Histological evaluation of bone response to pediatric endodontic pastes: an experimental study in guinea pig

Histological evaluation of bone response to pediatric endodontic pastes: an experimental study in guinea pig

Andréa Mara Lacativa; Adriano M. Loyola; Cassio José Alves Sousa

Department of Endodontics, Dental School, UFU - Federal University of Uberlândia, Uberlândia, MG, Brazil

^{Correspondence}

ABSTRACT

This study aimed to evaluate by the intra-osseous implant technique the most commonly used materials for pulp therapy in pediatric dentistry: calcium hydroxide (CH), Guedes Pinto paste and CTZ paste, according to FDI (1980) and ANSI/ADA (1982) recommendations. Thirty guinea pigs, 10 for each material, divided into experimental periods of 4 and 12 weeks received one implant on each side of the lower jaw symphysis. The external lateral tube wall served as control for the technique. At the end of the observation periods, the animals were euthanized and specimens were prepared for routine histological examination. It was observed that CH and CTZ paste induced severe inflammation, a large amount of necrotic tissue, lymphocytes, foreign body cells and bone resorption, while Guedes Pinto Paste induced little or no inflammation in the 4-week observation period. After 12 weeks, the reactions to CH and Guedes Pinto paste were also absent/mild, presenting a general pattern of replacement by recently formed bone tissue while a moderate to severe inflammatory response was observed with CTZ paste. Guedes Pinto paste presented acceptable biocompatibility levels in both analyzed periods; CH only showed acceptable biocompatibility in the 12-week period while CTZ paste showed no biocompatibility in both periods. Among the tested materials, only Guedes Pinto paste presented an acceptable biocompatibility.

Keywords: biocompatibility, dental apex, pediatric dentistry, pulp therapy, root filling.

RESUMO

A pesquisa teve como objetivo avaliar a biocompatibilidade através da técnica de implantes intra-ósseos dos materiais utilizados em odontopediatria para tratamento pulpar: hidróxido de cálcio, pastas Guedes Pinto e CTZ, de acordo com as recomendações da FDI (1980) e ANSI/ADA(1982). Trinta guinea pigs, dez para cada material, divididos em períodos experimentais de 4 e 12 semanas receberam um implante em cada lado da sínfise mandibular. A parede lateral externa do copo serviu como controle para a técnica. No final dos períodos experimentais, os animais foram sacrificados e os espécimes preparados para o exame histológico de rotina. Observou-se que o hidróxido de cálcio e a pasta CTZ mostraram reação inflamatória severa, grande quantidade de tecido necrosado, linfócitos, células de corpo estranho e reabsorção óssea; enquanto a pasta Guedes Pinto induziu pouca ou nenhuma inflamação no período de 4 semanas. Após 12 semanas as reações para o hidróxido de cálcio e pasta Guedes Pinto foram ausentes/suaves apresentando um padrão geral de substituição por tecido ósseo neoformado, enquanto uma resposta inflamatória de moderada a severa foi observada para a pasta CTZ. A pasta Guedes Pinto apresentou níveis aceitáveis de biocompatibilidade nos dois períodos analisados; hidróxido de cálcio apresentou biocompatibilidade aceitável somente no período de 12 semanas e a pasta CTZ não mostrou biocompatibilidade em ambos os períodos. Entre estes, apenas a pasta Guedes Pinto apresentou níveis de biocompatibilidade nos dois períodos analisados.

INTRODUCTION

Success in Endodontics depends on the complete removal of canal content, followed by apical closure using a material of adequate compatibility to avoid possible irritation to the remaining pulp and periapical tissues. The constant evolution of concepts is brought along with new methods, new techniques and the development of more effective materials and instruments. In pediatric dentistry, pulp therapy is performed due to pulp exposure or necrosis and aims at keeping the primary teeth in place until the exfoliation period (1,2). Although the concepts of endodontic treatment of primary and permanent teeth are different (1), biological behaviors of pulp and periapical tissues of primary teeth are similar to those of the permanent dentition, except for the physiological root resorption. In pediatric dentistry, biocompatibility with periapical tissues is of utmost importance, since any material which is not biologically acceptable may be responsible for failures (2,3).

A large number of materials have been proposed to achieve this goal; however, there is a lack of consensus concerning to when pulp therapy is required in primary teeth. Several techniques and materials have been empirically used for more than a century, often considering only the clinical aspects (1,2,4,5).

Particularly, iodophorm-based and calcium hydroxide (CH)-based pastes have been usually and successfully used in endodontic therapy for primary teeth, based specially on clinical application (3-5) and cytotoxicity assays (1). However, few studies focused on bone response to these materials (2).

