Reaction of Rat Connective Tissue to Implanted Dentin Tubes Filled with a White Mineral Trioxide Aggregate



Roberto HOLLAND1
Valdir de SOUZA1
Mauro Juvenal NERY1
Italo Medeiros FARACO JÚNIOR2
Pedro Felício Estrada BERNABÉ1


1Department of Restorative Dentistry, Faculty of Dentistry of Araçatuba, UNESP, Araçatuba, SP, Brazil
Department of Pediatric Dentistry, Faculty of Dentistry, Lutheran University of Brazil, Porto Alegre, RS, Brazil

Correspondence: Dr. Roberto Holland, Departamento de Odontologia Restauradora, Faculdade de Odontologia de Araçatuba, UNESP, R. José Bonifácio 1193, 16015-050, Araçatuba, SP, Brasil. Fax: +55-18-624-8490. e-mail:

Braz Dent J (2002) 13(1): 23-26  ISSN 0103-6440


The purpose of this paper was to study the reaction of rat subcutaneous connective tissue to the implantation of dentin tubes filled with white mineral trioxide aggregate (MTA), a material that will be marketed. The tubes were implanted into rat subcutaneous tissue and the animals were sacrificed after 7 and 30 days. The undecalcified pieces were prepared for histological analysis with polarized light and von Kossa technique for mineralized tissues. Granulations birefringent to polarized light and an irregular structure like a bridge were observed next to the material; both were von Kossa positive. Also, in the dentin wall tubules a layer of birefringent granulations was observed. The results were similar to those reported for gray MTA, indicating that the mechanisms of action of the white and gray MTA are similar.

Key Words: mineral trioxide aggregate, subcutaneous tissue implantation.


A mineral trioxide aggregate (MTA) was developed at Loma Linda University for the sealing between the tooth and the external surfaces (1). Various in vivo and in vitro investigations have shown good biological properties of this material (2,3). Deposition of neoformed hard tissue in direct contact with MTA was observed in cases of pulp capping (4), pulpotomy (5), repair of furcal perforations (6), root canal fillings (7) and root-end filling (8).

According Holland et al. (9), the mechanism of action of MTA, encouraging hard tissue deposition, is similar to that of calcium hydroxide. Implanting both materials in subcutaneous connective tissue, they observed granules birefringent to polarized light and next
to these granulations irregular tissue, like a bridge, that is von Kossa-positive. Torabinejad et al. (10) reported that MTA has calcium oxide and calcium phosphate in its composition. In contact with water, calcium oxide could form calcium hydroxide (9). The calcium ions from calcium hydroxide react with carbon dioxide from tissue to form calcite crystals that are birefringent to polarized light (11,12). Next to these granulations there is deposition of a hard tissue that is von Kossa-positive and resembles a bridge (9).

There is a commercially available MTA called ProRoot MTA (Dentsply, Tulsa, OK, USA) that has a gray powder. However, Glickman and Koch (13) reported that the manufacturer will place a white ProRoot MTA on the market. The biological difference between the gray and the white MTA, when implanted in subcutaneous tissues is unknown. Thus, the purpose of this study was to analyze the reaction of rat subcutaneous connective tissue to the implantation of dentin tubes filled with white MTA.


The white MTA (Loma Linda University, Loma Linda, CA) was prepared in distilled water and introduced into the root canal of human dentin tubes before being implanted in the subcutaneous connective tissue. The dentin tube preparation and the implantation technique were previously described by Holland et al. (9). The tubes were implanted in 10 rats and the animals were sacrificed after 7 and 30 days. The pieces were removed and fixed in 10% buffered formalin solution at pH 7.0. The undecalcified samples were embedded in paraffin-carnauba wax and serial sections were made at 10 µm intervals using a hard-tissue microtome, according to Holland et al. (14). Some sections were stained by the von Kossa technique for mineralized tissues and other sections, without staining, were examined with a polarized light microscope to locate the birefringent material.


The results observed at 7 and 30 days were the same. For this reason they will be described together. Numerous large granulations birefringent to polarized light and von Kossa positive were observed near the tube opening and generally in contact with the filling material (Figure 1). Next to these granulations, there were extensive areas of irregular tissue positive to the von Kossa technique, like a bridge (Figure 2). A highly birefringent structure was also observed in the interior of the dentin walls tubules (Figure 3). This structure formed a layer that was observed at different depths.


It may appear that in this paper we studied white MTA in subcutaneous tissue without a comparative analysis with a control gray MTA group. In reality the data presented here are an experimental group of another paper developed in rat subcutaneous connective tissue (15). That investigation studied the two kinds of MTA before the material was available commercially. When we observed that only the gray MTA was on the market, without any explanation about the matter, we believed white MTA disappeared for unknown reasons.

In view of this, we decided not to publish the results with white MTA. However, the paper of Glickman and Koch (13) gives new life to the white MTA, at least for us. Thus, the control group of this study is in the previously published paper (15).

The same results reported by Holland et al. (9,15) for gray MTA were observed in this experimentation. This fact suggests that some differences in the materials did not influence the results when they are implanted in rat subcutaneous connective tissue. The MTA employed by Holland et al. (9,15) and the one studied in this experimentation were provided by Dr. Torabinejad (Loma Linda University). We clinically observed two differences with these materials: the color and the setting time, which was greater in the white one.

A large number of papers about MTA were published in the literature before the material was put on the market (1-10); however, the color of the material was not described. Only Faraco (16) compared the two materials in dog dental pulp. He reported slightly better results with gray MTA than with white MTA. The white MTA we studied in this paper was 3 years old and we do not know how much time the material can be kept before its properties change.

