mello W, Bemal~ PFE, Otoboni JA. A histological study on the effect of calcium hydroxide in the treatment of pulpless teeth in dogs. J Br Endod Soc 1979;12:15-23. 7. Rasmussen P, Mj6r IA. Calcium hydroxide as an ectoplc bone inductor in rats. Scand J Dent Res 1971;79:24-30. 8. Sciaky I, Pisanti S. Localization of calcium placed over amputated pulp in dog's teeth. J Dent Res 1960;39:1126-32. 9. Pisanti S, Sciaky I. Odgin of calcium in the repair wall after pulp exposure in the dog. J Dent Res 1964;43:641-4. 10. Eda S. Histochemlcal analysis on the mechanism of dentin formation in dog's pulp. Bull Tokyo Dent Coil 1961 ;2:59-88. 11. Holland R, Pinheiro CE, de Mello W, Ned MJ, de Souza V. Histochemical analysis of the dog's dental pulp after pulp capping with calcium, barium ant! strontium hydroxides. J Endodon 1982;8:444-7. 12. Leonardo MR, Holland R. Healing process after vital pulp extirpation and immediate root canal filling with calcium hydroxide. Histological study in human teeth. Rev Fac Odontol Aracatuba 1974;3:159-69. 13. Holland R, Nery MJ, de Mello W, de Souza V, Bema.b~ PFE, Otoboni JA. Root canal treatment with calcium hydroxide. I. Effect of overfilling and refilling. Oral Surg 1979;47:87-92. 14. Holland R, Nery MJ, de Mello W, de Souza V, Bemab~ PFE, Otoboni JA. Root canal treatment with calcium hydroxide. II. Effect of instrumentation beyond the apices. Oral Surg 1979;47:93-6.
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15. Rowe AH. Effect of filling material on periapical tissue. Br Dent J 1967;122:98-102. 16. Hovland EJ, Dumsha TC. Leakage evaluation in vitro of the root canal sealer cement Sealapex. Int Ended J 1985;18:179-82. 17. Cohen T, Gutmann JL, Wagner M. An assessment in vitro of the sealing properties of calciobiotic root canal sealer. Int Endod J 1985;18:172-78. 18. Wilcko WM. Comparative study of two endodontic sealers in rats [MS Thesis]. Morgantown, WV: University of West Virginia, 1982. 19. Holland R, de Souza V. Ability of a new calcium hydroxide root canal filling material to induce hard tissue formation. J Endodon 1985;11:535-43. 20. Itoiz ME, Carranza FA Jr., Cabrini RL. Histologic and histometdc study of experimental occlusal trauma in rats. J Periodontol 1963;34:305-14. 21. Lbpez Otero R, Carranza FA Jr., Cabrini RL. Histometric study of age changes in interradlcular bone of Wistar rats. J Periodont Res 1967;2:40-5. 22. Carranza FA Jr., Cabrini RL. Histometric studies of periodontal tissues. Periodontics 1967;5:308-12. 23. Weibel ER, Elias H. Introduction to stereology and morphology. Quantitative methods in morphology. Berlin: Springer-Verlag, 1967:3-19. 24. Weibel ER, Kistler GS, Schede WF. Practical stereological methods for morphometdc cytology. J Cell Biol 1966;30:23-38. 25. Langeland K. Root canal sealants and pastes. Dent Clin North Am 1974;18:309-27. 26. Olsson B, Sliwkowski A, Langeland K. Subcutaneous implantation for the biological evaluation of endodontic materials. J Endodon 1981 ;7:355-69. 27. Langeland K, Guttuso J, Langeland LK, Tobon G. Methods in the study of biologic responses to endodontic materials. Oral Surg 1969;27:522-42. 28. Tomeck CD_ Reaction of rat connective tissue to polyethylene tube implants. Oral Surg 1966;21:379-87. 29. Spangberg L. Comparison between tissue reactions to gutta-percha and polytetrafluorethylene implanted in the mandible of the rat. Sven Tandlak Tidski 1968;61:705-15. 30. Browne RM, Friend LA. An investigation into the irritant properties of some root filling materials. Arch Oral Bio11968;13:1355-69. 