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Biocompatibility of New Calcium Aluminate Cement: Tissue Reaction and Expression of Inflammatory Mediators and Cytokines Lucas da Fonseca Roberti Garcia, DDS, MSc, PhD, Claudia Huck, DDS, MSc, Letıcia Menezes de Oliveira, Pedro Paulo Chaves de Souza, DDS, MSc, PhD, and Carlos Alberto de Souza Costa, DDS, MSc, PhD Abstract Introduction: The aim of this study was to evaluate the biocompatibility of a new calcium aluminate cement (EndoBinder) in subcutaneous tissue of rats in comparison with mineral trioxide aggregate and calcium hydroxide hard-setting cement. Methods: Polyethylene tubes (1.5 10 mm) containing the dental cements were implanted into dorsal subcutaneous tissue of 30 rats. After experimental periods of 7, 30, and 90 days, biopsies were performed for tissue response analysis under optical light microscope. The mRNA extraction was performed for molecular evaluation of the inflammatory process in the peri-implant tissue, which was submitted to quantitative real-time polymerase chain reaction analysis for inflammatory mediators and cytokines TNF-a, Ptges2, Il-1b, Il-4, and Il-10. Results: On the basis of the score used to grade the tissue reaction (0–3), EndoBinder (0) presented no inflammatory reaction after the 90-day period, a similar result to mineral trioxide aggregate and calcium hydroxide. The thickness of inflammatory capsules (mm) also presented significant decrease during the course of periods (P < .05). As regards expression of inflammatory mediators, Ptges2 and Il-10 were detected only at 7 and 30 days, with no statistically significant difference among the experimental groups (P > .05). Conclusions: EndoBinder induced limited inflammatory reaction. It was considered biocompatible when tested in subcutaneous tissue of rats. (J Endod 2014;40:2024–2029)
Key Words Biocompatibility, calcium aluminate cement, calcium hydroxide, mineral trioxide aggregate, tissue reaction
From the Department of Physiology and Pathology, Araraquara School of Dentistry, Univ Estadual Paulista, Araraquara, S~ao Paulo, Brazil. Address requests for reprints to Dr Carlos Alberto de Souza Costa, Department of Physiology and Pathology, Univ Estadual Paulista - UNESP, Araraquara School of Dentistry, Humaita Street, 1680, Araraquara, SP, Brazil 14801-903. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.08.015
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everal studies have reported that calcium hydroxide continues to be one of the most used materials for pulp/dentin complex protection, against which new products must be evaluated and compared, especially in biological tests involving animals and humans (1, 2). Despite the widespread use of calcium hydroxide, some questions with regard to its performance persist (3). Therefore, the constant development of new materials for pulp therapy application created mineral trioxide aggregate (MTA) (4). The MTA cement was initially used as material for retrograde filling and root and furca perforation treatments (4); however, because of its good clinical performance, it has also been used for several other clinical applications such as pulpotomy (5), treatment of teeth with incomplete apexification (6), root resorption (7), intracoronal barrier before bleaching, and apical plug (8), root canal filling (4), and pulp capping in conservative treatments (9). Among the advantages of MTA in comparison with calcium hydroxide, its higher mechanical strength and marginal sealing ability are outstanding (10). However, some of its negative properties such as poor handling characteristics (11), low flow ability (12), high rates of tooth structure staining (13), solubility in moist conditions (14), long setting time (15), and presence and release of arsenic (16) above the safe limits proposed by ISO 9917-1 specification (17) must be considered. The negative features observed for calcium hydroxide and MTA cements justify the development of new materials that incorporate the appropriate biological properties of both materials in a new product with easy handling, application, and adequate mechanical strength (15). Thus, a calcium aluminate-based cement, EndoBinder (Binderware, S~ao Carlos, SP, Brazil), was developed at the Federal University of S~ao Carlos (BrazilUFSCAR, patent number PI0704502-6). The laboratory synthesis process of EndoBinder provides several advantages in comparison with MTA such as greater control of impurity levels, especially Fe2O3, which promotes tooth staining (7, 13), and free MgO and CaO, responsible for an undesired expansion of the material in contact with moisture (18). The balance between stoichiometric phases rich in Al2O3 and CaCO3, responsible for the hydrophilic setting process of EndoBinder, promotes greater compatibility between living tissues and cement, as well as adequate physicochemical properties (12, 19). However, in vivo studies that prove the biological compatibility of EndoBinder are necessary before its validation as a material for clinical application. Thus, the aim of this study was to evaluate the biocompatibility of this new calcium aluminate cement, containing zinc oxide as radiopacifying agent, by means of tissue reaction analysis and expression of inflammatory mediators in rats in comparison with MTA and calcium hydroxide.
Materials and Methods Selection of Animals The study was developed in accordance with the determinations of the Research Ethics Committee on the Use of Animals - Araraquara School of Dentistry/UNESP (Process CEUA No. 3/2013), in compliance with the ethical principles for use of laboratory animals at all stages of the experiment. The animals were kept in plastic cages
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Basic Research—Technology (40 32 17 cm) in an acclimatized bioterium with established lighting (12 hours light/12 hours dark at temperature of 21 –25 C), receiving balanced rations (Nuvilab, Colombo, PR, Brazil) and water ad libitum.