The deposition of calcite crystals seems to be very important to the mechanism of action of calcium hydroxide formed after mixing MTA with water. Seux et al. (17) reported a rich extracellular network of fibronectin in close contact with these crystals on incubation in a culture medium without cells. They reported that fibronectin first came from the culture medium and later from the cells. The authors concluded that their findings strongly supported the role of calcite crystals and fibronectin as an initiating step in the formation of a hard tissue barrier. In our paper we observed a von Kossa-positive tissue barrier next to these crystals, remembering the hard tissue deposition in direct contact with MTA as described in a series of papers (6,8). This hard tissue is very irregular in subcutaneous tissue (9,15) but is similar to dentin in pulp tissue (4,5) and looks like cementum in furcal perforations (6) and periapical tissues (7).

In conclusion, the observed results and the mechanism of action of white MTA is very similar to those reported for gray MTA (9,15).


Holland R, Souza V, Nery MJ, Faraco Júnior IM, Bernabé PFE, Otoboni Filho JA, Dezan Junior E. Reação do tecido conjuntivo do rato ao implante de tubos de dentina obturados com um agregado de trióxido mineral branco. Braz Dent J 2002;13(1):23-26.

Foi objetivo deste trabalho estudar a reação do tecido conjuntivo subcutâneo do rato ao implante de tubos de dentina obturados com MTA branco, material a ser introduzido no mercado. Os tubos foram implantados no tecido conjuntivo subcutâneo e os animais sacrificados com 7 e 30 dias de períodos pós-operatório. As peças não descalcificadas foram preparadas para análise histológica com luz polarizada e ténica de von Kossa para tecidos mineralizados. Foi observado junto ao material estudado granulações birrefringentes à luz polarizada seguida de uma estrutura irregular, na forma de ponte, ambos von Kossa positivos. Foi observado também no interior dos túbulos dentinários uma camada de granulações birrefringentes à luz polarizada. Os resultados observados são similares aos descritos para o MTA cinza. Conclui-se que os mecanismos de ação do MTA branco e cinza são similares.

Unitermos: agregado de trióxido mineral, implante em tecido subcutâneo.


1. Lee SJ, Monsef M, Torabinejad M. Sealing ability of a mineral trioxide aggregate for repair of lateral root perforations. J Endodon 1993;19:541-544.

2. Torabinejad M, Hong CU, Pitt Ford TR, Kettering JD. Cytotoxicity of four root end filling materials. J Endodon 1995;21:489-492.

3. Torabinejad M, Hong CU, Pitt Ford TR, Kariyawasan SP. Tissue reaction to implanted Super-EBA and mineral trioxide aggregate in the mandible of guinea pigs: a preliminary report. J Endodon 1995;21:569-571.

4. Pitt Ford TR, Torabinejad M, Abedi HR, Bakland LK, Kariyawasam SP. Using mineral trioxide aggregate as a pulp-capping material. J Am Dent Assoc 1996;127:1491-1496.

5. Soares IML. Resposta pulpar ao MTA - agregado de trióxido mineral - comparado ao hidróxido de cálcio em pulpotomias: estudo histológico em dentes de cães. [Thesis], Brazil: Faculdade de Odontologia, UFSC; 1996.

6. Pitt Ford TR, Torabinejad M, McKendry DJ, Hong CU, Kariyawasam SP. Use of mineral trioxide aggregate for repair of furcal perforations. Oral Surg 1995;79:756-762.

7. Holland R, Souza V, Nery MJ, Otoboni Filho JA, Bernabé PFE, Dezan Junior E. Reactions of dogs´ teeth to root canal filling with mineral trioxide aggregate or a glass ionomer sealer. J Endodon 1999;25:728-730.

8. Torabinejad M, Hong CU, Lee SJ, Monsef M, Pitt Ford TR. Investigation of mineral trioxide aggregate for root-end filling in dogs. J Endodon 1995;21:603-608.

9. Holland R, Souza V, Nery MJ, Otoboni Filho JA, Bernabé PFE, Dezan Junior E. Reaction of rat connective tissue to implanted dentin tubes filled with mineral trioxide aggregate or calcium hydroxide. J Endodon 1999;25:161-166.

10. Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical properties of a new root-end filling material. J Endodon 1995;21:349-353.

11. Holland R. Histochemical response of amputed pulps to calcium hydroxide. Rev Bras Pesq Med Biol 1971;4:83-95.

12. Holland R, Pinheiro CE, Mello W, Nery MJ, Souza V. Histochemical analysis of the dogs´ dental pulp after pulp capping with calcium, barium, and strontium hydroxides. J Endodon 1982;8:444-447.

13. Glickman GN, Koch K. 21st-Century endodontics. J Am Dent Assoc 2000;131:39S-46S.

14. Holland R, Mello W, Nery MJ, Bernabé PFE, Souza V. Reaction of human periapical tissue to pulp extirpation and immediate root canal filling with calcium hydroxide. J Endodon 1977;3:63-67.

15. Holland R, Souza V, Nery MJ, Faraco Júnior IM, Bernabé PFE, Otoboni Filho JA, Dezan Junior E. Reaction of rat connective
tissue to implanted dentin tube filled with mineral trioxide aggregate, Portland cement or calcium hydroxide. Braz Dent J 2001;12:3-8.

16. Faraco Júnior IM. Avaliação histomorfológica da resposta da polpa de dentes de cães submetida ao capeamento com sistema adesivo, cimento de hidróxido de cálcio e dois tipos de agregado de trióxido mineral. [Doctorate thesis]. Araçatuba: Faculdade de Odontologia, UNESP; 1999.

17. 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;36:117-128.

Accepted August 7, 2001