Tissue Reactions to a New Mineral Trioxide Aggregate–containing Endodontic Sealer

Basic Research—Technology

Tissue Reactions to a New Mineral Trioxide Aggregate–containing Endodontic Sealer Cauana Oliva Tavares, DDS,* Daiana Elisabeth B€ ottcher, DDS, MSc,† Eloısa Assmann, DDS,† † Patrıcia Maria Poli Kopper, PhD, DDS, MSc, Jose Ant^ onio Poli de Figueiredo, PhD, DDS, MSc,* Fabiana Soares Grecca, PhD, DDS, MSc,† and Roberta Kochenborger Scarparo, DDS, MSc, PhD* Abstract Introduction: The purpose of this study was to analyze ^ the connective tissue reactions to MTA Fillapex (Angelus Industria de Produtos Odontlogicos Ltda, Londrina, Brazil) compared with a zinc oxide–based sealer (EndoFill; Dentsply Industria e Comerico Ltda, Petropolis, Brazil) and an epoxy resin–based material (AH Plus; Dentsply DeTrey GmbH, Konstanz, Germany) in Wistar rats. Methods: Polyethylene tubes containing the test materials and empty polyethylene tubes (control) were implanted in the subcutaneous tissue of 12 rats. Empty tubes were used as a negative control. After 7 and 60 days (n = 6 per period), observations were made for cellular inflammatory components, fibrous condensation, and abscess formation. Comparisons among the groups and between the experimental periods were made using 2-way analysis of variance and the Bonferroni post hoc test (P < .05). Results: At the end of the 7-day experimental period, all sealers scored higher than the control group for the variable lymphocytes, and MTA Fillapex presented lower fiber condensation compared with empty tubes. After 60 days, macrophages and lymphocytes scored significantly higher for MTA Fillapex and EndoFill compared with the negative control, and AH Plus showed similar results related to the empty tubes. Comparing the materials’ responses at the end of the 2 evaluated periods, for EndoFill samples the variable neutrophils was detected less after 60 days. Both EndoFill and MTA Fillapex presented increased fiber condensation after 60 days. Conclusions: Although none of the sealers promoted ideal tissue responses, AH Plus presented the best outcomes. Although MTA Fillapex contains MTA powder, it presented no biocompatibility advantages when compared with AH Plus and EndoFill. (J Endod 2013;39:653–657)

Key Words Biocompatibility, endodontics, endodontic sealers, MTA Fillapex, root canal filling

I

t is well documented that obturation procedures have a role in favoring and maintaining adequate conditions for endodontic repair (1, 2). In this regard, biocompatibility (3–5) and sealing ability (1) are of paramount importance, and several endodontic sealers have been constantly developed with the aim of improving these features (6). MTA Fillapex (^Angelus Industria de Produtos Odontlogicos Ltda, Londrina, Brazil) is an endodontic sealer that claims to take advantage of the biological and physical properties of mineral trioxide aggregate (MTA) (6–8). Nevertheless, apart from containing MTA compounds, this sealer is comprised of resins (ie, salicylate, diluting, and natural), radiopaque bismuth, nanoparticulated silica, and pigments (6, 7). Although some authors describe a similar tissue response when compared with the original MTA formulations (8), the biological responses to MTA Fillapex compared with the more commonly used endodontic sealers have not been explored yet. Moreover, a severe reduction of cell survival rates has been reported to be associated with MTA Fillapex (9), leading to uncertainties regarding its biological properties. Because of these conflicting results, the aim of the present study was to analyze the connective tissue reactions to MTA Fillapex compared with a zinc oxide–based sealer (EndoFill; Dentsply Industria e Comerico Ltda, Petropolis, Brazil) and an epoxy resin–based material (AH Plus; Dentsply DeTrey GmbH, Konstanz, Germany) in Wistar rats.

