Neste capítulo estão relatadas as complementações e as mudanças ocorridas no planejamento e execução dos experimentos desta pesquisa.
3.1 O projeto de qualificação de tese por mim apresentado a banca, se intitulava ―Revascularização de dentes com polpa necrosada e rizogênese incompleta: estudo clínico randomizado‖. No entanto, não estou apresentando esse trabalho como tese de doutorado devido a dificuldade de conseguir pacientes que se enquadrassem nos critérios de inclusão do estudo.
3.2 O trabalho apresentado como tese de doutorado foi qualificado durante o meu mestrado.
3.3 No teste de biocompatibilidade houve uma perda da amostra durante o processamento histológico, o que reduziu o nosso n de 18 animais para 15.
3.4 As mensurações de cor seriam prorrogadas por mais três meses, no entanto, o espectrofotômetro (Vita Easyshade) da Faculdade de Odontologia de Pelotas encontrava-se danificado. As mensurações foram realizadas, portanto, pelo tempo máximo de 9 meses, sendo que esse tempo foi suficiente para verificarmos a discoloração dentária ocorrida nos espécimes. É importante salientar que o uso de outro equipamento poderia trazer desvio de resultados.
4 Artigos 4.1 Artigo 1*
Tissue reaction to a new MTA-based root canal filling material for primary teeth
K. J. PILOWNIC1; A. P. N. GOMES1; A. C. FELIX2; A. R. ROMANO1& F. G. PAPPEN1
1
Graduate Program in Dentistry, Federal University of Pelotas, Pelotas, RS, Brazil.
2
Central Vivarium, Faculty of Veterinary, Federal University of Pelotas, Pelotas, Brazil
Correspondence address: Fernanda Geraldo Pappen Graduate Program, Dentistry, Federal University of Pelotas
R. Gonçalves Chaves, 457, sala 507, Pelotas, RS, Brasil. CEP: 96015-560 E-mail: [email protected]
Abstract
Purpose: This study evaluated the tissue reactions to the experimental MTA- based material in comparison to Zinc Oxide and Eugenol cement (ZOE); Vitapex; and Calen paste thickened with zinc oxide (Calen+ZO). Methods: Fifteen Wistar rats had received four implants containing the tested filling materials. The tubes had one of the ends closed with gutta-percha used as negative control. After 15, 30 and 60 days, the animals were euthanized and the tubes and surrounding tissue were removed and processed for histopathologic evaluation. The specimens were evaluated for intensity of inflammatory response, fibrous capsule thickness, and for the presence of calcification and giant cells. The results were analyzed by the Kruskal-Wallis test. Results: On day 15, thick fibrous capsule and moderate inflammatory response were observed in Vitapex and experimental MTA-based material, while in ZOE and Calen+ZO groups, the intensity of inflammatory reaction was moderate to severe. On day 30, most of experimental MTA-based material specimens showed mild inflammatory reaction and a thin fibrous capsule. After 60 days, Calen+ZO group still showed moderate inflammation, while the others showed predominantly mild inflammatory reaction and a thin fibrous capsule. Conclusion: Experimental MTA-based material had showed to be biocompatible, representing an alternative for endodontic filling of primary teeth. Key Words: primary teeth; root canal filling materials; biocompatibility
Introduction
An ideal endodontic filling material for primary teeth must have several properties that make it suitable for use, such as resorption at a rate similar to that of the primary root, be harmless to the periapical tissues and permanent tooth germ and readily resorb if pressed beyond the apex.1,2
Several materials have been used for endodontic filling in primary teeth. Zinc oxide (ZO) and zinc oxide eugenol (ZOE) have been widely used in the primary dentition. 1,2,3 Nevertheless, due to their irritating potential 1,2 and low resorption capacity3 the use of these materials have been substituted by iodoform containing materials, or calcium hydroxide pastes, because of their antibacterial activity, biocompatibility and easy resorption.2,4,5 Other studies have also suggested the use of association of calcium hydroxide and zinc oxide
6,7
as well as calcium hydroxide and iodoform. 4,6,7
Recently, an experimental MTA-based material, ready to use, endodontic filling material for primary teeth had been developed (Angelus, Londrina, PR, Brazil). According to the manufacturer, the experimental MTA-based material has in its composition mineral trioxide aggregate (MTA), calcium tungstate, polyethylene glycol, glycerin, silicate oxide e silicon. Unpublished data demonstrated that this material has acceptable radiopacity, low cytotoxity, antimicrobial efficacy and proper pH.
