JOURNAL OF ENDODONTICS Copyright © 2002 by The American Association of Endodontists Printed in U.S.A. VOL. 28, NO. 3, MARCH 2002 Calcium Salts Deposition in Rat Connective Tissue After the Implantation of Calcium Hydroxide-Containing Sealers Roberto Holland, PhD, Valdir de Souza, PhD, Mauro Juvenal Nery, PhD, Pedro Felicio Estrada Bernabé, PhD, José Arlindo Otoboni Filho, PhD, Eloi Dezan Junior, MS, and Sueli Satomi Murata, DDS This study was conducted to observe the rat subcutaneous connective tissue reaction to implanted dentin tubes that were filled with mineral trioxide aggregate, Sealapex, Calciobiotic Root Canal Sealer (CRCS), Sealer 26, and the experimental material, Sealer Plus. The animals were sacrificed after 7 and 30 days, and the specimens were prepared for histological analysis after serial sections with a hard-tissue microtome. The undecalcified sections were examined with polarized light after staining according to the Von Kossa technique for calcium. At the tube openings, there were Von Kossa-positive granules that were birefringent to polarized light. Next to these granulations, there was irregular tissue, like a bridge, that was Von Kossapositive. The dentin walls of the tubes exhibited a structure highly birefringent to polarized light, usually like a layer, in the tubules. These results were observed with all the studied materials, except the CRCS, which didn’t exhibit any kind of mineralized structure. The results suggest that among the materials studied, the CRCS could have the least possibility of encouraging hard tissue deposition. positive at the tube openings (4, 5). The same calcite crystals are observed in the interior of the dentin wall tubules (5). These observations demonstrated that the implantation of dentin tubes in rat connective tissue, and the later analysis of undecalcified sections, was a good method to study the mechanism of calcium hydroxide action. Employing this methodology, Holland et al. (5) demonstrated that the mechanism of action of mineral trioxide aggregate (MTA) is probably the same as that of calcium hydroxide. Nevertheless, there is a lack of studies about the mechanism of action of calcium hydroxide-containing root canal filling materials. The purpose of this study was to observe the reaction of rat subcutaneous connective tissue to the implantation of dentin tubes filled with calcium hydroxide-containing sealers, employing the methodology already described (5). MATERIALS AND METHODS Dentin tubes were prepared from human tooth roots, according to the specification described by Holland et al. (5). The dentin tubes were thoroughly irrigated with EDTA and sodium hypochlorite and then washed in distilled water before being autoclaved. The tubes were filled with MTA (Loma Linda University, Loma Linda, CA), Sealapex (Kerr Corporation, Romulus, MI), Calciobiotic Root Canal Sealer (CRCS) (Hygenic-Dental Ind. Com. Ltda, Rio de Janeiro, Brazil), Sealer 26 (Dentsply Ind. Com. Ltda., Petrópolis, Brazil), and the experimental filling material, Sealer Plus (6) (Dentsply Ind. Com. Ltda., Petrópolis, Brazil) and immediately implanted subcutaneously in the dorsal region, on each side of the midline, in 60 rats. For control purposes, empty dentin tubes were implanted in 10 additional animals. The animals were killed after 7 and 30 postoperative days. The tubes and surrounding tissues were removed and fixed in 10% buffered formalin solution at pH 7.0. The undecalcified samples were embedded in a mixture of paraffin (95%) and carnauba wax (5%), according to Holland et al. (2). The sectioning was performed serially at 10-␮m intervals by using a hard-tissue microtome. The sections were obtained one by one, always after the application of paraffin wax on the sample surface. This layer of paraffin wax holds the sections flat and makes fitting them onto the heated glass slide with albumen easier. Some sections were When calcium hydroxide is applied over the exposed dental pulp (1) or pulp stump (2) it releases calcium and hydroxyl ions. The calcium ions react with the carbon dioxide from the tissue, giving rise to granulations of calcite crystals birefringent to polarized light (1). Seux et al. (3) observed a rich extra-cellular network of fibronectin in close contact with these crystals. They also reported that their findings strongly support the role of calcite crystals and fibronectin as an initiating step in the formation of a hard tissue barrier. After the implantation of dentin tubes filled with calcium hydroxide in rat connective tissue, there is deposition of calcite crystals and irregular tissue, like a bridge, which is Von Kossa173 174 Holland et al. Journal of Endodontics FIG. 1. Control group: 30 days. Decalcified section. Dentin tube surrounded by a thin fibrous capsule (hematoxylin and eosin; original magnification ⫻100.) FIG. 2. CRCS: 7 days. Undecalcified section. Dentin (D), CRCS (FM), and