Materials and Methods The present study was approved by the Research and Ethics Committee of the School of Dentistry of the Federal University of Rio Grande do Sul, Porto Alegre, Brazil (no. 13534). The inflammatory reactions of 12 animals to MTA Fillapex, AH Plus, and EndoFill were evaluated after 2 experimental periods (ie, 7 and 60 days, n = 6 per period). The sealers were inserted into autoclaved polyethylene tubes approximately 10 mm long and 1.5 mm in diameter (Abbott Laboratorios do Brasil, S~ao Paulo, Brazil) to be implanted in the animals’ dorsal subcutaneous tissue. An empty tube was used as the negative control. The animals were anesthetized with 0.008 mL/100 g ketamine and 0.004 mL/100 g 2% xylazine hydrochloride (Virbac do Brasil Industria e Comercio Ltda, S~ao Paulo, Brazil). Dorsal trichotomy was manually performed in an area comprising approximately 10 cm2. Disinfection with an alcohol-iodine solution was performed. Four 0.5-cm-long incisions were made in the animals’ backs 2 cm from the spine and at least 2 cm apart from one another. Using blunt-tipped scissors, the lateral tearing of the subcutaneous tissue provided 4 surgical cavities displayed in quadrants equidistant from the center of the animals’ backs. The materials were prepared according to the manufacturers’ instructions and introduced into their respective tubes using sterile

From the *Clinical Department, School of Dentistry, Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, Rio Grande do Sul, Brazil; and Conservative Dentistry Department, School of Dentistry, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil. Address requests for reprints to Dr Daiana Elisabeth B€ottcher, Av Ramiro Barcelos, 2492, Cep: 90035-003, Porto Alegre, RS, Brazil. E-mail address: daibottcher@ hotmail.com 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.10.009 †

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Basic Research—Technology insulin syringes (Injex Industria Cirurgica Ltda, Ourinhos, Brazil). The tubes filled with the materials were immediately inserted into the surgical cavities parallel to the incision. The position in which each sealer was implanted was standardized. The incisions were closed using a 3-0 silk thread (Johnson & Johnson Produtos Profissionais Ltda, S~ao Jose dos Campos, Brazil). At the end of each experimental period, the animals were euthanized by CO2 inhalation. Trichotomy was performed once more, and an excisional biopsy of the implant area was performed

with a safety margin of 1 cm. The samples were placed on paper discs and fixed in 10% formalin for 24 hours. Then, the polyethylene tubes were removed from the samples, set in paraffin blocks, and processed for histologic analysis. Sections with a thickness of 3 to 4 mm were cut along the axis of each tube, mounted on slides, and stained with hematoxylin-eosin. Three sections were obtained per sample. The slices were examined under a light microscope by a single-blinded, calibrated examiner (k > 6 for all evaluated variables).

Figure 1. A comparison among the study groups and between the 7- and 60-day experimental periods regarding the following variables: neutrophils, lymphocytes, macrophages, giant cells, eosinophils, fibers, and abscesses. Statistically significant differences were detected in the study groups compared with the control (*P < .05, **P < .01). The same materials also showed different scores between the 2 experimental periods (+P < .05, ++++P < .0001).

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Basic Research—Technology Slides were analyzed qualitatively according to the criteria described by Figueiredo et al (10) and adapted for other studies (3, 4). The cellular inflammatory component was determined by the presence of neutrophils, lymphocytes, eosinophils, macrophages, and giant cells. Cellular events were classified according to the following scale: 1. Absent: Inflammation was either absent or within blood vessels. 2. Mild: Cells were present although sparse or in reduced clusters. 3. Moderate: Cells were present but did not dominate the microscopic field. 4. Intense: Cells were present in the form of an infiltrate close to the material used. Fiber condensation was classified according to the following scale: 1. The absence of collagen fibers 2. The presence of a thin layer of collagen fibers 3. The presence of a thick layer of collagen fibers Abscess formation was classified as follows: 1. The absence of an abscess 2. The presence of an abscess in contact with the surgical cavity in which the material had been inserted 3. The presence of abscess areas far from the surgical cavity in which the material had been inserted Differences among the groups and between the 2 experimental periods were evaluated by using 2-way analysis of variance in conjunction with the Bonferroni post hoc test. The significance level was set at .05.