Subcutaneous Implant Thirty male rats (Rattus novergicus; Wistar Institute, Philadelphia, PA) were selected, with weight ranging between 250 and 300 g, and 10 specimens were used for each of the evaluation time intervals (7, 30, and 90 days). Polyethylene tubes measuring 1.5 mm in internal diameter and 10 mm long were obtained according to the methodology described by Souza et al (20). The tubes were immersed in 70% alcohol for 120 minutes and abundantly washed with sterilized distilled water, and after this they were autoclaved. The materials tested were as follows: EndoBinder + ZnO (radiopacifying agent, 20% by weight), white MTA (^Angelus, Londrina, PR, Brazil), and calcium hydroxide (Biodin^amica, Ibipor~a, PR, Brazil). All the materials were manipulated in accordance with the manufacturers’ recommendations, with the proportion of 1 g powder to 0.21 mL distilled water being used for EndoBinder and for MTA 1 dose of powder (0.15 mg) to 1 drop (0.5 mL) of distilled water. For calcium hydroxide P.A., 1 g powder was placed on a glass plate, which was manipulated with 1 mL physiological solution at 0.9% (1:1) until a homogeneous paste was obtained. After manipulation, the previously sterilized polyethylene tubes were filled with the cements by using sterile lentulo spirals (Dentsply/ Maillefer, Ballaigues, Switzerland) compatible with the internal diameter of the tubes. For the surgical procedures, the animals were anesthetized by intraperitoneal administration of a solution composed of 10% ketamine chloride (Ketamina Agener; Uni~ao Quımica Farmac^eutica Nacional S/A, Embu-Guac¸u, SP, Brazil) and xylazine (Dopazer; Laboratorios Calier S/A, Barcelona, Spain) in the proportion of 75 mg/kg and 10 mg/kg, respectively. After trichotomy and disinfection of the operative field with sterile gauze imbibed in a solution of 5% povidone-iodine, 2 central incisions, one on the left and the other on the right side (5 mm long each), were performed in the dorsum of each animal by using a no. 15 scalpel blade. Lateral divulsion of the subcutaneous tissue was carefully performed starting from each central incision by using a blunt-tipped scissors for this purpose. Thus, 2 surgical recesses with a mean depth of 20 mm were obtained, in which the polyethylene tubes filled with the test materials were implanted longitudinally, remaining parallel to the incisions. Two polyethylene tubes filled with MTA or EndoBinder were implanted in 15 rats. In another 15 rats, tubes without any material (negative control) or filled with calcium hydroxide were implanted. After this, suturing was performed with 3-0 silk thread (Ethicon, S~ao Jose dos Campos, SP, Brazil). After the time intervals of 7, 30, and 90 days had elapsed, the animals were killed by anesthetic overdose, and biopsies were obtained with sufficient safety margin around the polyethylene tubes. At one of the extremities, tissue samples were collected for molecular evaluation of the inflammatory process, with mRNA being extracted and submitted to quantitative real-time polymerase chain reaction analysis (qPCR) for inflammatory mediators and cytokines. The histopathological analysis was performed in the connective tissue adjacent to the other extremity of the polyethylene tube. For this purpose, the surgical parts (n = 5) were immediately immersed in a 10% formalin solution (Merck, Darmstadt, Germany), in which they remained in a fixation process for the period of 24 hours. After this, they were submitted to routine laboratory processing. Semi-serial 5-mm-thick histologic cuts were obtained and stained with hematoxylin-eosin (Merck). JOE — Volume 40, Number 12, December 2014
Histopathological Analysis Histopathological analysis was performed by using an optical light microscope Axio Star Plus (Carl Zeiss, Oberkochen, Germany) at 32, 64, 125, and 320 magnifications. The histopathological events evaluated were the following: inflammatory infiltrate (polymorphonuclear and mononuclear cells), cellularity (fibroblasts), vascularization (blood vessels), and macrophagic activity (macrophages and multinuclear giant cells independently scored). On the basis of the tissue responses stimulated by the cements and by the control group and in accordance with the standard ISO 7405 (21), a score was used to quantify the absence or presence of these events; the inflammatory reaction was classified as (0) absent, (1) discrete, (2) moderate, and (3) severe. The thickness of the inflammatory capsule (mm) was gauged with the aid of the software program AxioVision 4.6 (Carl Zeiss, http://www.zeiss.de/microscopy). qPCR Samples of the tissues submitted to biopsy in the different time intervals of analysis (7, 30, and 90 days) were used for total RNA extraction in accordance with the protocol for the RNAqueous-4PCR kit (Ambion; Life Technologies, Grand Island, NY). For each total RNA sample extracted from the samples, the complementary DNA (cDNA) was synthetized for the qPCR reaction. For this purpose, the protocol of the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used. The qPCR was performed in StepOnePlus Real-Time PCR System equipment (Applied Biosystems) by using cDNA as template, a set of TaqMan primers and probes designed to detect the genes encoding to interleukin 1b (Il-1b), tumor necrosis factor-a (TNF-a), interleukin-4 (Il-4), interleukin 10 (Il-10), and prostaglandin E synthase 2 (Ptges2), Taqman Universal Master Mix (Applied Biosystems), and nuclease-free water. To control the amount of cDNA input, the expression of all genes was normalized against the gene encoding to the housekeeping gene b-actin (Actb). For evaluation of the inflammatory response, real-time PCR was performed for the proinflammatory cytokines; for evaluation of the resolution of inflammation, the mRNA expression of the anti-inflammatory cytokines Il-4 and Il-10 was evaluated. Statistical Analysis The normal distribution of data was tested by the KolmogorovSmirnov test, and the values obtained were statistically compared (2-way analysis of variance, the Bonferroni test, P < .05) with the aid of Graphpad Prism 4.0 Software (GraphPad Software, La Jolla, CA).
Results Histopathological Analysis The values obtained for all the histopathological events evaluated in each time interval are presented in Table 1. Generally the tissue reactions diminished during the course of time for all tested materials. Only one severe score was determined for MTA with regard to blood vessels at the 7-day period; all other scores were moderate or lower. Period of 7 Days For EndoBinder, at the tubular opening was observed the formation of an ample loose inflammatory capsule, still disorganized, with moderate mixed inflammatory infiltrate, characterized by the presence of neutrophils and lymphocytes. Moderate fibroangioblastic proliferation and discrete local collagenization, associated with the presence of mononuclear phagocytes (macrophages), and multinuclear giant cells Biocompatibility of Calcium Aluminate Cement
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Basic Research—Technology TABLE 1. Mean Values of Histopathologic Events Observed in Each Group at Different Time Intervals of the Study 7 days
30 days
90 days
Histopathological events
C
MTA
CH
EB
C
MTA
CH
EB
C
MTA
CH
EB
Inflammatory infiltrate Fibroblasts Blood vessels Macrophage Multinuclear giant cells General inflammatory index
2 1 1 1 1 1
2 2 3 2 2 2
2 2 2 2 2 2
2 2 2 2 2 2
1 1 1 1 1 1
1 2 2 1 1 1
1 2 1 1 1 1
1 2 1 1 1 1
0 1 0 0 0 0
0 1 0 0 0 0
0 1 0 0 0 0
0 1 0 0 0 0
C, control; CH, calcium hydroxide; EB, EndoBinder. Scores: 0, absent; 1, discrete; 2, moderate; 3, severe. N = 5.
encircling the residual material were also observed in the main area of analysis (Fig. 1A). For the MTA cement, there was evidence of disorganized inflammatory capsule formation, with discrete fibrous content and moderate mixed inflammatory infiltrate. Congested blood vessels in the midst of areas of edema and intense fibroangioblastic proliferation were also observed. Moderate quantities of mononuclear phagocytes and multinucleated giant cells in the process of phagocytosis of dispersed
material residues at the tubular opening were detected. Discrete necrotic areas were observed in the main area of analysis (Fig. 1B). For calcium hydroxide, the reactive events in the tissue were similar to those of EndoBinder and MTA; however, a larger area of tissue necrosis was observed adjacent to the tubular opening (Fig. 1C). Moderate inflammatory infiltrate, characterizing the formation of loose tissue at the tubular opening, was observed for control group (Fig. 1D).