31. Wennberg A, Bergdahl M, Spangberg L Biologic effect of polyisobutylene on HeLa cells and on subcutaneous tissue in guinea pigs. Scand J Dent Res 1974;82:813-7. 32. Zmener O, Dominguez FV. Corrosion of silver cones in the subcutaneous connective tissue of the rat. A preliminary scanning electron microscope, electron microprobe, and histological study. J Endodon 1985;11:55-61. 33. Safavi KE, Pascon EA, Langeland K. Evaluation of tissue reaction to endodontlc materials. J Endodon 1983;9:421-9. 34. Erausquin J. Pedapical tissue reaction to root canal fillings with zinc, titanium, lead and aluminum oxides. Oral Surg 1970;30:545-54. 35. Benatti-Neto C, Bramante CM, Berbert A, Lia RCC. Rea(;:~odo tecido conjuntivo subcutaneo de rato ante a implanta~o dos materials componentes do cimento AH-26. Rev Bras Odontol 1982;39:11-20. 36. Rappaport HM, Lilly GE, Kapsismalis P. Toxicity of endodontic filling matedals. Oral Surg 1964;18:785-802. 37. Friend LA, Browne RM. Tissue reactions to some root filling materials. Br Dent J 1968;125:291-8. 38. Holland R, Souza V, Nery MJ, Bernab~ PFE. Comportamento do tecido conjuntivo subcutaneo do rato ao implante de tubos de polietileno preenchidos parcial ou totalmente con alguns materials obturadores de canal. Rev Bras Odonto11971 ;28:197-201.
Printed in U.S.A. VOL. 14, NO. 5, MAY 1988
Biocompatibility of Two Calcium Hydroxide-based Endodontic Sealers: A Quantitative Study in the Subcutaneous Connective Tissue of the Rat Osvaldo Zmener, DDS, Dr. Odont., Maria B. Guglielmotti, DDS, Dr. Odont., and Romulo L. Cabrini, MD
In this study, the biocompatibility of two calcium hydroxide-based endodontic sealers was investigated. Silicone tubes containing freshly mixed Sealapex or CRCS were implanted in the dorsal subcutaneous connective tissue of the rat. Equal size solid silicone rods were also implanted and used as controis. The tissue reaction to test and control materials was histometrically and quantitatively analyzed under light microscopy. After 7, 30, and 90 days of implantation, different grades of tissue reaction to the tested materials were recorded at the end of the tubes. A granulomatous tissue containing foreign body giant cells and macrophages with engulfed material in their cytoplasm as well as many fibroblasts and vessels was initially observed in contact with Sealapex. This reaction increased progressively at the 30- and 90-day observation period. An acute inflammation was detected in tissues in contact with CRCS. However, the severity of this reaction decreased with time and it seemed to be resolved at the 90-day observation period. Taking into account the limitations of the experimental model used in this study, we consider that more extensive experiences will be necessary prior to extrapolating these findings to the actual clinical situation.
able calcium hydroxide-based root canal sealers, i.e. Sealapex (Kerr, Romulus, MI) and CRCS (Hygenic Co., Akron, OH), were analyzed from the point of view of some of their physical characteristics (16, 17). Information about their biological properties, however, is scarce (18, 19). Consequently, we feel that their implantation in small animals will constitute a suitable source of initial information on the biocompatibility of these materials. The purpose of this study was to compare quantitatively the tissue response to both Sealapex and CRCS when they were implanted in the subcutaneous connective tissue of the rat.