Results The study results are summarized in Figure 1. The histologic parameters used in assessing the results over the experimental periods

are shown in Figures 2 and 3. After 7 days, statistically significant differences were detected among the groups. All sealers scored higher than the control group for the variable lymphocytes. MTA Fillapex presented lower fiber condensation compared with the other groups’ responses (Fig. 2A and B). Although significant differences were not observed for the variables macrophages and neutrophils, at the end of the 7-day experimental period, these cells were more intensely detected in the reaction to the sealers than in the control group. Similarly, EndoFill and MTA Fillapex were likely to present higher scores for giant cells and abscesses (Fig. 2C), whereas eosinophils were more frequently detected in the EndoFill samples. At the end of the 60-day experimental period, significant differences were observed for macrophages and lymphocytes in the MTA Fillapex (Fig. 3A) and EndoFill (Fig. 3B) groups compared with the negative control. In contrast to the results obtained after the 7-day experimental period, AH Plus showed similar results related to the empty polyethylene tubes after 60 days (Fig. 3C and D). Also, at the end of the 60-day experimental period, there was a decrease in the number of neutrophils for EndoFill samples compared with the number of neutrophils at the end of the 7-day experimental period. EndoFill and MTA Fillapex produced increased fiber condensation at the end of the 7-day experimental period (Figs. 2 and 3).

Discussion The present investigation showed that although it was expected that a sealer containing MTA acted as a repairer, no advantage could be obtained regarding the tissue responses to MTA Fillapex compared with the epoxy resin–based sealer AH Plus or the zinc oxide–based sealer EndoFill. In this regard, significant differences were observed when MTA Fillapex and AH Plus were compared, and the MTA-containing material scores of inflammatory cells and abscesses were only slightly lower compared with EndoFill. Moreover, after the 7-day experimental

Figure 2. Features observed 7 days after the surgical procedure: connective tissue responses to (A) the control and (B and C) MTA Fillapex (100). Empty tube implants (ET) promoted the formation of a vascularized tissue presenting few inflammatory cells (*) and fibrous condensation (arrows). On the contrary, MTA Fillapex (FL) produced an intense and extended inflammatory response, mainly comprised of lymphocytes (arrows) and not limited by fibrous condensation. In some samples, an abscess formation could also be observed next to the experimental material (>).

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Figure 3. Tissue responses to the sealers. (A) After 60 days, MTA Fillapex (FL) showed inflammatory infiltrate (*), and giant cells (arrows) were identified in some of the samples (200). (B) At the same period, Endofill promoted the maintenance of an inflammatory response, mainly comprised of lymphocytes and macrophages (arrows) (400). AH Plus (AHP) showed a moderate inflammatory response (*) (C) after 7 days (100), which became mild and well limited for fibrous condensation (D) after 60 days (40).

period, MTA Fillapex showed limited fiber condensation (which had an influence on the extension of the inflammatory reaction), and the variables lymphocytes and macrophages scored higher than the control group throughout the experiment. Although not statistically significant, taken together with the presence of macrophages for long periods, the detection of giant cells emphasizes MTA Fillapex’s potential for tissue irritation. Therefore, MTA Fillapex does not have the claimed biological advantages over other available products. These results probably are associated with MTA Fillapex’s chemical composition, which is mainly resin based but also contains MTA compounds. A previous in vitro study (9) showed that white MTA was able to keep cell viability and increase its proliferation, whereas MTA Fillapex was severely cytotoxic. The current results showing intense and extensive inflammation in response to MTA Fillapex corroborate these findings. On the contrary, in earlier studies (4, 11) Endo-CPM sealer, which also attempts to maintain the MTA biological properties, showed a more favorable connective tissue response (4, 11), which reflects the material’s composition. Apart from containing the setting accelerator calcium chloride, Endo-CPM sealer basically presents the same compounds of the original MTA formulation (7, 12). On the other hand, although reformulated to improve handling characteristics, Endo-CPM sealer has some disadvantages regarding its physical properties, leading to failures in root canal fillings radiographically detected as voids and gaps (7). For this reason, the attempt to combine physical properties from resin-based sealers with the MTA biological and sealing features is fully justified. The results of this study point out that the more commonly used epoxy resin–based sealer AH Plus promoted more favorable results than MTA Fillapex and EndoFill; this is also corroborated by other investigations comparing endodontic sealers (13, 14). In a previous study (3), AH Plus was the only sealer that exhibited similar inflammatory infiltration compared with the control group after longer periods. 656