Figure 1. (A) EndoBinder 7 days: inflammatory infiltrate (circle) of moderate amplitude and congested blood vessels (arrow). Hematoxylin-eosin; original magnification, 125. (B) MTA 7 days: moderate inflammatory infiltrate associated with dilated and congested blood vessels (arrow). Observe presence of material residues, with small area of necrosis close (box). Hematoxylin-eosin; original magnification, 125. (C) Calcium hydroxide 7 days: large quantity of material residue dispersed within the tissue. Observe ample area of necrosis close to material (box), more accentuated than areas found for EndoBinder and MTA. Hematoxylin-eosin; original magnification, 125. (D) Control 7 days: moderate infiltrate of polymorphous inflammatory and mononuclear cells, characterizing formation of loose tissue at tubular opening. Hematoxylin-eosin; original magnification, 125. (E) EndoBinder 30 days: area adjacent to tubular opening with more organized and delimited inflammatory capsule. Note decrease in number of polymorphous and mononuclear cells and discrete macrophagic activity. Hematoxylin-eosin; original magnification, 64. (F) MTA 30 days: significant reduction in capsule thickness and inflammatory infiltrate. Significant decrease in number of polymorphous and mononuclear cells as well as discrete local macrophagic activity. Hematoxylin-eosin; original magnification, 320. (G) Calcium hydroxide 30 days: evidence of decrease in inflammatory infiltrate, with discrete local macrophagic activity. Hematoxylin-eosin; original magnification, 320. (H) Control 30 days: significant decrease in inflammatory infiltrate and discrete macrophagic activity because of absence of material capable of inducing local inflammatory process. Hematoxylin-eosin; original magnification, 320. (I) EndoBinder 90 days: accentuated reduction in inflammatory capsule thickness and inflammatory infiltrate. Hematoxylin-eosin; original magnification, 64. (J) MTA 90 days: accentuated reduction in dense inflammatory capsule thickness (arrow) with reduced inflammatory infiltrate. Hematoxylin-eosin; original magnification, 32. (K) Calcium hydroxide 90 days: dense fibrous capsule presented as very thin (bar). Hematoxylin-eosin; original magnification, 64. (L) Control 90 days: absence of inflammatory infiltrate with discrete fibroangioblastic proliferation, indicative of reparative process. Hematoxylin-eosin; original magnification, 125.
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Figure 2. (A and B) Graphic representation of mean values (mm) of inflammatory capsule thickness at different periods of study. (C) Graphic representation of inflammatory mediator Il-10 expression according to experimental periods. (D) Graphic representation of inflammatory mediator Ptges2 expression according to experimental periods. (E and F) Graphic representation of inflammatory mediator and cytokine expression in function of period of analysis. Different lower case letters over bars indicate statistically significant difference (2-way analysis, Bonferroni test, P < .05). n = 5. Gapdh, glyceraldehyde-3-phosphate dehydrogenase.
Period of 30 Days For EndoBinder cement, an important reduction in inflammatory infiltrate was observed, associated with discrete macrophagic activity mediated by giant cells. The inflammatory capsule present at the tubular opening presented as fibrous and well-organized, characterizing a tissue repair process in evolution. No foci of mineralization or necrotic tissue were found close to the tubular opening (Fig. 1E). For MTA, the findings were similar to those of EndoBinder; however, a discrete necrotic area still persisted in this intermediate experimental period (Fig. 1F). For calcium hydroxide, the reduction in the intensity of tissue response was similar to that observed for the other cements. The notable reduction in the area of necrosis adjacent to the tubular opening should be pointed out (Fig. 1G). For control group, a significant decrease in inflammatory infiltrate and discrete macrophagic activity was observed (Fig. 1H). Period of 90 Days At 90 days, EndoBinder tissue samples exhibited no inflammatory infiltrate. Moreover, no mononuclear phagocytes and multinucleated giant cells were observed. A dense fibrous capsule, in continuity with that formed on the lateral surface of the tube, was present at the tubular opening, characterizing the local occurrence of complete tissue repair (Fig. 1I). For the MTA cement, a discrete fibroangioblastic proliferation still persisted, with no tissue necrosis areas being detected. A thin, dense fibrous capsule was observed at the tubular opening, characterizing complete tissue repair in contact with the implanted material (Fig. 1J). A similar tissue repair response was observed for calcium hydroxide in this period (Fig. 1K). In control group, absence of inflammatory infiltrate with discrete fibroangioblastic proliferation was observed JOE — Volume 40, Number 12, December 2014
(Fig. 1L). The general inflammatory index for the 3 materials in this period was established as 0 (absent), determining acceptable biologic compatibility.
Inflammatory Capsule The mean values obtained for inflammatory capsule thickness of the different materials in the different experimental time intervals are seen in Figure 2A and B. During the course of the experimental periods of analysis, significantly decreasing amplitude of inflammatory capsule formation was observed for all the groups (P < .05). The reactive capsule formed adjacent to EndoBinder cement (133.18 mm) in the period of 7 days was significantly thinner than those observed for calcium hydroxide (153.53 mm) and MTA (142.60 mm) in the same period (P < .05). However, this capsule was thicker when compared with the control group (86.05 mm) (P < .05). The trend toward reduction in capsule thickness remained in the intermediate period of analysis (30 days); however, the thickness of capsules formed by EndoBinder and MTA no longer presented statistically significant difference (P > .05). In the period of 90 days, all the groups presented significant decrease in the amplitude of dense fibrous capsules (P < .05), with EndoBinder (30.66 mm), MTA (30.83 mm), and calcium hydroxide (31.26 mm) being statistically similar (P > .05) but differing from that of the control group (16.99 mm) (P < .05). qPCR The mean values obtained for each of the inflammatory mediators and cytokines expressed in the different experimental time intervals may be seen in Figure 2C–F. Biocompatibility of Calcium Aluminate Cement
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Basic Research—Technology Among the analyzed genes, only Il-10 and Ptges2 transcripts could be detected after 7 and 30 days of implantation of the materials. In the period of 90 days, it was not possible to detect the expression of any of the mediators. At 7 days, EndoBinder presented the highest values for Ptges2 and Il-10 mRNA expression, with statistically significant difference in comparison with the control and MTA groups (P < .05), however, similar to the values of calcium hydroxide (P > .05). At 30 days, all the materials presented a reduction in the values of expression of both mediators; nevertheless, this reduction was statistically significant (P < .05) only for EndoBinder.