MATERIALS AND METHODS In this study, autoclaved silicone tubes (Raholin SRL; V. Madero, Buenos Aires, Argentina), 10-mm long and 1 m m in internal diameter, were filled flush with freshly mixed Sealapex or CRCS. They were immediately implanted into the subcutaneous connective tissue of 30 white male Wistar rats that weighed 90 g each. According to the manufacturer, Sealapex contains calcium hydroxide, barium sulfate, zinc oxide, titanium dioxide, and zinc stearate in a matrix blend of poly(methylene methyl salicylate) resin, isobutyl salicylate, methyl salicylate, ethyl toluene sulfonamide isomers, and pigments. The formulation of CRCS is not totally available; however, it is believed to contain a calcium hydroxide and zinc oxide-eugenol mixture in which approximately 40 to 50% of the eugenol content is replaced by eucaliptol. In addition, solid silicone rods, equal in size, were also implanted and used as inert controls. The implantation procedures were as follows: The animals were anesthetized by intraperitoneal administration of sodium pentobarbital (0.025 g per 1,000g wt); and the dorsal skin was shaved and disinfected with 5% iodine in alcohol. Under aseptic conditions an incision approximately 15-mm long was made through the skin using a scalpel and three separate subcutaneous pockets were prepared by blunt dissection to a depth of 20 mm. Then, three implants (one of each experimental material and one control) were carefully placed into the pockets prepared in each rat. Care was taken to prevent smearing of the test materials on the outside areas of the tubes. After implantation, the wounds were sutured.
Calcium hydroxide has been widely used in endodontics and the evidence shows that it does stimulate or promote hard tissue repair in many instances (1-6). Interestingly, it appears that a direct contact between calcium hydroxide and the tissues is necessary for an adequate inductive action (7) but the actual mechanisms by which calcium hydroxide may act on the surrounding tissues still remain unclear (8-I 1). Despite apical closure by hard tissue deposition found under different experimental conditions when calcium hydroxide was used as a root canal filling (4-6, 12), some complications have been observed. This occurred in some cases in which this material was in direct contact with periapical tissues ( 13-15). Recently, two new commercially avail-
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The animals were killed in groups of 10 each after 7, 30, and 90 days by ether suffocation. The implants along with the surrounding tissues were removed in rectangular blocks and then fixed in 10% formalin solution. After fixation the tissues were processed for paraffin embedding and then longitudinally sectioned through the implants. Serial sections approximately 7-#m thick were obtained from each specimen and then stained with hematoxylin and eosin. In order to obtain a quantitative estimation of the tissue reaction around both ends of the tubes, three sections belonging to the central areas of each specimen were examined. Histometric measurements based on standard stereological methods (20-24) were made on tracings obtained from projections of the histological sections. These projections were made using a projection head mounted on a standard light microscope (Carl Zeiss, Oberkochen, West Germany) at a magnification of x25. The areas of tissue reaction to the implanted materials were measured over the image of each tracing using a transparent celluloid grid marked with equidistant dots every 7 mm. The dots within the outlines of reactive areas and those of the total field were counted and then the areas of reaction were calculated. These values represent the volume proportion of the areas measured on the tracings (24). The values obtained from both ends of each tube were similar in each individual case for all tested materials and for all of the observation periods. For this reason the area of reaction which was finally evaluated in each of the three sections from each individual specimen was randomly selected. Then, a mean value from these sections was calculated. Final values were obtained by transferring the results of the measurements performed on the tracings to the actual size of tissue reaction observed on the histological slides, which were expressed in square millimeters. For cell quantitation, the total number of inflammatory cells, i.e. lymphocytes, polymorphonuclear leukocytes, macrophages, multinucleated giant cells, plasmocytes, and fibroblasts, which were observed in the selected areas of tissue reaction were recorded by counting cells in three separate fields of vision (7.5 mm 2 each) at x400. They were located and spaced at regular intervals within the outlines of these areas. The total number of cells observed in the same sections used for the histometric measurement of the reactive areas of each specimen was counted twice by two different examiners. Since no substantial differences between the two readings were observed, a mean value for these records was obtained. Final results and their standard deviations were derived from the mean values obtained from the total number of specimens of each material examined in each observation period. The data obtained from the measurements of the areas of tissue reaction and from the cell counts were subjected to an analysis of variance and to Duncan's multiple range test. Because of problems inherent to laboratory procedures, some specimens from each observation period and for each material had to be excluded from the study. In these instances, final values were obtained from fewer readings. Definitive data from the number of specimens studied in each observation period are shown in Table 1.