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The differences between MTA Fillapex and AH Plus suggest that some of the compounds of the first material have the potential to cause tissue aggression, impairing MTA biological properties. Thus, although not presenting ideal tissue responses at short experimental periods, AH Plus should still be considered the gold standard because of its biocompatibility. Accordingly, the response to MTA Fillapex was similar to that observed with zinc oxide–based sealers, which are recognized for their potential to bring about greater and longer-lasting inflammatory reactions (3–15). Unlike the results of this study, MTA Fillapex promoted favorable reactions of the connective tissue in a previous investigation (8). Apart from the differences in the study groups, the conflicting results are probably related to details in the experimental procedures and the evaluation methodology. In the present study, after the preparation of the sealers, they were immediately inserted into the implants and placed into the surgical cavities in such a way that allowed their contact with the rat connective tissue to set. Moreover, the scores adopted herein made it possible to supplement an objective analysis of the results with the description of the inflammatory events, considering details regarding the predominant cell type and its role in the different stages of tissue reaction (3, 10). On the contrary, the study by Gomes-Filho et al (8) used scores that considered inflammatory cell counts, regardless of their type, in conjunction with a less detailed classification of the host response intensity. Taking into account that several studies emphasize that the quantification of the tissue response is only possible in materials with very distinct degrees of irritability (3, 10, 16), this evaluation criterion could have masked more subtle differences.

Conclusion The current results showed that although none of the sealers promoted ideal tissue responses, AH Plus presented the best outcomes, which was evidenced by the similarities between this sealer and the JOE — Volume 39, Number 5, May 2013

Basic Research—Technology control group after longer periods of time. Despite containing MTA, MTA Fillapex presented no biocompatibility advantages when compared with AH Plus and EndoFill.

Acknowledgments The authors deny any conflicts of interest related to this study.

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7. Assmann E, Scarparo RK, B€ottcher DE, Grecca FS. Dentin bond strength of two mineral trioxide aggregate–based and one epoxy resin–based sealers. J Endod 2012;38:219–21. 8. Gomes-Filho JE, Watanabe C, Lodi CS, et al. Rat tissue reaction to MTA Fillapex. Dent Traumatol 2011 Dec 8. doi: 10.1111/j.1600-9657.2011.01096.x. [Epub ahead of print]. 9. Bin CV, Valera MC, Camargo SEA, et al. Cytotoxicity and genotoxicity of root canal sealers based on mineral trioxide aggregate. J Endod 2012;38: 495–500. 10. Figueiredo JA, Pesce HF, Gioso MA, et al. The histological effects of four endodontic sealers implanted in the oral mucosa: submucous injection versus implant in polyethylene tubes. Int Endod J 2001;34:377–85. 11. Gomes-Filho JE, Watanabe S, Bernabe PFE, et al. A mineral trioxide aggregate sealer stimulated mineralization. J Endod 2009;35:256–60. 12. Camilleri J. Evaluation of selected properties of mineral trioxide aggregate sealer cement. J Endod 2009;35:1412–7. 13. Batista RF, Hidalgo MM, Hernandes L, et al. Microscopic analysis of subcutaneous reactions to endodontic sealer implants in rats. J Biomed Mater Res 2007;81: 171–7. 14. Leonardo MR, da Silva LA, Almeida WA, et al. Tissue response to an epoxy resinbased root canal sealer. Endod Dent Traumatol 1999;15:28–32. 15. G€orduysus MO, Etikan I, G€ok€oz A. Histopathological evaluation of the tissue reactions to Endo-Fill root canal sealant and filling material in rats. J Endod 1998;24: 194–6. 16. Olsson B, Sliwkowiski A, Langeland K. Subcutaneous implantation for the biological evaluation of endodontic materials. J Endod 1981;7:355–67.

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