Considering the biological properties of endodontic materials, there is a broad range of characteristics which may be considered and methodologies available for this purpose, including initial tests, secondary tests and usage studies. The initial evaluation comprises some basic ways of assessing biological properties with in vitro methods. The secondary assessments are performed in vivo in laboratory animals and implantation as an experiment belongs to this category. Usage studies are applications of materials in therapeutic procedures on primates or humans (6,7).

The aim of this study was to compare the biocompatibility of CH, Guedes Pinto paste and CTZ paste with the intra-osseous implant technique.

MATERIAL AND METHODS

The materials tested in this study were CH, Guedes Pinto paste (composition: iodoform, PMCC and rifocort) and CTZ paste (composition: zinc oxide, tetracycline, chloramphenicol and eugenol). All materials were prepared according to the manufacturers' instructions for its clinical use and inserted in a polytetrafluorethylene cup (Teflon^®; DuPont, HABIA, Knivsta, Sweden), ensuring that air was not entrapped.

The intra-osseous implant in the guinea pig mandible proposed by Spångberg (8,9) was used in this study, following standardized methods to evaluate the biological properties recommended by Technical Report #9 (6) and by Document #41 (7).

Thirty guinea pigs weighing about 800 g were used in the experiment. Each animal received two implants of the same material, with a total of 10 specimens for each material and observation period. Additionally, the connective tissue response along the lateral wall outside the cup served as a negative control for the technique. According to Technical Report #9 (6) and Document #41 (7) the reactions along the external periphery of the Teflon^® cup reflect the trauma caused by surgical procedures required for the introduction of the Teflon^® cup and its contents. Teflon^® itself was proven to cause only insignificant irritation to tissues (10).

Animals were anesthetized intramuscularly with 0.6 mL of ketamine chlorohydrate (100 mg/mL; Francotar; Virbac do Brasil Indústria e Comércio Ltda. Santo Amaro, SP, Brazil) and 0.5 mg/mL of 2% xylazine chlorhydrate (Dorcipec; Vallée S/A Produtos Veterinários, Montes Claros, MG, Brazil). Local anesthesia was administered in the mucobuccal fold, in the area of the mandibular incisors, with 0.6 mL of 2% xylocaine with epinephrine (1:100,000) to prevent any local discomfort. The guinea pigs were shaved in the submandibular area and the skin was disinfected with a 5% tincture of iodine. An incision of about 15 mm was made in the midline along the symphysis in the triangle periosteal elevator used to expose the mandibular bone. On the side of the symphysis in the triangle between the symphysis and the constantly developing incisor, 2x2 mm diameter cylindrical holes were prepared with special burs under saline irrigation. The cups filled with the materials under sterile conditions were inserted into the bone cavities. The periosteum was pulled over the cup and the incision was sutured with 3-0 black silk. The observation periods were 4 and 12 weeks. After the guinea pigs were euthanized, the mandible was dissected out and the bone adjacent to the cups in situ was cut into 10 mm blocks. The specimens were immersed in 10% neutral buffered formalin and prepared for routine histological examination. The slides were stained with hematoxylin-eosin (HE) for cellular recognition.

The interface at the opening of the cup, between the material and the bone, was examined and evaluated for the intensity of inflammation, the presence or absence of necrosis and/or resorption, possible replacement of the tested material by bone and osteolytic, osteosclerotic or osteoclastic activity. The tissue reactions were classified as: absentto slight tissue reaction, moderate tissue reaction or severe tissue reaction, according to the criteria defined by FDI (6) and ANSI/ADA (7) protocols. It was considered as biologically acceptable the material that showed none to slight reaction at both experimental periods of 4 and 12 weeks, or a moderate reaction at 4 weeks that diminished at 12 weeks.

The present investigation was developed according to the Ethical Principles on Animal Research as directed by the Brazilian National Advice of Control and Animal Experimentation (CONCEA) and approved by the Ethics Committee on the Use of Animals (CEUA) from the Federal University of Uberlândia, Brazil.

RESULTS

The histological evaluations of the materials at 4 and 12 weeks are summarized in Table 1 and illustrated in Figures 1, 2, 3, 4, 5 and 6.

Negative Control

There was bone growth toward the grooves and a thin layer of connective tissue, without any inflammatory reaction between the cup and bone, at all observation periods in all tested specimens.

4 Weeks

CH and CTZ Paste: The reaction was considered severe, showing bone necrosis, hemorrhage and a neutrophil inflammatory infiltrate in close contact with the material (Figs. 1 and 2).

Guedes Pinto Paste: The inflammatory response was classified as absent to slight. There was formation of new healthy bone in close contact with the material. In some cases, there was a narrow layer of connective tissue between the implanted material and the newly formed bone tissue with no presence of inflammatory response (Fig. 3).

12 Weeks

CH and Guedes Pinto Paste: The inflammatory response was classified as absent to slight. There was new formation and apposition of healthy bone which tended to enclose the entire implant. In some cases, the connective tissue around the cup showed abundant fibroblasts and vessels, with no inflammatory cells (Figs. 4 and 5).