Discussion Biological compatibility is one of the most important properties of endodontic sealing cements, because toxic components may come in contact with adjacent tissues and induce irritation (1, 20). The results of this study demonstrated absence of inflammatory reaction for all the tested materials at the end of the 90-day period of analysis. In a similar study, Aguilar et al (19) demonstrated that after 42 days, MTA still presented discrete inflammatory reaction, indicative of a persistent chronic inflammatory process. Nevertheless, in accordance with the ISO 7405 standard (21), a moderate inflammatory reaction 15 days after implant placement with a trend toward decrease after 12 weeks is considered acceptable. It has been demonstrated that materials in intimate contact with live tissues are irritants; however, according to Shahi et al (22), more important than the irritant potential of materials is the duration of this effect on tissues to which they are applied. Yaltirik et al (23) reported the occurrence of a chronic inflammatory process promoted by MTA cement 60 days after subcutaneous implantation of the material; this was a different result to that found in the present study, in which 30 days after implantation, all the cements tested presented, at most, a discrete inflammatory reaction at the tubular opening. Moreover, the infiltrate of polymorphous and mononuclear cells presents significant reduction in comparison with the initial period of analysis, which is different from the findings of previous studies, in which macrophages and multinucleated giant cells were observed phagocytosing particles of MTA dispersed in the connective tissue (19, 23). Furthermore, the results obtained in the present research as regards the expression of inflammatory mediators by means of qPCR analysis proved the results of the histopathological analysis. Only Ptges2, the gene encoding cycloogenase-2, the enzyme responsible for the metabolism of arachidonic acid and production of prostanoids, and Il-10, a cytokine expressed during chronic inflammatory reactions, were detected in the initial periods (7 and 30 days) of evaluation. It should be pointed out that several previous studies have corroborated the results of the present study, because they demonstrated the excellent biological properties of MTA (4, 24), with these being related to the capacity of releasing calcium ions and, consequently, to the alkaline pH produced by the material (24). According to Garcia et al (14), the MTA and EndoBinder hardsetting cements present similar solubility and disintegration, with a mass loss index of greater than 3%, a value higher than the limit proposed by specification no. 57 of American National Standards Institute/American Dental Association (25). Nevertheless, it has been demonstrated that such solubilization indices do not contraindicate the use of these materials, because their good biological performance is related to their capacity to release calcium and hydroxyl ions into the medium during their hydration process (4). Other studies have proved that these ions are the main components detected in soluble and insoluble residues released by MTA, demonstrating that the solubility of the cement is an important factor for 2028
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the delivery of calcium and hydroxyl ions in periodontal and pulp tissue (4, 5, 9). It has been demonstrated that moisture conditions may improve the biological performance of MTA in shorter periods of time, favoring the separation of cement particles by solubilization and disintegration process, which come into contact with the tissues, initiating an inflammatory process that leads to their repair (4). The same could be observed for EndoBinder, because it concerns a cement with hydraulic setting, such as MTA, in which the physical-chemical interaction of the material with the humid environment is fundamental, particularly with regard to its reparative capacity (24, 26, 27). During the setting of MTA, the tricalcium silicate hydrates and releases calcium hydroxide. The calcium hydroxide releases calcium ions into the moist areas adjacent to the placement of MTA (24). These ions are produced from the calcium hydroxide present and from the decomposition of calcium silicate hydrate, which is released in a lower speed in comparison with that of calcium hydroxide (26). However, the hydration process of calcium aluminate cements such as EndoBinder leads to the formation of calcium aluminate and aluminum hydroxide hydrate (14, 19). Therefore, the release of calcium ions by the cement occurs as a result of the decomposition of the calcium aluminate hydrate in a lower speed than that in MTA (14, 19). In spite of the lower release of calcium ions of EndoBinder (14, 19), in the present research this cement presented a tissue reaction similar to that caused by MTA and calcium hydroxide in all the experimental time intervals. The materials always presented an inflammatory reaction with chronic evolution during the course of the periods of analysis, with progressive collagenization from the initial period until the end, inflammatory infiltrate with predominance of polymorphous and mononuclear cells, and a reduced quantity of macrophages and giant cells phagocytosing dispersed material residues. Therefore, on the basis of the methodology used in this in vivo study, it was possible to conclude that from the biologic point of view, EndoBinder becomes a promising option in endodontic therapy. Nevertheless, it is valid to emphasize that further research related to other biologic and physicochemical properties of this new cement must be conducted before its validation and indication as an endodontic treatment option for clinical situations in humans.
Acknowledgments The authors acknowledge the Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo-FAPESP (grant 2012/18479-8), the Fundac¸~ao para o Desenvolvimento da UNESP-FUNDUNESP (grant 0024/021/13-PROPe-CDC), and the Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico-CNPq (grant 301029/2010-1) for financial support. The authors deny any conflicts of interest related to this study.
References 1. De Souza Costa CA, Lopes do Nascimento AB, Teixeira HM, Fontana UF. Response of human pulps capped with a self-etching adhesive system. Dent Mater 2001;17: 230–40. 2. Accorinte ML, Holland R, Reis A, et al. Evaluation of mineral trioxide aggregate and calcium hydroxide cement as pulp-capping agents in human teeth. J Endod 2008; 34:1–6. 3. Stanley HR. Pulp capping: conserving the dental pulp—can it be done? is it worth it? Oral Surg Oral Med Oral Pathol 1989;68:628–39. 4. Torabinejad M, Parirokh M. Mineral trioxide aggregate: a comprehensive literature review–part II: leakage and biocompatibility investigations. J Endod 2010;36: 190–202. 5. Shahravan A, Jalali SP, Torabi M, et al. A histological study of pulp reaction to various water/powder ratios of white mineral trioxide aggregate as pulp-capping
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material in human teeth: a double-blinded, randomized controlled trial. Int Endod J 2011;44:1029–33. Simon S, Rilliard F, Berdal A, Machtou P. The use of mineral trioxide aggregate in one-visit apexification treatment: a prospective study. Int Endod J 2007;40:186–97. Jacobovitz M, de Lima RK. Treatment of inflammatory internal root resorption with mineral trioxide aggregate: a case report. Int Endod J 2008;41:905–12. Orosco FA, Bramante CM, Garcia RB, et al. Sealing ability, marginal adaptation and their correlation using three root-end filling materials as apical plugs. J Appl Oral Sci 2010;18:127–34. Leye Benoist F, Gaye Ndiaye F, Kane AW, et al. Evaluation of mineral trioxide aggregate (MTA) versus calcium hydroxide cement (Dycal) in the formation of a dentine bridge: a randomised controlled trial. Int Dent J 2012;62:33–9. Islam I, Chng HK, Yap AU. Comparison of the physical and mechanical properties of MTA and Portland cement. J Endod 2006;32:193–7. Ber BS, Hatton JF, Stewart GP. Chemical modification of ProRoot MTA to improve handling characteristics and decrease setting time. J Endod 2007;33:1231–4. Bortoluzzi EA, Broon NJ, Bramante CM, et al. Sealing ability of MTA and radiopaque Portland cement with or without calcium chloride for root-end filling. J Endod 2006;32:897–900. Garcia Lda F, Aguilar FG, Rossetto HL, et al. Staining susceptibility of new calcium aluminate cement (EndoBinder) in teeth: a 1-year in vitro study. Dent Traumatol 2013;29:383–8. Garcia Lda F, Chinelatti MA, Rossetto HL, Pires-de-Souza Fde C. Solubility and disintegration of new calcium aluminate cement (EndoBinder) containing different radiopacifying agents. J Endod 2014;40:261–5. Parirokh M, Torabinejad M. Mineral trioxide aggregate: a comprehensive literature review—part I: chemical, physical, and antibacterial properties. J Endod 2010;36: 16–27.