RESULTS Macroscopic examination showed that wound healing was satisfactory in all observation periods. Histological evaluation
of the implants revealed that they were surrounded by fibrous connective encapsulation of irregular thickness. It could be easily distinguished from the reaction of the tissues in contact with the experimental materials at both ends of the tubes. The quantitative measurements of the areas of necrosis or inflammatory responses are summarized in Table 2. The results of cell quantitation are shown in Tables 3 to 5. At the 7-day observation the extent of tissue reaction to Sealapex and CRCS was similar. In this respect no significant differences were found. However, these results strongly differed from those obtained from the controls which were much less. At the ends of the tubes filled with Sealapex and CRCS, some amounts of necrotic tissue as well as the presence of macrophages were observed in direct contact with these materials. Whereas a concentration of lymphocytes and polymorphonuclear leukocytes were present only in contact with CRCS (Table 3). Some foreign body giant cells with engulfed particles of the test materials were also seen in the areas surrounding the ends of the tubes filled with Sealapex and CRCS (Fig. l, A to E). In addition, a fibroblastic proliferation was also observed around both of these materials. In general, a thin fibrous connective tissue composed of young fibroblasts and few inflammatory cells was observed in contact with the controls (Fig. 1F). At the 30-day observation, significant differences between the areas of tissue reaction to Sealapex and CRCS as well as between these two materials and the controls were observed. As is noted in Table 2, the values obtained from the measurements made on CRCS and the control specimen projections were less than those obtained from Sealapex. In the areas in contact with Sealapex, some fragments of this material were surrounded by granulomatous tissue that appeared to be invaginated into the lumen of the tubes (Fig. 2A). Macrophages and foreign body giant cells containing many dark particles in their cytoplasm were seen in contact or at some distance from the end of the tubes (Fig. 2B). In contact with CRCS, the inflammatory reaction subsisted (Fig. 3) but it was much less than those observed at the 7-day period (Table 4). A consistent fibroblastic proliferation at the ends of the specimens was also detected. In contact with the
TABLE 1. Observation periods and number of analyzed
specimens Days
Sealapex CRCS Control
7
30
90
9 8 10
9 9 9
10 10 8
TABLE 2. Areas of tissue reaction to the implanted materials*
Days 7 30 90
Sealapex
CRCS
Control
(mm2)
p
0.66 ___0.311" 0.82 -+ 0.36 1.23 _ 0.54
0.68 - 0.281" 0.63 _ 0.25 0.30 _+ 0.12
0.31 _ 0.08 0.33 _ 0.16 0.00 - 0.00
<0,05 <0.05 <0.01
(mm2)
(mm2)
*The results of the histometric measurements were expressedas the actual size in square millimeters of tissue reaction obeervedon the histological slides. 1"No significant differences were observed between Sealapex and CRCS speomens (+SD).
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TABLE 3. Differential cell counts, 7 days after implantation* Cell Type
Lymphocytes Polymorphonuclear leukocytes Macrophages Giant cells Piasmocytes Fibroblasts
Sealapex
3.0 2.0 22.0 3.5 0.0 51.0
-+ 0.7 _+ 1.5 _+ 7.0 -+ 1.3 + 0.0 -+ 25.5
CRCS
Control
p
19.0 _+ 8.0 24.0 _+ 20.0 30.0 +- 14.0 1.0 _+ 2.0 0.4 _+ 0.5 45.0 + 18.0
9.0 -+ 6.0 6.0 _+ 5.0 9.0 -+ 6.0 0.0 -+ 0.0 0.0 -+ 0.0 58.0 -+ 23.0
<0.01 <0.01 <0.01 >0.05 >0.05
9The resultsof the cellcountswereexpressedas the meanvalueobtained from the total numberof cells whichwere countedin all specimensof eachmaterial(+ SD).