The biocompatibility of all experimental dental materials that might come in contact with tissues should be examined. Biological properties are amongst the most important aspects of endodontic filling materials because the release of substances from the materials may generate reactions in the periapical
tissue. The subcutaneous connective tissue implantation method is one of the main methods used to evaluate the biocompatibility of a material.10-13
Therefore, the purpose of this study was to evaluate the tissue reaction induced by an experimental MTA-based material when implanted in subcutaneous connective tissues, in comparison with others endodontic filling materials used for primary teeth.
Materials and Methods
The materials tested were the experimental MTA-based material (Angelus, Londrina, PR, Brazil); Zinc Oxide and Eugenol cement (ZOE) (S.S.White Artigos Dentários Ltda., Rio de Janeiro, RJ, Brazil); Vitapex, a premixed calcium hydroxide and iodoform paste (Neo Dental International Inc, Federal Way, WA, USA); Calen, a premixed calcium hydroxide and polyethylene glycol-based paste (S.S.White Artigos Dentários Ltda.), thickened with zinc oxide (ZO) (1:1)(Biodinâmica Química e Farmacêutica Ltda.,Ibiporã, PR, Brazil).
The materials were inserted into sterile polyethylene tubes measuring 10 mm long and 1.0 mm internal diameter (Abbott Laboratorios do Brasil, São Paulo, Brazil). To avoid the materials extrusion, each tube had one of the ends closed with gutta-percha. The side of the tube containing gutta-percha was used as negative control. After complete filling, the tubes were immediately implanted on animal‘s dorsal subcutaneous tissue.
The Research Ethics Committee for Animal Use of the Federal University of Pelotas approved this study (Protocol #8977; 02/04/14). All procedures were carried out in accordance with institutional guidelines for animal care and use. Fifteen Wistar rats (Rattusnorvegicus; age, 6 months; weight, ~250 g) were
used in this study. The animals‘ tails were marked for individual identification. The rats were housed in plastic cages (twoper cage) placed in ventilated racks (Alesco, Monte Mor, SP, Brazil) at 22°C with a 12 hours light⁄dark cycle (lights on at 7:00 am, off at 7:00 pm). During the experiments, the rats were provided with a standard diet of rat chow (Nuvilab, Colombo, PR, Brazil) and filtered water ad libitum.
The animals were anesthetized by intraperitoneal injection of ketamine (80mg kg-1 by weight) combined with xylazine (4mg kg-1 by weight; both from SDC ECommerce for Agricultural Products LTDA, Marilia, SP, Brazil). Dorsal trichotomy and antisepsis was performed using iodinated alcohol and sterile distilled water. 14
Four 2-cm incisions were made on the shaved backs in a head-to-tail orientation using a #15 Bard Parker blade (LamedidSolidor, Osasco, SP, Brazil). Using blunt-tipped scissors, the lateral tearing of the subcutaneous tissue provided four surgical cavities displayed in quadrants equidistant from the center of the animals' backs. Each animal received four implants, two in the scapular region and two in the caudal region. The tubes filled with materials were implanted into the spaces created by blunt dissection, according to a previously established placement order and site rotation, and the skin was closed using a 4-0 silk suture (Johnson & Johnson Produtos Profissionais Ltda, São José dos Campos, SP, Brazil). Sterile instruments and aseptic techniques were used throughout the experiments. The animals were placed in individual cages until they recovered from anesthesia. To aid recovery, paracetamol (0.06 mg g-1day-1) was added to their drinking water for 72 h.15
The animals were euthanized at 15, 30 and 60 days after the dorsal implantation (n=5/group at each time point). They were anesthetized with an intraperitoneal injection of chloral hydrate (350 mg/kg) (Sigma Aldrich, St. Louis, MI, USA), and physiological saline (Sigma Aldrich, St. Louis, MI, USA) followed by 10% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) (Sigma Aldrich, St. Louis, MI, USA) was perfused transcardially. The tubes and the surrounding tissues were removed and fixed in 10% buffered formalin at a pH of 7.0. The tubes were bisected transversely, and both halves were subsequently cut longitudinally with a sharp blade to allow the surfaces to maintain contact with the processing solutions. The specimens were paraffin embedded, sectioned serially to 3-mm slices, and stained with hematoxylin and eosin.