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16. Schembri M, Peplow G, Camilleri J. Analyses of heavy metals in mineral trioxide aggregate and Portland cement. J Endod 2010;36:1210–5. 17. International Standards Organization. Water-based cements: part 1—powder/liquid acid-base cements. ISO 9917-1 2007;1:20. 18. Kazemi RB, Safavi KE, Sp angberg LS. Dimensional changes of endodontic sealers. Oral Surg Oral Med Oral Pathol 1993;76:766–71. 19. Aguilar FG, Roberti Garcia LF, Panzeri Pires-de-Souza FC. Biocompatibility of new calcium aluminate cement (EndoBinder). J Endod 2012;38:367–71. 20. Souza PP, Aranha AM, Hebling J, et al. In vitro cytotoxicity and in vivo biocompatibility of contemporary resin-modified glass-ionomer cements. Dent Mater 2006;22: 838–44. 21. International Standards Organization. Dentistry: evaluation of biocompatibility of medical devices used in dentistry. ISO 7405 2007;1:34. 22. Shahi S, Rahimi S, Lotfi M, et al. A comparative study of the biocompatibility of three root-end filling materials in rat connective tissue. J Endod 2006;32: 776–80. 23. Yaltirik M, Ozbas H, Bilgic B, Issever H. Reactions of connective tissue to mineral trioxide aggregate and amalgam. J Endod 2004;30:95–9. 24. Dammaschke T, Wolff P, Sagheri D, et al. Mineral trioxide aggregate for direct pulp capping: a histological comparison with calcium hydroxide in rat molar teeth. Quintessence Int 2010;41:e20–30. 25. ANSI/ADA specification no. 57. Endodontic Sealing Material. Chicago: ANSI/ADA; 2000. 26. Camilleri J. Characterization of hydration products of mineral trioxide aggregate. Int Endod J 2008;41:408–17. 27. Ozdemir HO, Ozcelik B, Karabucak B, Cehreli ZC. Calcium ion diffusion from mineral trioxide aggregate through simulated root resorption defects. Dent Traumatol 2008;24:70–3.
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Biocompatibility of New Calcium Aluminate Cement: Tissue Reaction and Expression of Inflammatory Mediators and Cytokines Lucas da Fonseca Roberti Garcia, DDS, MSc, PhD, Claudia Huck, DDS, MSc, Letıcia Menezes de Oliveira, Pedro Paulo Chaves de Souza, DDS, MSc, PhD, and Carlos Alberto de Souza Costa, DDS, MSc, PhD Abstract Introduction: The aim of this study was to evaluate the biocompatibility of a new calcium aluminate cement (EndoBinder) in subcutaneous tissue of rats in comparison with mineral trioxide aggregate and calcium hydroxide hard-setting cement. Methods: Polyethylene tubes (1.5 10 mm) containing the dental cements were implanted into dorsal subcutaneous tissue of 30 rats. After experimental periods of 7, 30, and 90 days, biopsies were performed for tissue response analysis under optical light microscope. The mRNA extraction was performed for molecular evaluation of the inflammatory process in the peri-implant tissue, which was submitted to quantitative real-time polymerase chain reaction analysis for inflammatory mediators and cytokines TNF-a, Ptges2, Il-1b, Il-4, and Il-10. Results: On the basis of the score used to grade the tissue reaction (0–3), EndoBinder (0) presented no inflammatory reaction after the 90-day period, a similar result to mineral trioxide aggregate and calcium hydroxide. The thickness of inflammatory capsules (mm) also presented significant decrease during the course of periods (P < .05). As regards expression of inflammatory mediators, Ptges2 and Il-10 were detected only at 7 and 30 days, with no statistically significant difference among the experimental groups (P > .05). Conclusions: EndoBinder induced limited inflammatory reaction. It was considered biocompatible when tested in subcutaneous tissue of rats. (J Endod 2014;40:2024–2029)
Key Words Biocompatibility, calcium aluminate cement, calcium hydroxide, mineral trioxide aggregate, tissue reaction
From the Department of Physiology and Pathology, Araraquara School of Dentistry, Univ Estadual Paulista, Araraquara, S~ao Paulo, Brazil. Address requests for reprints to Dr Carlos Alberto de Souza Costa, Department of Physiology and Pathology, Univ Estadual Paulista - UNESP, Araraquara School of Dentistry, Humaita Street, 1680, Araraquara, SP, Brazil 14801-903. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.08.015
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everal studies have reported that calcium hydroxide continues to be one of the most used materials for pulp/dentin complex protection, against which new products must be evaluated and compared, especially in biological tests involving animals and humans (1, 2). Despite the widespread use of calcium hydroxide, some questions with regard to its performance persist (3). Therefore, the constant development of new materials for pulp therapy application created mineral trioxide aggregate (MTA) (4). The MTA cement was initially used as material for retrograde filling and root and furca perforation treatments (4); however, because of its good clinical performance, it has also been used for several other clinical applications such as pulpotomy (5), treatment of teeth with incomplete apexification (6), root resorption (7), intracoronal barrier before bleaching, and apical plug (8), root canal filling (4), and pulp capping in conservative treatments (9). Among the advantages of MTA in comparison with calcium hydroxide, its higher mechanical strength and marginal sealing ability are outstanding (10). However, some of its negative properties such as poor handling characteristics (11), low flow ability (12), high rates of tooth structure staining (13), solubility in moist conditions (14), long setting time (15), and presence and release of arsenic (16) above the safe limits proposed by ISO 9917-1 specification (17) must be considered. The negative features observed for calcium hydroxide and MTA cements justify the development of new materials that incorporate the appropriate biological properties of both materials in a new product with easy handling, application, and adequate mechanical strength (15). Thus, a calcium aluminate-based cement, EndoBinder (Binderware, S~ao Carlos, SP, Brazil), was developed at the Federal University of S~ao Carlos (BrazilUFSCAR, patent number PI0704502-6). The laboratory synthesis process of EndoBinder provides several advantages in comparison with MTA such as greater control of impurity levels, especially Fe2O3, which promotes tooth staining (7, 13), and free MgO and CaO, responsible for an undesired expansion of the material in contact with moisture (18). The balance between stoichiometric phases rich in Al2O3 and CaCO3, responsible for the hydrophilic setting process of EndoBinder, promotes greater compatibility between living tissues and cement, as well as adequate physicochemical properties (12, 19). However, in vivo studies that prove the biological compatibility of EndoBinder are necessary before its validation as a material for clinical application. Thus, the aim of this study was to evaluate the biocompatibility of this new calcium aluminate cement, containing zinc oxide as radiopacifying agent, by means of tissue reaction analysis and expression of inflammatory mediators in rats in comparison with MTA and calcium hydroxide.
Materials and Methods Selection of Animals The study was developed in accordance with the determinations of the Research Ethics Committee on the Use of Animals - Araraquara School of Dentistry/UNESP (Process CEUA No. 3/2013), in compliance with the ethical principles for use of laboratory animals at all stages of the experiment. The animals were kept in plastic cages
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Basic Research—Technology (40 32 17 cm) in an acclimatized bioterium with established lighting (12 hours light/12 hours dark at temperature of 21 –25 C), receiving balanced rations (Nuvilab, Colombo, PR, Brazil) and water ad libitum.
