Reaction of Rat Connective Tissue to Implanted Dentin Tube Filled with Mineral Trioxide Aggregate, Portland Cement or Calcium Hydroxide


Roberto HOLLAND1,2
Valdir de SOUZA1,2
Mauro Juvenal NERY1
Italo Medeiros FARACO JÚNIOR3
Pedro Felício Estrada BERNABÉ1
José Arlindo OTOBONI FILHO1
Eloi DEZAN JUNIOR1
 
 

1Faculty of Dentistry of Araçatuba, UNESP, Araçatuba, SP, Brazil
2Faculty of Dentistry, UNIPAR, Umuarama, PR, Brazil
3Faculty of Dentistry, Lutheran University of Brazil, Porto Alegre, RS, Brazil


Correspondence: Professor Roberto Holland, Rua José Bonifácio 1193, 16015-050 Araçatuba, SP, Brasil. e-mail: rholland@foa.unesp.br


Braz Dent J (2001) 12(1): 3-8 ISSN 0103-6440

INTRODUCTION | MATERIAL AND METHODS | RESULTS | DISCUSSION | RESUMO | REFERENCES



The subject of this study was to observe the rat subcutaneous connective tissue reaction to implanted dentin tubes filled with mineral trioxide aggregate, Portland cement or calcium hydroxide. The animals were sacrificed after 7 or 30 days and the undecalcified specimens were prepared for histological analysis with polarized light and Von Kossa technique for mineralized tissues. The results were similar for the studied materials. At the tube openings, there were Von Kossa-positive granules that were birefringent to polarized light. Next to these granulations, there was an irregular tissue like a bridge that was Von Kossa-positive. The dentin walls of the tubes exhibited in the tubules a structure highly birefringent to polarized light, usually like a layer and at different depths. The mechanism of action of the studied materials has some similarity.

Key Words: calcium hydroxide, mineral trioxide aggregate, Portland cement.


INTRODUCTION

A mineral trioxide aggregate (MTA) has been developed at Loma Linda University to seal communications between the tooth and the external surfaces (1). This material was studied in a series of in vivo and in vitro investigations, which reported good sealing ability (2) and tissue behavior (3,4). Formation of new cementum over the material was reported in experimentally perforated furcation (5), in root end filling (6) and root canal filling of dogs teeth (4). Bridge-like dentin was observed in cases of pulp capping (7) and pulpotomy (8) in monkey and dog teeth.

When MTA was implanted in rat connective tissue, in dentin tubes, the observation of Von Kossa-positive granules, birefringent to polarized light was reported (3). Next to these granulations, there was also
irregular tissue like a bridge that was Von Kossa-positive. The dentin walls of the tube exhibited in the tubules a structure highly birefringent to polarized light, usually like a layer and at different depths. Similar results were reported for calcium hydroxide (CH) (9-11).

Wucherpfenning and Green (12) reported that both MTA and Portland cement (PC) seem almost identical macroscopically, microscopically and by X-ray defraction analysis. They reported that both substances support matrix formation in a similar fashion in cultures of osteoblast-like cells, and also as apposition of reparative dentin when used as direct pulp capping material in rat teeth.

Estrela et al. (13) studied the antimicrobial and chemical properties of some materials, including MTA and PC. The analysis of chemical elements present in MTA and in two samples of PC were performed with a spectrometer of fluorescence of X-ray. They reported that PC contains the same principle chemical elements as MTA, except that MTA also contains bismuth. They also reported that PC had pH and antimicrobial activity similar to MTA.

Considering the reported results, the purpose of this study was to analyze the reaction of rat subcutaneous connective tissue to the implantation of dentin tubes filled with MTA, PC or CH, in order to observe if the calcified structures reported by Holland et al. (3) occur with these materials.


MATERIAL AND METHODS

Dentin tubes were prepared from human teeth roots. The canals were enlarged up to reamer #35, over-instrumenting the apical foramen about 2 mm. The length of the tubes was 7 mm, and the thickness of their outer walls about 0.5 mm. 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), PC (Cia Portland Cement Itaú, Itaú de Minas, MG, Brazil) or CH (Reagen, Quimibrás Indústrias Químicas S/A, Rio de Janeiro, RJ, Brazil), all in distilled water, and immediately implanted subcutaneously in the dorsal region, on each side of the midline in 30 rats. For control purposes, empty dentin tubes were implanted in 10 additional animals.

The rats were sacrificed after 7 or 30 postoperative days. The tubes and surrounding tissues were removed and fixed in 10% buffered formalin at pH 7.0. The samples were embedded in a mixture of paraffin (95%) and carnauba wax (5%), according to Holland et al. (14). The sectioning was done serially at 10 µm intervals 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 stained according to the Von Kossa technique; other sections without staining were examined under a polarized light microscope to locate the birefringent material. Some sections were decalcified for 10 minutes in EDTA before staining by hematoxylin and eosin.