Tissue reactions in contact with the material at the opening of the tube were scored as follow: 0, no or few inflammatory cells and no reaction; 1, less than 25 cells and mild reaction; 2, between 25 and 125 cells and moderate reaction; and 3, 125 or more cells and severe reaction. Fibrous capsules were classed as ‗‗thin‘‘ or ‗‗thick‘‘ if the thickness was <150 mm or >150 mm, respectively. Calcification was recorded as present or absent. Also the presence of giant cells was recorded. In a random order, only one control in each animal was evaluated. The observer, who was a pathologist, was blinded to the treatment. The results were analyzed by the Kruskal-Wallis test at a 5% significance level.
Results
On day 15, thick fibrous capsule formation and moderate cell inflammatory infiltration by lymphocytes and macrophages were observed with
all the Vitapex and experimental MTA-based material specimens, while in ZOE and Calen+ZO groups, the intensity of inflammatory reaction was moderate to severe (p = 0.001). The capsule thickness surrounding the tubes was significantly thinner in the control specimens when compared with the experimental materials (p < 0.001) and the presence of giant cells in this period varied from 20 to 60% in the experimental groups (p > 0.005). The presence of calcificated precipitates was noticed in 40% of specimens from Calen+ZO and Vitapex on day 15, and it was not observed in ZOE and experimental MTA- based material groups (Table 1).
The Vitapex group showed a large capsule extension, with evident presence of macrophage foam cells on day 15 and 30. Mild to moderate inflammatory cell infiltration and a reduction in the thickness of the fibrous capsule was evident from day 30 onward (Fig.1C;H).
On day 30, moderate inflammatory cell infiltration was observed in 100% of Vitapex and Calen+ZO, and 80% of ZOE specimens, while most experimental MTA-based material specimens showed mild inflammatory reaction only (p = 0.001). The fibrous capsule thickness was also significantly thinner in the experimental MTA-based material group (p = 0.001). On this period, the presence of giant cells was mostly observed in Vitapex specimens, but was still noticed in the other groups (p > 0.005). Vitapex and Calen+ZO induced the deposition of calcified tissue more frequently than the other materials (p = 0.02).
In the control group, mild inflammatory reaction with lymphocytes and macrophages was observed at 15 and 30 days. After 60 days, inflammatory
response was absent in the areas in contact with gutta-percha. There was no giant cells or calcificated precipitated in the specimens from control group.
At 60 days, the inflammatory reaction was mild in most specimens of ZOE, experimental MTA and Vitapex, while 80% of specimens from Calen+ZO group still showed moderate inflammatory infiltrate (p = 0.001). The capsule extension was predominantly thin for all the materials (p > 0.005), and the presence of giant cells were recorded only in few ZOE specimens (p > 0.005). The occurrence of calcification, was more common in specimens from ZOE and Calen+ZO (p = 0.003).
Discussion
The biomechanical endodontic treatment of primary teeth remains far from ideal, especially due to their anatomical features and difficulties in obtaining a good radiographic view of the apex for determining the working length.16Due to the physiological root resorption process, materials used in endodontic treatment of primary teeth should be harmless to the periapical tissues and permanent tooth germs.17The experimental MTA-based material has been developed as an alternative to be used in the endodontic filling for primary teeth, and in the present study demonstrated promising results for the material in terms of subcutaneous implantation.