Subcutaneous Implant Thirty male rats (Rattus novergicus; Wistar Institute, Philadelphia, PA) were selected, with weight ranging between 250 and 300 g, and 10 specimens were used for each of the evaluation time intervals (7, 30, and 90 days). Polyethylene tubes measuring 1.5 mm in internal diameter and 10 mm long were obtained according to the methodology described by Souza et al (20). The tubes were immersed in 70% alcohol for 120 minutes and abundantly washed with sterilized distilled water, and after this they were autoclaved. The materials tested were as follows: EndoBinder + ZnO (radiopacifying agent, 20% by weight), white MTA (^Angelus, Londrina, PR, Brazil), and calcium hydroxide (Biodin^amica, Ibipor~a, PR, Brazil). All the materials were manipulated in accordance with the manufacturers’ recommendations, with the proportion of 1 g powder to 0.21 mL distilled water being used for EndoBinder and for MTA 1 dose of powder (0.15 mg) to 1 drop (0.5 mL) of distilled water. For calcium hydroxide P.A., 1 g powder was placed on a glass plate, which was manipulated with 1 mL physiological solution at 0.9% (1:1) until a homogeneous paste was obtained. After manipulation, the previously sterilized polyethylene tubes were filled with the cements by using sterile lentulo spirals (Dentsply/ Maillefer, Ballaigues, Switzerland) compatible with the internal diameter of the tubes. For the surgical procedures, the animals were anesthetized by intraperitoneal administration of a solution composed of 10% ketamine chloride (Ketamina Agener; Uni~ao Quımica Farmac^eutica Nacional S/A, Embu-Guac¸u, SP, Brazil) and xylazine (Dopazer; Laboratorios Calier S/A, Barcelona, Spain) in the proportion of 75 mg/kg and 10 mg/kg, respectively. After trichotomy and disinfection of the operative field with sterile gauze imbibed in a solution of 5% povidone-iodine, 2 central incisions, one on the left and the other on the right side (5 mm long each), were performed in the dorsum of each animal by using a no. 15 scalpel blade. Lateral divulsion of the subcutaneous tissue was carefully performed starting from each central incision by using a blunt-tipped scissors for this purpose. Thus, 2 surgical recesses with a mean depth of 20 mm were obtained, in which the polyethylene tubes filled with the test materials were implanted longitudinally, remaining parallel to the incisions. Two polyethylene tubes filled with MTA or EndoBinder were implanted in 15 rats. In another 15 rats, tubes without any material (negative control) or filled with calcium hydroxide were implanted. After this, suturing was performed with 3-0 silk thread (Ethicon, S~ao Jose dos Campos, SP, Brazil). After the time intervals of 7, 30, and 90 days had elapsed, the animals were killed by anesthetic overdose, and biopsies were obtained with sufficient safety margin around the polyethylene tubes. At one of the extremities, tissue samples were collected for molecular evaluation of the inflammatory process, with mRNA being extracted and submitted to quantitative real-time polymerase chain reaction analysis (qPCR) for inflammatory mediators and cytokines. The histopathological analysis was performed in the connective tissue adjacent to the other extremity of the polyethylene tube. For this purpose, the surgical parts (n = 5) were immediately immersed in a 10% formalin solution (Merck, Darmstadt, Germany), in which they remained in a fixation process for the period of 24 hours. After this, they were submitted to routine laboratory processing. Semi-serial 5-mm-thick histologic cuts were obtained and stained with hematoxylin-eosin (Merck). JOE — Volume 40, Number 12, December 2014
Histopathological Analysis Histopathological analysis was performed by using an optical light microscope Axio Star Plus (Carl Zeiss, Oberkochen, Germany) at 32, 64, 125, and 320 magnifications. The histopathological events evaluated were the following: inflammatory infiltrate (polymorphonuclear and mononuclear cells), cellularity (fibroblasts), vascularization (blood vessels), and macrophagic activity (macrophages and multinuclear giant cells independently scored). On the basis of the tissue responses stimulated by the cements and by the control group and in accordance with the standard ISO 7405 (21), a score was used to quantify the absence or presence of these events; the inflammatory reaction was classified as (0) absent, (1) discrete, (2) moderate, and (3) severe. The thickness of the inflammatory capsule (mm) was gauged with the aid of the software program AxioVision 4.6 (Carl Zeiss, http://www.zeiss.de/microscopy). qPCR Samples of the tissues submitted to biopsy in the different time intervals of analysis (7, 30, and 90 days) were used for total RNA extraction in accordance with the protocol for the RNAqueous-4PCR kit (Ambion; Life Technologies, Grand Island, NY). For each total RNA sample extracted from the samples, the complementary DNA (cDNA) was synthetized for the qPCR reaction. For this purpose, the protocol of the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used. The qPCR was performed in StepOnePlus Real-Time PCR System equipment (Applied Biosystems) by using cDNA as template, a set of TaqMan primers and probes designed to detect the genes encoding to interleukin 1b (Il-1b), tumor necrosis factor-a (TNF-a), interleukin-4 (Il-4), interleukin 10 (Il-10), and prostaglandin E synthase 2 (Ptges2), Taqman Universal Master Mix (Applied Biosystems), and nuclease-free water. To control the amount of cDNA input, the expression of all genes was normalized against the gene encoding to the housekeeping gene b-actin (Actb). For evaluation of the inflammatory response, real-time PCR was performed for the proinflammatory cytokines; for evaluation of the resolution of inflammation, the mRNA expression of the anti-inflammatory cytokines Il-4 and Il-10 was evaluated. Statistical Analysis The normal distribution of data was tested by the KolmogorovSmirnov test, and the values obtained were statistically compared (2-way analysis of variance, the Bonferroni test, P < .05) with the aid of Graphpad Prism 4.0 Software (GraphPad Software, La Jolla, CA).
Results Histopathological Analysis The values obtained for all the histopathological events evaluated in each time interval are presented in Table 1. Generally the tissue reactions diminished during the course of time for all tested materials. Only one severe score was determined for MTA with regard to blood vessels at the 7-day period; all other scores were moderate or lower. Period of 7 Days For EndoBinder, at the tubular opening was observed the formation of an ample loose inflammatory capsule, still disorganized, with moderate mixed inflammatory infiltrate, characterized by the presence of neutrophils and lymphocytes. Moderate fibroangioblastic proliferation and discrete local collagenization, associated with the presence of mononuclear phagocytes (macrophages), and multinuclear giant cells Biocompatibility of Calcium Aluminate Cement
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Basic Research—Technology TABLE 1. Mean Values of Histopathologic Events Observed in Each Group at Different Time Intervals of the Study 7 days
30 days
90 days
Histopathological events
C
MTA
CH
EB
C
MTA
CH
EB
C
MTA
CH
EB
Inflammatory infiltrate Fibroblasts Blood vessels Macrophage Multinuclear giant cells General inflammatory index
2 1 1 1 1 1
2 2 3 2 2 2
2 2 2 2 2 2
2 2 2 2 2 2
1 1 1 1 1 1
1 2 2 1 1 1
1 2 1 1 1 1
1 2 1 1 1 1
0 1 0 0 0 0
0 1 0 0 0 0
0 1 0 0 0 0
0 1 0 0 0 0
C, control; CH, calcium hydroxide; EB, EndoBinder. Scores: 0, absent; 1, discrete; 2, moderate; 3, severe. N = 5.
encircling the residual material were also observed in the main area of analysis (Fig. 1A). For the MTA cement, there was evidence of disorganized inflammatory capsule formation, with discrete fibrous content and moderate mixed inflammatory infiltrate. Congested blood vessels in the midst of areas of edema and intense fibroangioblastic proliferation were also observed. Moderate quantities of mononuclear phagocytes and multinucleated giant cells in the process of phagocytosis of dispersed
material residues at the tubular opening were detected. Discrete necrotic areas were observed in the main area of analysis (Fig. 1B). For calcium hydroxide, the reactive events in the tissue were similar to those of EndoBinder and MTA; however, a larger area of tissue necrosis was observed adjacent to the tubular opening (Fig. 1C). Moderate inflammatory infiltrate, characterizing the formation of loose tissue at the tubular opening, was observed for control group (Fig. 1D).