CTZ Paste: The reaction was still considered severe, showing a mononuclear inflammatory infiltrate with a large number of lymphocytes, macrophages and giant foreign body cells. The presence of clastic cells containing material in the cytoplasm and necrotic tissue was common. There was more collagen fiber deposition near the bone tissue with the presence of many inflammatory cells. The irregularities at the bone surface demonstrated osteoclastic activity (Fig. 6).

DISCUSSION

Compatibility with living tissues in the periapical region is one of the most important properties of a material used in root filling of primary teeth because it will be in permanent contact with these tissues during the physiological root resorption. Furthermore, a possible injury to the germ of permanent teeth is a constant concern.

Most scientific investigations providing the experimental basis for these therapeutic procedures began in the last three decades. Considering the amount of materials recommended for pulp therapy in primary teeth and the lack of consensus as to their indications, this work aimed to evaluate the biocompatibility of some of them.

The implant test in guinea pig bone tissue, a secondary test recommended by the FDI Technical Report #9 (6), allows testing of a material as it is used in clinical situations and prepared following the manufacturer's recommendations. Although the results cannot be directly extrapolated to humans, the test is standardized and allows for a direct comparison between materials. The literature in this field includes research done in different laboratories at different times with the same materials, thus the results of the present study could be compared and confirmed (11-14). The obtained results confirmed the findings of those authors and indicate that any material placed in contact with vital tissues could elicit the development of a foreign body reaction. The bone in contact with the walls along the cup did not show inflammatory reaction in any of the observation periods, although in some cases there was a slender connective fibrous capsule and giant cells between the bone and the cup. The grooves were filled by healthy bone.

Hume (15) demonstrated that any material that contains eugenol elicits a severe tissue reaction because of the depression of cellular respiration. It has also been shown that the severity of inflammation increases with time because the reaction between the material and tissue fluids forms hydroxyl ions that release eugenol from the material (4,16-18). These results differ from those reported by Bernabé (19) and Sousa et al. (14) that showed a severe reaction to ZOE-based materials after 30 days, but the severity decreased considerably after 90 days and bone growth occurred at the material/tissue interface. Among all tested materials, the inflammatory response to CTZ paste was significantly higher for both observation periods in this study. Such a severe response to CTZ paste was previously described in the literature (20,21). It is thought to be due to the eugenol in the composition of CTZ paste.

Several studies demonstrated the acceptable use of CH in permanent and deciduous dentition (2,18,22). The results of this study corroborate the findings of a previous investigation (24) regarding tissue response to CH. These authors tested CH in vivo, demonstrating its biocompatibility potential. The present results indicate that CH showed a high degree of toxicity within the 4-week experimental period, but this changed significantly at 12 weeks, when it presented biocompatibility characteristics with no significant inflammatory response.

Previous clinical studies have demonstrated the efficacy of Guedes Pinto paste in the endodontic treatment of deciduous teeth (2,3,24). However, there are no publications using intra-osseous implant methods with Guedes Pinto paste. The obtained results confirm the previously published clinical findings. The Guedes Pinto paste presented the best results among all tested materials in the two experimental periods analyzed in the present study. This could be explained because the presence of Rifocort corticosteroid promotes a decrease in the inflammatory reaction to the other antiseptic drugs (3,24). Previous studies have shown that iodoform-based materials are easily absorbed and do not cause a foreign body reaction when compared with ZOE-based materials (17,20).

When a new material is introduced to the market, or an existing material is proposed for a different application, its properties must be investigated and the results should, if possible, be compared with tests performed by other authors. It was concerning that not all of the studies referred to in this paper emphasized the long-term potential systemic effect of these materials. In this way, within the technical limitations and the acceptability of the technique, it was possible to compare the tested materials in the same situation and to verify the degree of the bone tissue response in guinea pigs in a characteristic location (the alveolar ridge near the periodontal and dental structures), thus providing evidence for the intensity of the inflammatory response to these materials.

The results obtained in this study yielded the following conclusions: Guedes Pinto paste demonstrated acceptable biocompatibility levels in the two analyzed periods when in close contact with bone tissue; CH presented a high degree of toxicity in the 4-week experimental period, but this profile changed significantly at 12 weeks, when CH presented evidence of biocompatibility; CTZ paste presented no acceptable biocompatibility levels in the two analyzed periods. According to the FDI (6) and ANSI/ADA (7) criteria, CH and CTZ should not be used in contact with periapical tissue due the highly irritating potential of these materials.

ACKNOWLEDGEMENTS

The authors wish to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) for the financial support.

Received May 22, 2012

Accepted December 5, 2012

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Publication Dates

• Publication in this collection
  18 Jan 2013
• Date of issue
  2012

History

• Received
  22 May 2012
• Accepted
  05 Dec 2012