controls, a thin fibrous capsule containing some scattered inflammatory cells could be observed. At the 90-day observation, the specimens of Sealapex showed the greatest areas of tissue reaction whereas the values obtained from the measurements of CRCS and control specimens were markedly reduced (Table 2). The results of cell quantitation (Table 5) showed that macrophages and foreign body giant cells with important amounts of engulfed material persist at the ends of the tubes filled with Sealapex. The majority of these specimens showed some extent of granulomatous tissue invagination into the lumen of the tubes (Fig. 4A). In addition, fragments of the filling material that appeared to be surrounded by macrophages and multinucleated giant cells were detected in the invaginated tissues, especially in the areas of the specimen end (Fig. 4B). At these sites the granulomatous tissues were generally walled off by fibrous encapsulation. Interestingly, the accumulation of acute inflammatory cells that was noted in the areas of contact with CRCS decreased as the observation period increased. In spite of these findings, some lymphocyte concentration persisted (Fig. 5,4). On the other hand, a consistent fibrous connective tissue capsule with the presence of only occasional lymphocytes was detected in contact with the controls (Fig. 5B).
DISCUSSION In conjunction with previous reports (25, 26), our results showed that the histological response to endodontic materials implanted in the subcutaneous connective tissue of the rat could be used as a preliminary source of information on their biocompatibility. The introduction of experimental materials into tubes is amply recognized as a suitable method for the implantation of these materials. This methodology allows a precise control of the amount of material that will be in contact with the tissues (27). However, in spite of handling all implants carefully to prevent smearing of the test materials on the outside of the tubes, our results revealed that both of these materials, i.e. Sealapex and CRCS, easily flowed out of the tubes or broke up. In this respect, particles of these materials were seen in the tissues surrounding the end of the tubes and this could be the main cause of the extent of the tissue reaction during the short observation periods. Different materials such as polyethylene or Teflon are normally used for implantation purposes. Since these materials show no irritation properties on the surrounding tissues (28, 29), the reactions at both ends of the implants are normally related to the toxicity of the material that is contained in the lumen of the tubes. In this study, we used silicone tubes in order to eliminate some methodological problems. For example, the tissues in
contact with polyethylene or Teflon implants have the tendency to adhere to the implant surfaces. Therefore, the interface adjacent to these materials is frequently destroyed or removed during the laboratory procedures. In this study, the silicone tubes or the solid silicone rods were found to be surrounded by a thin fibrous connective capsule without signs of adherence to the material surface. In addition, there was no evidence of the presence of foreign body giant cells in contact with the lateral walls of the tubes or at the ends of the solid silicone rods. These cells were occasionally seen in contact with polyethylene or Teflon as reported elsewhere (30-32). The use of quantitative methods for the evaluation of the tissue reaction to freshly prepared materials has been subjected to criticism (33). However, we consider the use of histometric procedures to quantificate some of the histological parameters related to the toxicity of endodontic materials to be an objective method of evaluation. There were no evident differences in simple observation which could be detected. The results of the histometric evaluation showed that the greatest areas of tissue reaction were seen in contact with Sealapex. At the 7-day observation no significant differences between this material and CRCS were observed, but the amount of tissue reaction to Sealapex increased after 30 and 90 days. In the majority of these cases, Sealapex was fragmented or it was lost from the inside of the tubes and the test material was progressively replaced by invagination of the granulomatous tissues which were initially located at the external areas of the end of the implants (Figs. 2A and 4A). Consequently, significant areas of tissue reaction measured in surface units resulted from the analysis of Sealapex specimens at the 30- and 90-day observations. In this respect the statistical analysis of the data showed that the differences observed between both Sealapex and CRCS or between these two materials and the controls were significant (p < 0.01), especially in the late observation period. The results of cell quantitation revealed that a granulomatous tissue composed by macrophages and foreign body giant cells with engulfed material in their cytoplasm as well as a high number of fibroblasts and vessels were observed in contact with Sealapex, and that this reaction increased severely at the 30- and 90-day observation periods. The presence of particles of the material in macrophages and foreign body giant cells or in vessels in areas located at some distance from the end of the tubes revealed transportation of the filling material to sites far away from the implants. This was probably due to some of the components of Sealapex as titanium dioxide which can be easily dispersed into the surrounding tissues, provoking an intense foreign body reaction (34, 35). In contact with CRCS, an initial acute inflammation was observed. In this respect, the presence of polymorphonuclear leukocytes at the 7-day observation or their persistence through other