exudate with neutrophils (INF). (Von Kossa technique; original magnification ⫻40.) stained, according to the Von Kossa technique, for calcium; other sections, without staining, were examined under a polarized light microscope to locate the birefringent material. Some sections were decalcified for 10 min in EDTA before being stained with hematoxylin and eosin. RESULTS Control Group At 7 days, a layer of exudate with neutrophils was observed around the implanted tubes. Next to this area, the cellular population was represented by young fibroblasts and chronic inflammatory cells. The dentin tubes were filled with an eosinophilic mass containing neutrophils. Thirty days after implantation, the samples showed ingrowth of connective tissues containing some inflammatory cells, filling all the tube space in some cases. Outside, the tubes were surrounded by a thin fibrous capsule exhibiting a mild chronic inflammatory reaction in some cases (Fig. 1). Experimental Groups CRCS was the only material that didn’t exhibit any calcified structures that were Von Kossa-positive or birefringent to polarized light (Fig. 2). At 7 days, there was a layer of exudate with neutrophils around the implanted materials. Thirty days after implantation, the cellular population next to the filling material was represented by young fibroblasts and a moderate chronic inflammatory reaction. All the remaining materials exhibited calcified structures at 7 and 30 days. The only difference among the materials was the amount of calcified structures. At the tube openings, there were large granulations positive to Von Kossa technique and birefringent to polarized light. These granulations were generally in contact with the filling materials and usually more numerous with MTA and Sealapex than with the other materials (Figs. 3 and 4). Next to these granulations were extensive areas of irregular tissue, like a bridge, and more positive to the Von Kossa technique than to the birefringent granulations (Fig. 5). These Von Kossa-positive structures were larger and more extensive with MTA and Sealapex FIG. 3. Sealer 26: 7 days. Undecalcified section. Dentin (D), Sealer 26 (FM), and birefringent granulations (arrows). (Polarized light; original magnification ⫻70.) than with the other materials. A highly birefringent structure, localized in the interior of the dentin tubules, was also observed. This structure formed a layer, generally next to the filling materials, and was larger with MTA than with the other materials (Figs. 6 and 7). The decalcified sections exhibited an irregular and basophilic structure in the same places that the highly Von Kossa-positive tissue was observed. The connective tissue around these structures showed fibroblast proliferation, a mild-to-moderate chronic inflammatory reaction, and some giant cells at 7 days. After 30 days, the Von Kossa-positive structure was observed as a basophilic and irregular tissue with cell inclusions. Around this structure, there was a fibrous connective tissue with a mild chronic inflammatory reaction and some giant cells. DISCUSSION The dentin tubes for subcutaneous implantation were used according to the suggestion of de Souza et al. (4) and Holland et al. (5). The results in the control group are in agreement with previous reports (4, 5). We could have used polyethylene or Teflon tubes, Vol. 28, No. 3, March 2002 Filling Materials in Subcutaneous Tissue 175 FIG. 4. Sealapex: 30 days. Undecalcified section. Dentin (D), Sealapex (FM), and birefringent granulations (G). (Polarized light; original magnification ⫻70.) FIG. 6. MTA: 7 days. Undecalcified section. Dentin (D), MTA (FM), and layer of a highly birefringent structure localized in the dentin tubules (L). (Polarized light; original magnification ⫻70.) FIG. 5. Sealapex: 30 days. Undecalcified section. Dentin (D), Sealapex (FM), and Von Kossa-positive irregular tissue (K). (Von Kossa technique; original magnification ⫻40.) FIG. 7. Sealer 26: 7 days. Undecalcified section. Dentin (D), Sealer 26 (FM), and layer of birefringent structure within the dentin tubules (arrow). (Polarized light; original magnification ⫻70.) but we preferred dentin tubes to observe the action of the materials on dentin walls. CRCS was the only material that didn’t exhibit any kind of calcified structure, which was observed with the other materials. This result supports the one reported by Tagger and Tagger (7) in monkey teeth after root canal filling with CRCS. It is possible that the occurrence of mineralized tissues has some relation to the presence of calcium ions released from the material. Silva et al. (8) reported that CRCS releases calcium ions. However, Tagger et al. (9) reported that CRCS released calcium ions, but they reacted immediately with free eugenol from the material forming calcium eugenolate. The results observed in this study support those of Tagger et al. (9). All the other studied