Figure 1. (A) EndoBinder 7 days: inflammatory infiltrate (circle) of moderate amplitude and congested blood vessels (arrow). Hematoxylin-eosin; original magnification, 125. (B) MTA 7 days: moderate inflammatory infiltrate associated with dilated and congested blood vessels (arrow). Observe presence of material residues, with small area of necrosis close (box). Hematoxylin-eosin; original magnification, 125. (C) Calcium hydroxide 7 days: large quantity of material residue dispersed within the tissue. Observe ample area of necrosis close to material (box), more accentuated than areas found for EndoBinder and MTA. Hematoxylin-eosin; original magnification, 125. (D) Control 7 days: moderate infiltrate of polymorphous inflammatory and mononuclear cells, characterizing formation of loose tissue at tubular opening. Hematoxylin-eosin; original magnification, 125. (E) EndoBinder 30 days: area adjacent to tubular opening with more organized and delimited inflammatory capsule. Note decrease in number of polymorphous and mononuclear cells and discrete macrophagic activity. Hematoxylin-eosin; original magnification, 64. (F) MTA 30 days: significant reduction in capsule thickness and inflammatory infiltrate. Significant decrease in number of polymorphous and mononuclear cells as well as discrete local macrophagic activity. Hematoxylin-eosin; original magnification, 320. (G) Calcium hydroxide 30 days: evidence of decrease in inflammatory infiltrate, with discrete local macrophagic activity. Hematoxylin-eosin; original magnification, 320. (H) Control 30 days: significant decrease in inflammatory infiltrate and discrete macrophagic activity because of absence of material capable of inducing local inflammatory process. Hematoxylin-eosin; original magnification, 320. (I) EndoBinder 90 days: accentuated reduction in inflammatory capsule thickness and inflammatory infiltrate. Hematoxylin-eosin; original magnification, 64. (J) MTA 90 days: accentuated reduction in dense inflammatory capsule thickness (arrow) with reduced inflammatory infiltrate. Hematoxylin-eosin; original magnification, 32. (K) Calcium hydroxide 90 days: dense fibrous capsule presented as very thin (bar). Hematoxylin-eosin; original magnification, 64. (L) Control 90 days: absence of inflammatory infiltrate with discrete fibroangioblastic proliferation, indicative of reparative process. Hematoxylin-eosin; original magnification, 125.
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Figure 2. (A and B) Graphic representation of mean values (mm) of inflammatory capsule thickness at different periods of study. (C) Graphic representation of inflammatory mediator Il-10 expression according to experimental periods. (D) Graphic representation of inflammatory mediator Ptges2 expression according to experimental periods. (E and F) Graphic representation of inflammatory mediator and cytokine expression in function of period of analysis. Different lower case letters over bars indicate statistically significant difference (2-way analysis, Bonferroni test, P < .05). n = 5. Gapdh, glyceraldehyde-3-phosphate dehydrogenase.
Period of 30 Days For EndoBinder cement, an important reduction in inflammatory infiltrate was observed, associated with discrete macrophagic activity mediated by giant cells. The inflammatory capsule present at the tubular opening presented as fibrous and well-organized, characterizing a tissue repair process in evolution. No foci of mineralization or necrotic tissue were found close to the tubular opening (Fig. 1E). For MTA, the findings were similar to those of EndoBinder; however, a discrete necrotic area still persisted in this intermediate experimental period (Fig. 1F). For calcium hydroxide, the reduction in the intensity of tissue response was similar to that observed for the other cements. The notable reduction in the area of necrosis adjacent to the tubular opening should be pointed out (Fig. 1G). For control group, a significant decrease in inflammatory infiltrate and discrete macrophagic activity was observed (Fig. 1H). Period of 90 Days At 90 days, EndoBinder tissue samples exhibited no inflammatory infiltrate. Moreover, no mononuclear phagocytes and multinucleated giant cells were observed. A dense fibrous capsule, in continuity with that formed on the lateral surface of the tube, was present at the tubular opening, characterizing the local occurrence of complete tissue repair (Fig. 1I). For the MTA cement, a discrete fibroangioblastic proliferation still persisted, with no tissue necrosis areas being detected. A thin, dense fibrous capsule was observed at the tubular opening, characterizing complete tissue repair in contact with the implanted material (Fig. 1J). A similar tissue repair response was observed for calcium hydroxide in this period (Fig. 1K). In control group, absence of inflammatory infiltrate with discrete fibroangioblastic proliferation was observed JOE — Volume 40, Number 12, December 2014
(Fig. 1L). The general inflammatory index for the 3 materials in this period was established as 0 (absent), determining acceptable biologic compatibility.
Inflammatory Capsule The mean values obtained for inflammatory capsule thickness of the different materials in the different experimental time intervals are seen in Figure 2A and B. During the course of the experimental periods of analysis, significantly decreasing amplitude of inflammatory capsule formation was observed for all the groups (P < .05). The reactive capsule formed adjacent to EndoBinder cement (133.18 mm) in the period of 7 days was significantly thinner than those observed for calcium hydroxide (153.53 mm) and MTA (142.60 mm) in the same period (P < .05). However, this capsule was thicker when compared with the control group (86.05 mm) (P < .05). The trend toward reduction in capsule thickness remained in the intermediate period of analysis (30 days); however, the thickness of capsules formed by EndoBinder and MTA no longer presented statistically significant difference (P > .05). In the period of 90 days, all the groups presented significant decrease in the amplitude of dense fibrous capsules (P < .05), with EndoBinder (30.66 mm), MTA (30.83 mm), and calcium hydroxide (31.26 mm) being statistically similar (P > .05) but differing from that of the control group (16.99 mm) (P < .05). qPCR The mean values obtained for each of the inflammatory mediators and cytokines expressed in the different experimental time intervals may be seen in Figure 2C–F. Biocompatibility of Calcium Aluminate Cement
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Basic Research—Technology Among the analyzed genes, only Il-10 and Ptges2 transcripts could be detected after 7 and 30 days of implantation of the materials. In the period of 90 days, it was not possible to detect the expression of any of the mediators. At 7 days, EndoBinder presented the highest values for Ptges2 and Il-10 mRNA expression, with statistically significant difference in comparison with the control and MTA groups (P < .05), however, similar to the values of calcium hydroxide (P > .05). At 30 days, all the materials presented a reduction in the values of expression of both mediators; nevertheless, this reduction was statistically significant (P < .05) only for EndoBinder.