The method of implantation of endodontic filling materials in the subcutaneous tissue of rats, in part simulates the situation of the root canal, and is one of the most appropriate tests to evaluate materials biocompatibility.12- 13
biocompatibility of experimental MTA-based material. Besides, the inert nature of polyethylene tubes and their ability to expose a test material to living tissue in a controlled and effective manner justify its choice.18In the present methodology, one end of the tubes were sealed with gutta-percha in order to avoid material extrusion. Also, since gutta-percha is well tolerated by connective tissues, this tube extremity was used as negative control.9,13
Both, the control and the tested material specimens presented significantly less inflammation severity at longer time intervals (30 and 60 days) in comparison to 15 days specimens. Similarly, the capsule extension was mostly thick in all groups on day 15 and it declined by over the time, demonstrating that every tested material was well tolerated by the subcutaneous tissues. It was possible to observe collagen fiber formation at the area in contact with the all the tested materials. Studies had described that there is a meaningful relationship between inflammation and fibrous capsule thickness.19, 20, 21 Makkes et al.19 concluded that decreasing capsule thickness is a sign of biocompatibility, and the deferred harmful effects of a material are considered to be more important than its initial effects in biocompatibility tests.22
On day 60, the inflammatory reaction was absent in control group; mild in most specimens of ZOE, experimental MTA-based material and Vitapex, while most specimens from Calen+ZO group still showed moderate inflammatory infiltrate. Contrary to our findings, Nelson-Filho et al.23 and Queiroz et al.24 had reported that zinc oxide does not interfere the biocompatibility of calcium hydroxide. When used alone, Calen paste present recognized biocompatibility23,25, thus the persistent inflammation observed in Calen+ZO group may be attributed to the higher concentration of zinc in the medicament,
since zinc has the capacity to influence in the inflammatory process for reducing phagocytic capacity of macrophages and to interfere in the membrane of the lysosomes.10, 11
The tissue reaction demonstrated by ZOE in this study was similar to those described by other authors for ZOE and other ZOE-based sealers.26, 27 In the initial periods, ZOE demonstrated severe inflammation which had decline by over the time. ZOE cement is initially highly toxic because of its traces of unreacted eugenol, exerting a toxic reaction to cells.28,29 This profile tends to change significantly along the time, when the material becomes more biocompatible, due to the reduced availability of unreacted eugenol.
Although the conventional MTA is well known by its excellent biocompatibility,30,31 The composition of experimental MTA-based material differs from the traditional MTA: besides a portion of MTA, the experimental filling material also has in its composition calcium tungstate, polyethylene glycol, glycerin, silicate oxide and silicon. Our results demonstrated an optimal tissue reaction to the experimental MTA-based material, what had occurred probably due to the presence of MTA in its composition and the absence of toxic components. Also, despite the optimal clinical and radiographic success rates of Vitapex as a endodontic filling material of primary teeth,2,32 there are few information regarding its biocompatibility. Similarly to our results, Huang et al.4 also showed Vitapex to be biocompatible, since it resulted in high survival rate of cells after the experimental period.
In all the experimental groups, it was possible to observe the presence of giant cells, mainly within 15 days. The presence of giant cells may be
associated with the presence of material that the organism hardly breaks down31. However, the number of particles of materials present in the subcutaneous tissue had decreased by over the time, suggesting the capacity of these filling materials to be reabsorbed when in contact with connective tissue. The ability of the material to be resorbable is considered one of the main requirements for the indication of a endodontic filling material for primary teeth. The material resorption must occur simultaneously to root resorption during exfoliation, allowing normal eruption of permanent teeth. Also in cases of over- filling, the material should be resorbable and non-toxic to the periapical tissues and germ of the permanent.1,2
Calcific precipitation was observed around implantation sites in all experimental groups, but more frequently in Calen+ZO specimens. The production of calcific structures in subcutaneous investigations is a sign of osteoinductivity of the material.33The osteoinductivity and conductivity the materials may be attributed to the release of calcium and phosphorous as well as the formation of hydroxyapatite crystals over the material.25,34 Similar to our study, other authors had also reported the presence of calcification in conventional MTA and Calcium hydroxide based materials.30MTA does not have calcium hydroxide in its composition, but it has calcium oxide that could react with tissue fluids to form calcium hydroxide, what can explain the similar behavior of experimental-MTA, Calen+ZO and Vitapex regarding the induction of calcification. However, no prior study demonstrated the osteoinductivity of ZOE.