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FiG 1. Tissue reaction to implanted materials at the end of the tubes after 7 days of implantation. A and B, Sealapex. A, Low-power photomicrograph showing the tissue response at the areas of tissue-material contact (hematoxylin and eosin; original magnification x70). B, Higher magnification of tissues in direct contact with Sealapex showing the presence of particles of the filling material in foreign body giant cells and macrophages. Note also the presence of fibroblasts and blood vessels (hematoxylin and eosin; original magnification x650). C, Low-power photomicrograph showing the tissue response at the end of a tube filled with CRCS. There is a dense cell concentration at the areas of tissuematerial contact (hematoxylin and eosin; original magnification • D, Higher magnification of C showing the tissue-material interface with lymphocytes, polymorphonuclear leukocytes, fibroblasts, macrophages, and some foreign body giant cells (hematoxylin and eosin; original magnification • E, Foreign body giant cell (arrow) and macrophages containing particles of CRCS in the cytoplasm (hematoxylin and eosin; original magnification • F, Silicone rod (control). Note the presence of a thin fibrous connective tissue capsule and a mild tissue response in the areas surrounding the implant (hematoxylin and eosin; original magnification •
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Vol. 14, No. 5, May 1988
233
observation periods may reveal that the material still remains toxic even after 30 days of implantation. This irritative quality may be due to the presence of eugenol in the composition of CRCS. In spite of the manufacturer's claims that eugenol is present in low concentrations in CRCS, our results agreed with those of previous reports in which other sealers containing eugenol showed some irritative action in the surrounding tissues (30, 36-38). In addition, the presence of dark granules in the cytoplasm of macrophages which persists in the lumen of the tubes or into the tissues surrounding the end of the implants may also reveal that there is a permanent transportation of the material. However, the severity of this reaction decreased with time and it seemed to be resolved after 90 days of implantation. The histological evaluation of the controls initially showed a delicate fibrous capsule and a mild inflammatory reaction in contact with the implants, probably as a result of the
FIG 2. Tissue reaction at the end of a silicone tube filled with Sealapex. Time of implantation, 30 days. A, Granulomatous tissue invagination into the lumen of the tube. Within this, note the presence of many dark particles of the filling material (hematoxylin and eosin; original magnification x70). B, Higher magnification of the tissues in direct contact with Sealapex showing multinucleated foreign body giant cells and macrophages containing particles of the filling material in their cytoplasm (hematoxylin and eosin; original magnification x1,000).
F=G 3. Photomicrograph of an inflammatory reaction in the tissues in contact with CRCS after 30 days of implantation showing lymphocytes, polymorphonuclear leukocytes, and macrophages as well as many fibroblasts and newly formed vessels (hematoxylin and eosin; original magnification x400).
TABLE 4. Differential cell counts, 30 days after implantation* Cell Type
Sealapex
Lymphocytes Polymorphonuclear leukocytes Macrophages Giant cells Plasmocytes Fibroblasts
13.0 0.0 35.0 4.0 0.0 58.0
+_ 8.0 _ 0.0 _+ 16.0 _+ 1.5 __.0.0 _+8.0
CRCS
Control
p
12.0 +_ 3.5 8.5 _+ 1.5 27.0 -F 12.0 0.3 -+ 0.5 1.0 -+ 1.0 48.0 +_ 18.0
11.0 +- 2.0 1.0 - 1.5 4.0 _+ 5.0 0.0 +- 0.0 1.0 -+ 0.0 74.0 _ 37.0
>0.05 <0.01 <0.01 <0.01 >0.05 >0.05
9 The results of the cell counts were expressed as the mean value obtained from the total number of ceils which were counted in all specimens of each material (•
TABLE 5. Differential cell counts, 90 days after implantation* Cell Type Lymphocytes Polymorphonuclear leukocytes Macrophages Giant cells Plasmocytes Fibroblasts
Sealapex 16.0 1.3 41.0 3.0 0.0 83.0
_+ 5.0 +_ 1.5 _ 6.5 _+ 2.0 +_ 0.0 _+27.0
CRCS
Control
p
32.0 _+ 6.0 3.5 -+ 1.5 5.0 _ 1.0 0.0 -+ 0.0 0.0 +--0.0 74.0 _+ 35.0
4.3 -+ 3.0 0.0 -+ 0.0 0.0 -+ 0.0 0.0 -+ 0.0 0.0 +--0.0 62.0 -+ 30.0
<0.01 >0.05 <0.01 <0.05 >0.05
* The results of the cell counts were expressed as the mean value obtained from the total number of cells whiCh were counted in all speornens of each material (_+SD).