Discussion Biological compatibility is one of the most important properties of endodontic sealing cements, because toxic components may come in contact with adjacent tissues and induce irritation (1, 20). The results of this study demonstrated absence of inflammatory reaction for all the tested materials at the end of the 90-day period of analysis. In a similar study, Aguilar et al (19) demonstrated that after 42 days, MTA still presented discrete inflammatory reaction, indicative of a persistent chronic inflammatory process. Nevertheless, in accordance with the ISO 7405 standard (21), a moderate inflammatory reaction 15 days after implant placement with a trend toward decrease after 12 weeks is considered acceptable. It has been demonstrated that materials in intimate contact with live tissues are irritants; however, according to Shahi et al (22), more important than the irritant potential of materials is the duration of this effect on tissues to which they are applied. Yaltirik et al (23) reported the occurrence of a chronic inflammatory process promoted by MTA cement 60 days after subcutaneous implantation of the material; this was a different result to that found in the present study, in which 30 days after implantation, all the cements tested presented, at most, a discrete inflammatory reaction at the tubular opening. Moreover, the infiltrate of polymorphous and mononuclear cells presents significant reduction in comparison with the initial period of analysis, which is different from the findings of previous studies, in which macrophages and multinucleated giant cells were observed phagocytosing particles of MTA dispersed in the connective tissue (19, 23). Furthermore, the results obtained in the present research as regards the expression of inflammatory mediators by means of qPCR analysis proved the results of the histopathological analysis. Only Ptges2, the gene encoding cycloogenase-2, the enzyme responsible for the metabolism of arachidonic acid and production of prostanoids, and Il-10, a cytokine expressed during chronic inflammatory reactions, were detected in the initial periods (7 and 30 days) of evaluation. It should be pointed out that several previous studies have corroborated the results of the present study, because they demonstrated the excellent biological properties of MTA (4, 24), with these being related to the capacity of releasing calcium ions and, consequently, to the alkaline pH produced by the material (24). According to Garcia et al (14), the MTA and EndoBinder hardsetting cements present similar solubility and disintegration, with a mass loss index of greater than 3%, a value higher than the limit proposed by specification no. 57 of American National Standards Institute/American Dental Association (25). Nevertheless, it has been demonstrated that such solubilization indices do not contraindicate the use of these materials, because their good biological performance is related to their capacity to release calcium and hydroxyl ions into the medium during their hydration process (4). Other studies have proved that these ions are the main components detected in soluble and insoluble residues released by MTA, demonstrating that the solubility of the cement is an important factor for 2028
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the delivery of calcium and hydroxyl ions in periodontal and pulp tissue (4, 5, 9). It has been demonstrated that moisture conditions may improve the biological performance of MTA in shorter periods of time, favoring the separation of cement particles by solubilization and disintegration process, which come into contact with the tissues, initiating an inflammatory process that leads to their repair (4). The same could be observed for EndoBinder, because it concerns a cement with hydraulic setting, such as MTA, in which the physical-chemical interaction of the material with the humid environment is fundamental, particularly with regard to its reparative capacity (24, 26, 27). During the setting of MTA, the tricalcium silicate hydrates and releases calcium hydroxide. The calcium hydroxide releases calcium ions into the moist areas adjacent to the placement of MTA (24). These ions are produced from the calcium hydroxide present and from the decomposition of calcium silicate hydrate, which is released in a lower speed in comparison with that of calcium hydroxide (26). However, the hydration process of calcium aluminate cements such as EndoBinder leads to the formation of calcium aluminate and aluminum hydroxide hydrate (14, 19). Therefore, the release of calcium ions by the cement occurs as a result of the decomposition of the calcium aluminate hydrate in a lower speed than that in MTA (14, 19). In spite of the lower release of calcium ions of EndoBinder (14, 19), in the present research this cement presented a tissue reaction similar to that caused by MTA and calcium hydroxide in all the experimental time intervals. The materials always presented an inflammatory reaction with chronic evolution during the course of the periods of analysis, with progressive collagenization from the initial period until the end, inflammatory infiltrate with predominance of polymorphous and mononuclear cells, and a reduced quantity of macrophages and giant cells phagocytosing dispersed material residues. Therefore, on the basis of the methodology used in this in vivo study, it was possible to conclude that from the biologic point of view, EndoBinder becomes a promising option in endodontic therapy. Nevertheless, it is valid to emphasize that further research related to other biologic and physicochemical properties of this new cement must be conducted before its validation and indication as an endodontic treatment option for clinical situations in humans.
Acknowledgments The authors acknowledge the Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo-FAPESP (grant 2012/18479-8), the Fundac¸~ao para o Desenvolvimento da UNESP-FUNDUNESP (grant 0024/021/13-PROPe-CDC), and the Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico-CNPq (grant 301029/2010-1) for financial support. The authors deny any conflicts of interest related to this study.
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16. Schembri M, Peplow G, Camilleri J. Analyses of heavy metals in mineral trioxide aggregate and Portland cement. J Endod 2010;36:1210–5. 17. International Standards Organization. Water-based cements: part 1—powder/liquid acid-base cements. ISO 9917-1 2007;1:20. 18. Kazemi RB, Safavi KE, Sp angberg LS. Dimensional changes of endodontic sealers. Oral Surg Oral Med Oral Pathol 1993;76:766–71. 19. Aguilar FG, Roberti Garcia LF, Panzeri Pires-de-Souza FC. Biocompatibility of new calcium aluminate cement (EndoBinder). J Endod 2012;38:367–71. 20. Souza PP, Aranha AM, Hebling J, et al. In vitro cytotoxicity and in vivo biocompatibility of contemporary resin-modified glass-ionomer cements. Dent Mater 2006;22: 838–44. 21. International Standards Organization. Dentistry: evaluation of biocompatibility of medical devices used in dentistry. ISO 7405 2007;1:34. 22. Shahi S, Rahimi S, Lotfi M, et al. A comparative study of the biocompatibility of three root-end filling materials in rat connective tissue. J Endod 2006;32: 776–80. 23. Yaltirik M, Ozbas H, Bilgic B, Issever H. Reactions of connective tissue to mineral trioxide aggregate and amalgam. J Endod 2004;30:95–9. 24. Dammaschke T, Wolff P, Sagheri D, et al. Mineral trioxide aggregate for direct pulp capping: a histological comparison with calcium hydroxide in rat molar teeth. Quintessence Int 2010;41:e20–30. 25. ANSI/ADA specification no. 57. Endodontic Sealing Material. Chicago: ANSI/ADA; 2000. 26. Camilleri J. Characterization of hydration products of mineral trioxide aggregate. Int Endod J 2008;41:408–17. 27. Ozdemir HO, Ozcelik B, Karabucak B, Cehreli ZC. Calcium ion diffusion from mineral trioxide aggregate through simulated root resorption defects. Dent Traumatol 2008;24:70–3.
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