RESULTS

Control Group

The implanted tubes were surrounded by a layer of exudate with neutrophils at 7 days. Over this area there were young fibroblasts and chronic inflammatory cells. There was an eosinophilic structure with a mild neutrophilic infiltrate in the tubes. In the 30-day group, there was growth of connective tissue with mild chronic inflammatory cells in the tube space. Outside, the tubes were surrounded by a thin fibrous capsule with a mild chronic inflammatory reaction in 5 cases (Figure 1).

Calcium Hydroxide

At 7 and 30 days, the undecalcified sections exhibited similar results. Numerous large granulations, birefringent to polarized light and positive to the Von Kossa technique, were observed near the tube opening (Figure 2). Next to these granulations, there were extensive areas of irregular tissue, intensively positive to the Von Kossa technique (Figure 3). In the interior of the dentin wall tubules there was a highly birefringent structure forming a layer at different depths (Figure 4).
 


Figure 1. Control group, 30 days. Note dentin wall (D), ingrowth of connective tissue with chronic inflammatory cells (IN) and a fibrous capsule and connective tissue outside of the tube (OT) (H & E, 100X).

Figure 2. Calcium hydroxide, 30 days. Note dentin (D) and numerous granulations (arrows), birefringent to polarized light (40X).

Figure 3. Calcium hydroxide, 30 days. Observe extensive areas of irregular tissues (arrows) highly positive to the Von Kossa technique (40X).

Figure 4. Calcium hydroxide, 7 days. Note calcium hydroxide (CH), dentin (D) and a layer (L) of a highly birefringent structure localized in the dentin tubules (polarized light, 72X)
 

The decalcified sections showed some irregular basophilic areas corresponding to the highly Von Kossa-positive one observed in the undecalcified sections. These areas exhibited cellular nucleus inclusions in their mass. Surrounding this area, there was initially (7 days) connective tissue with a mild or moderate chronic inflammatory reaction and some giant cells. At 30 days, the connective tissue around the calcified areas was fibrous, with some giant cells and a mild chronic in
flammatory reaction.

MTA and Portland Cement

The results observed with MTA and PC were similar and almost the same as that observed with CH at 7 and 30 days. The undecalcified sections exhibited granulations positive to Von Kossa and birefringent to polarized light (Figures 5-7). These granulations were usually less numerous than those of calcium hydroxide specimens and generally in contact with the filling material. There were also extensive and irregular areas, highly positive to Von Kossa technique, next to the birefringent granulations (Figures 8-11). A birefringent structure was also observed in the dentinal wall tubules forming a layer located at different depths, but generally next to the filling material (Figures 12-15). The decalcified sections stained by hematoxylin and eosin showed the same results described for calcium hydroxide at 7 and 30 days.
 


Figure 5. MTA, 7 days. Observe birefringent granulations (arrow) in direct contact with MTA (72X).

Figure 6. Portland cement, 7 days. Birefringent granulations (arrow) are observed at the tube opening (polarized light, 72X).

Figure 7. MTA, 7 days. Note birefringent granulations (arrow) in direct contact with MTA. A birefringent structure (BS) was observed in the dentin tubules (polarized light, 72X).

Figure 8. MTA, 7 days. Observe Von Kossa-positive irregular tissue (VK) located near the tube opening (100X).

Figure 9. MTA, 30 days. A layer of a structure positive to Von Kossa (VK) is observed at the tube opening (40X).

Figure 10. Portland cement, 7 days. Von Kossa-positive irregular tissue (VK) is observed near the implanted material (72X).

Figure 11. Portland cement, 30 days. Observe dentin wall (D), filling material (PC) and an irregular structure (VK) positive to Von Kossa technique (40X).

Figure 12. MTA, 7 days. Note the filling material (MTA), dentin (D) and a structure localized in the dentin tubules (PL) highly birefringent to polarized light (72X).

Figure 13. MTA, 30 days. Observe MTA, dentin (D) and the same birefringent structure (PL) reported in figure 12 (polarized light, 72X).

Figure 14. Portland cement, 7 days. Note PC and a highly birefringent structure (PL) in the dentin wall (D) tubules (polarized light, 72X).

Figure 15. Portland cement, 30 days. A highly birefringent structure (PL) localized in the dentin wall (D) tubules is observed near the filling material (PC), as reported in figure 14 (polarized light, 72X).


DISCUSSION

The dentin tubes for subcutaneous implantation were used according to Souza et al. (10) to observe the action of the studied materials not only on the connective tissue but also on dentin walls. The results in the control group are in agreement with previous reports (3,10).