Within the limits of this study, our results showed that all endodontic filling materials were well tolerated by the subcutaneous tissues. The
experimental-MTA based material showed to be biocompatible, representing an alternative for the endodontic filling of primary teeth.
REFERENCES
1. Kubota K, Golden BE, Penugonda B. Root canal filling materials for primary teeth: A review of the literature. J Dent Child.1992;59(3):225–7. 2. Mortazavi M, Mesbahi M. Comparison of zinc oxide and eugenol, and
Vitapex for root canal treatment of necrotic primary teeth. Int J Paediatr Dent 2004;14(6):417– 24.
3. Fuks AB, Eidelman E. Pulp therapy in the primary dentition. CurrOpin Dent 1991;1(5):556 –63.
4. Huang TH, Ding SJ, Kao CT. Biocompatibility of various formula root filling materials for primary teeth. J Biomed Mater Res B Appl Biomater.2007; 80b(2):486-90.
5. Ranly DM, Garcia-Godoy F. Current and potential pulp therapies for primary and young permanent teeth. J Dent. 2000;28(3):153–61
6. Chawla HS, Mathur VP, Gauba K, Goyal A. A mixture of Ca(OH)2 paste and ZnO powder as a root canal filling material for primary teeth: a preliminary study. J IndianSocPedodPrevDent.2001;19(3):107-9.
7. Queiroz AM, Nelson-Filho P, Silva LA, Assed S, Silva RA, Ito IY.Antibacterial activity of root canal filling materials for primary teeth: zinc oxide and eugenolcement, Calen paste thickened with zinc oxide, Sealapex and EndoREZ. Braz Dent J. 2009; 20(4):290-6.
8. Kaplan AE, Ormaechea MF, Picca M, Canzobre MC, Ubios AM. Rheological properties and biocompatibility of endodontic sealers. IntEndod J. 2003;36(8):527–32.Ho YC, Huang FM, Chang YC. Mechanisms of cytotoxicity of eugenol in human osteoblast cells in vitro. IntEndod J.2006;39 (5):389–93.
9. Pinto DN, Sousa DL, Rocha RB, Moreira JJ. Eighteen-month clinical and
radiographic evaluation of two root canal-filling materials in primary teeth with pulp necrosis secondary to trauma. Dent Traumatol.2011;27(3):221– 4.
10. Silva, LAB, Leonardo MR, Oliveira DS, Silva RA, Queiroz AM, Hernández PG, Nelson-Filho P. Histopathological evaluation of root canal filling materials for primary teeth. Braz Dent J.2010; 21(1):38–45.
11. Olsson B, Sliwkowsky A, Langeland K. Subcutaneous implantation for the biological evaluation of endodontic materials. J Endod. 1981;7(8):355–67. 12. Torneck CD. Reaction of rat conective tissue to polyethylene tube
implants.Oral Surg.1966;21(3):379-87.
13. Sobrinho AP, Barros MH, Nicoli JR,Carvalho MA, Farias LM, Bambirra EA.et al. Experimental root canal infections in conventional and germ-free mice. J Endod.1998; 24(6), 405-8.
14. Simon S, Cooper P, Smith A, Picard B, Ifi CN, Berdal A. Evaluation of a new laboratory model for pulp healing: preliminary study. Int Endod J.2008; 41(9): 781-90.
15. Özalp N, Saroglu I, Sönmez H. Evaluation of various root canal filling materials in primary molar pulpectomies: an in vivo study. Amer J Dent 2005;18(6):347-50.
16. Mortazavi M, Mesbahi M. Comparison of zinc oxide and eugenol, and Vitapex for root canal treatment of necrotic primary teeth. Int J Paediatr Dent. 2004;14:417-24.
17. Langeland K, Olsson B, Pascon EA. Biological evaluation of hydron. J Endo.1981;7(5):196-204.
18. Makkes PC, van Velzen SK, Wesselink PR, deGreeve PC. Polyethylene tubes as a model for the root canal. Oral Surg, Oral Med, Oral Pathol.