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Zmener et al.
Journal of Endodontics
FtG 4. Sealapex, 90 days. A, Low-power photomicrograph showing significant tissue invagination into the lumen of the tube and fragments of
the filling material (hematoxylin and eosin; original magnification • B, Higher magnification of A showing particles of the filling material in the granulomatous tissue at the end of the tube. Note fibrous encapsulation of these particles (arrow) and the presence of many foreign body giant cells and macrophages containing particles of engulfed material in their cytoplasm (hematoxylin and eosin; original magnification x400).
Our results indicate that after 90 days of implantation, the tissues adjacent to CRCS will heal progressively whereas in those in contact with Sealapex the granulomatous reaction persists even in the late observation period. However, the limitations of the experimental model used in this study must be seriously considered. In this respect we feel that more extensive investigations on this issue will be necessary prior to extrapolation of the above observations to the actual clinical situation. These experiments are now in progress. SUMMARY
FtG 5. A, CRCS, 90 days. Low-power photomicrograph showing
resolution of acute inflammation at the end of the tube. Inflammatory infiltrate was progressively replaced by fibrous encapsulation (hematoxylin and eosin; original magnification • B, Silicone rod, 90 days. A continuous fibrous connective tissue capsule without signs of inflammation is observed in contact with the implant (hematoxylin and eosin; original magnification •
surgical trauma. With time this picture appeared to be resolved and a well-organized fibrous capsule which was free of inflammatory cells was seen around the implants, especially at the 90-day observation period.
In this study, the biocompatibility of two calcium hydroxide-based endodontic sealers was investigated. Silicone tubes, 10-mm long and l mm in internal diameter, containing freshly mixed Sealapex or CRCS were implanted into the subcutaneous connective tissue of 30 white male Wistar rats. Solid silicone rods equal in size were likewise implanted as controls. The observation periods were 7, 30, and 90 days, after which the areas of tissue reaction to the implanted materials were histometrically analyzed. In addition, the cellular population of these reactions was quantitated. Our results showed that different grades of tissue reaction among the tested materials were recorded at the ends of the tubes. Quantitative results revealed that a granulomatous tissue with many multinucleate foreign body giant cells and macrophages with engulfed material was observed in contact with Sealapex. This reaction increased in severity in the late observation periods. In spite of the occurrence of an initial acute inflammation when in direct contact with CRCS, the severity of this reaction decreased with time and it seemed to be resolved after 90 days of implantation. On the other hand, a wellorganized fibrous tissue capsule was seen around the control implants, especially at the 90-day observation period. We wish to thank Dr. R. L. Mechi for the valuable collaboration in the statistical analysis and Miss O. Chiessa and P. Mandalunis for their technical assistance,
Calcium Hydroxide-based Sealers
Vol. 14, No. 5, May 1988 Dr. Zmener is affiliated with the Department of Oral Pathology, Faculty of Odontology, University of Buenos Aires, Buenos Aires, Argentina, and in pdvate practice limited to endodonUcs. Dr. Guglielmotti is an associate researcher, Department of Radiobiology, National Atomic Commission and Department of Oral Pathology, Faculty of Odontology, University of Buenos Aires. Dr. Cabrini is a head professor, Department of Radiobiology, National Atomic Commission and Department of Oral Pathology, Faculty of Odontology, University of Buenos Aires. Address requests for repdnts to Dr. Osvaldo Zmener, Cfitedra de Anatomia Patolbglca, Facultad de Odontologia, Universidad de Buenos Aires. Marcelo T. de Alvear 2142 (1122), Buenos Aires, Rep=3blicaArgentina.
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