The present results with CH are similar to those described by Souza et al. (10) and Holland et al. (3) in subcutaneous implantation of dentin tubes filled with this material. The described granulations birefringent to polarized light were previously reported in dental pulp (9,15) and periapical tissues (14,15) when the filling material was CH. According to Holland (9), these granulations are calcite crystals originated from a reaction of the calcium from the CH with the carbon dioxide from the tissue. The same phenomenon has been described by Seux et al. (16) in an in vitro experiment. These authors observed a rich extracellular network of fibronectin in close contact with calcite crystals on incubation in a culture medium without cells. They reported that fibronectin came from the culture medium and later from the cells. Seux et al. (16) concluded that their findings strongly support the role of calcite crystals and fibronectin as an initiating step in the formation of a hard tissue barrier. In our experiment, we observed the crystals and a calcified tissue that resembles a barrier at the opening of the tubes. The same observation was also reported by Souza et al. (10) and Holland et al. (3) in similar studies.

In this experiment, we also observed in the dentin wall tubules a birefringent structure highly similar to the one reported on dentin by Holland et al. (3,17) when they used CH. Mjör and Furseth (18), studying the action of CH on dentin, reported mineral crystals in the tubules, which could be responsible for the minor permeability on dentin described by Pashley et al. (19) after CH use.

The same results reported for CH were observed with MTA and PC in the present study. The question is how this same phenomenon occurs if MTA and PC cements do not have CH in their composition. According to Torabinejad et al. (20), MTA shows specific phases throughout the material after the reaction with water. All MTA was divided into calcium oxide and calcium phosphate. Although MTA does not have CH, it does have calcium oxide that could react with tissue fluids to form CH. The sequence of the reaction was already described. We do not know if PC has calcium oxide in its composition, after the reaction with water, but the similarity of results between PC and MTA suggests it is possible. The results of this research support those described previously by Wucherpfenning and Green (12) and Estrela et al. (13) that suggest MTA and PC are almost the same material.

In conclusion, it seems that the mechanism of action of MTA and PC, encouraging hard tissue deposition, has some similarity to that of CH. The same can be concluded about the mechanism of action between MTA and PC. Of course, other experiments are necessary to confirm the observed data.


RESUMO

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 agregado de trióxido mineral, cimento Portland ou hidróxido de cálcio. Braz Dent J 2001;12(1)3-8.

Foi objetivo deste trabalho observar a reação do tecido subcutâneo do rato ao implante de tubos de dentina obturados com agregado de trióxido mineral, cimento Portland ou hidróxido de cálcio. Os animais foram sacrificados após 7 ou 30 dias e os espécimes, não descalcificados, foram preparados para análise histológica com luz polarizada e técnica de Von Kossa para tecidos mineralizados. Os resultados foram similares para os 3 materiais estudados. Próximo às aberturas dos tubos foram observadas granulações Von Kossa positivas, birrefringentes à luz polarizada. Junto dessas granulações foi observado um tecido irregular na forma de uma ponte, também Von Kossa positivo. As paredes de dentina dos tubos exibiram uma estrutura altamente birrefringente à luz polarizada, no interior dos túbulos, formando uma camada em diferentes profundidades. Diante do observado, é possível que os mecanismos de ação dos materiais estudados sejam similares entre si.

Unitermos: hidróxido de cálcio, agregado de trióxido mineral, cimento Portland.


REFERENCES

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, Watson TF, Pitt Ford TR. Sealing ability of a
mineral trioxide aggregate when used as a root end filling material. J Endodon 1993;19:591-595.

3. Holland R, Souza V, Nery MJ, Otoboni Filho JÁ, 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.

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

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

6. 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.

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

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

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

10. Souza V, Holland R, Mello W, Nery MJ. Reaction of rat connective tissue to the implant of calcium hydroxide pastes. Rev Fac Odontol Araçatuba 1977;6:69-79.

11. 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.

12. Wucherpfennig AL, Green DB. Mineral trioxide vs Portland cement: two biocompatible filling materials. J Endodon 1999;25:308 (Abstract).

13. Estrela C, Bammann LL, Estrela CRA, Silva RS, Pécora JD:.Antimicrobial and chemical study of MTA, Portland cement, calcium hydroxide paste, Sealapex and Dycal. Braz Dent J 2000;11:19-27.

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, Mello W, Russo MC. Healing process of the pulp stump and periapical tissue in dog teeth. III. Histopathological findings following root filling with calcium hydroxide. Rev Fac Odontol Araçatuba 1978;7:25-38.

16. Seux D, Couble ML, Hartmann DJ, Gauthier JP, Magloise 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.

17. Holland R, Souza V, Nery MJ, Bernabé PFE, Otoboni Filho JA. The effect of calcium hydroxide in dentin. Rev Fac Odontol Araçatuba 1978;7:177-183.

18. Mjör IA, Furseth R. The inorganic phase of calcium hydroxide and corticosteroid-covered dentin studied by electron microscopy. Arch Oral Biol 1968;13:755-763.

19. Pashley DH, Kalathoor S, Burnham D. The effects of calcium hydroxide on dentin permeability. J Dent Res 1986;65:417-420.

20. 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.


Accepted August 30, 2000
Braz Dent J 12(1) 2001


BACK TO CONTENTS