materials exhibited deposition of mineralized structures birefringent to polarized light or Von Kossa-positive, with some quantitative differences. In our experiment, the results with MTA agree with the results reported by Holland et al. (5), who observed similar results for MTA and calcium hydroxide. It is known that MTA has no calcium hydroxide in its formulation (10). However, mixing the powder with water results in a structure that contains basically calcium oxide and calcium phosphate (11). The calcium oxide could react with tissue fluids to form calcium hydroxide. The birefringent granulations observed next to MTA and into the dentin wall tubules are probably calcite crystals originating from the reaction of the calcium from the material with the carbon dioxide from the connective tissue (5). Seux et al. (3) reported that these crystals, together with fibronectin, can be an initiating step in the formation of a hard tissue barrier. In this experiment, we observed the birefringent crystals and a calcified tissue that resembles a barrier at the opening of the tubes. The same observation was reported by Holland et al. (5). In this experiment, the same structures reported for MTA were observed with Sealapex, Sealer Plus, and Sealer 26. The birefringent granulations and the Von Kossa-positive tissue structure were more evident with Sealapex than with Sealer Plus and Sealer 26. It is possible that this observation has some relation to the solubility and the manner of release of calcium ions from these materials. Silva et al. (8) and Duarte et al. (12) reported that Sealapex releases more calcium ions than Sealer 26. Tagger et al. (9) reported Sealapex is more soluble than CRCS and other materials that they have studied. In conclusion, with the exception of CRCS, it is possible that the mechanism of action of the materials in encouraging hard tissue deposition is similar to the one described by Holland et al. (5) for 176 Holland et al. Journal of Endodontics calcium hydroxide. The results also suggest that among these materials CRCS sealer could have the least possibility of encouraging hard tissue deposition after root canal filling. Tagger and Tagger (7) didn’t observe closure of the apical foramen with CRCS. However, with the other materials, studied closure of the apical foramen has been reported (6, 13–15). We wish to express our gratitude to Dr. Mahmoud Torabinejad for providing the mineral trioxide aggregate for this research. Drs. Holland, de Souza, Nery, Bernabé, Otoboni Filho, Dezan Junior and Murata are affiliated with the Department of Endodontics, Faculdade de Odontologia de Araçatuba, UNESP, São Paulo, Brazil. Adress request for reprints to Dr. Roberto Holland, Department of Endodontics, Faculdade de Odontologia de Araçatuba, UNESP, Rua José Bonifácio 1193, CEP 16015050, Araçatuba, São Paulo, Brazil. References 1. Holland R. Histochemical response of amputed pulps to calcium hydroxide. Rev Bras Pesq Med Biol 1971;4:83–95. 2. Holland R, de Mello W, Nery MJ, Bernabé PF, de Souza V. Reaction of human periapical tissue to pulp extirpation and immediate root canal filling with calcium hydroxide. J Endodon 1977;3:63–7. 3. Seux D, Couble ML, Hartmann DJ, Gauthier JP, Magloire H. Odontoblast-like cytodifferentiation of human dental pulp cells in vitro in the presence of a calcium hydroxide-containing cement. Arch Oral Biol 1991;36:117–28. 4. de Souza V, Holland R, de Mello W, Nery MJ. Reaction of rat connective tissue to the implant of calcium hydroxide pastes. Rev Fac Odontol Araçatuba 1977;6:69 –79. 5. Holland R, de Souza V, Nery MJ, Otoboni Filho JA, Bernabé PF, Dezan Junior E. Reaction of rat connective tissue to implanted dentin tubes filled with mineral trioxide aggregate or calcium hydroxide. J Endodon 1999;25:161– 6. 6. Holland R, de Souza V, Otoboni Filho JA, et al. Comportamento dos tecidos apicais e periapicais de dentes de cães à obturação de canal com o cimento experimental Sealer Plus. Rev Bras Odontol 2000;57:114 – 6. 7. Tagger M, Tagger E. Periapical reactions to calcium hydroxide-containing sealers and AH26 in monkeys. Endod Dent Traumatol 1989;5:139 – 46. 8. Silva LAB, Leonardo MR, Silva RS, Assed S, Guimaräes LFL. 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Estudo ‘‘in vitro’’ da infiltração marginal apical em dentes humanos e estudo ‘‘in vivo’’ do comportamento dos tecidos apicais e periapicais de dentes de cães a obturação de canal com os cimentos Sealer 26 e Sealer 26 modificado [master’s thesis]. Marı́lia, Brazil: University of Marilia, 1999. 14. Holland R, de Souza V. Ability of a new calcium hydroxide root canal filling material to induce hard tissue formation. J Endodon 1985;1:535– 43. 15. Holland R, de Souza V, Nery MJ, Otoboni Filho JA, Bernabé PFE, Dezan Junior RE. Reaction of dog’s teeth to root canal filling with mineral trioxide aggregate or a glass ionomer sealer. J Endodon 1999;25:728 –30.