Basic Research—Biology Tissue Response and Immunoexpression of Interleukin 6 Promoted by Tricalcium Silicate–based Repair Materials after Subcutaneous Implantation in Rats Aline S. Andrade, MSc,* Guilherme F. Silva, PhD,* Josette Camilleri, PhD,† Estela S. Cerri, PhD,‡ Juliane M. Guerreiro-Tanomaru, PhD,* Paulo S. Cerri, PhD,‡ and M�ario Tanomaru-Filho, PhD* Abstract Significance The evaluation of the inflammatory response induced by experimental tricalcium silicate cement with 20%zirconium oxide andMTA Plus showed a similar subcutaneous reaction in rats, suggesting that they are biocompatible to be used as repara- tive materials. Introduction: The aim of the present study was to eval- uate the inflammatory response induced by experi- mental tricalcium silicate cement with 20% zirconium oxide (TSC) and MTA Plus (MTAP; Avalon Biomed Inc, Bradenton, FL) in rat subcutaneous tissues. Methods: Polyethylene tubes were filled with TSC (n = 20) and MTAP (n = 20) and implanted in the dorsal subcutane- ous tissues of 32 rats. Empty tubes were used as the control (control group [CG], n = 20). After 7, 15, 30, and 60 days, the tubes with connective tissue were removed, and the inflammatory cells and immunola- beled cells for interleukin 6 (IL-6) were counted. Data were statistically analyzed using analysis of variance and the Tukey test (P # .05). Results: An increased number of inflammatory and immunolabeled cells for IL-6 were observed at 7 days. The number of inflamma- tory cells was higher for TSC and MTAP than the CG (P < .001) at 7 days; after 30 and 60 days, no significant differences were observed among the TSC, MTAP, and CG (P = .955). The number of immunolabeled cells for IL-6 was similar for TSC, MTAP, and CG at all evaluated periods. A gradual and significant decrease was observed in the number of inflammatory cells and IL- 6–immunopositive cells. At 60 days, the capsules adja- cent to TSC and MTAP exhibited fibroblasts and bundles of collagen fibers. Conclusions: TSC and MTAP caused a similar subcutaneous reaction in rats, suggesting that they are biocompatible and present similar immune re- sponses. (J Endod 2018;44:458–463) Key Words Biocompatibility, immunohistochemistry, interleukin 6, tricalcium Silicate, zirconium oxide From the Departments of *Restorative Dentistry and ‡Morpholo †Institute of Clinical Sciences, College of Medical and Dental Scienc Address requests for reprints to Dr M�ario Tanomaru-Filho, A Araraquara, SP, Brazil. E-mail address: tanomaru@uol.com.br 0099-2399/$ - see front matter Copyright ª 2017 American Association of Endodontists. https://doi.org/10.1016/j.joen.2017.12.006 458 Andrade et al. Mineral trioxide aggre- gate (MTA) is a tricalcium silicate–based cement containing bismuth oxide (Bi2O3) as a radio- pacifier (1). Tricalcium silicate–based biomate- rials have shown proper biological and physico- chemical properties (2–5). The replacement of Bi2O3 by other radiopacifiers has been proposed (6–8) because Bi2O3 interferes in the hydration of MTA (9), resulting in an increase in porosity and a reduction of material strength (10). Furthermore, bis- muth oxide interferes in cellular proliferation (11), and calcium silicate–based materials containing Bi2O3 induce an inflammatory response in rat subcutaneous tissue (12). Another disadvantage of someMTAmaterials is the presence of Portland cement as a major component, which may contain heavy metal contamination (13). MTA Plus (MTAP; Avalon Biomed Inc, Bradenton, FL) is composed of tricalcium silicate and di- calcium silicate added to Bi2O3 (14). As a material used in pulpotomies, MTAP pro- duced calcium hydroxide and did not exhibit dental discoloration (15). Moreover, MTAP promoted a low immunoexpression of interleukin 1 beta and 1 alpha, inflamma- tory cytokines, and allowed mineralized tissue formation over the pulp tissue of rats (16). Gomes-Corn�elio et al showed that MTAP presents biocompatibility and bioactivity in human osteoblastlike cells (17). A manufactured tricalcium silicate cement ([CaO]3.SiO2; Mineral Research Pro- cessing, Meyzieu, France) associated with zirconium oxide (ZrO2) as a radiopacifier is used as reparative material (18). The association of 80% pure tricalcium silicate cement (TSC) and 20% ZrO2 presents proper properties, such as solubility, radiopacity, compressive strength, setting time, and microhardness (3). Furthermore, this cement in contact with simulated tissue fluid showed bioactivity (3, 19). Despite these facts, until now, there is no other evidence of the biological in vivo response of this material reported in the literature. The aim of the present study was to evaluate the tissue response promoted by TSC and MTAP in rat subcutaneous tissues. Morphologic and morphometric analyses and gy, Araraquara Dental School, UNESP–S~ao Paulo State University, Araraquara, S~ao Paulo, Brazil; es, University of Birmingham, Birmingham, UK. raraquara Dental School, UNESP– Univ Estadual Paulista, Rua Humait�a 1680, CEP 14801-903 JOE — Volume 44, Number 3, March 2018 Delta:1_given name Delta:1_surname Delta:1_given name Delta:1_surname Delta:1_given name Delta:1_surname Delta:1_given name mailto:tanomaru@uol.com.br https://doi.org/10.1016/j.joen.2017.12.006 http://crossmark.crossref.org/dialog/?doi=10.1016/j.joen.2017.12.006&domain=pdf TABLE 1. Materials Evaluated and Used Proportions Group Material Composition Proportion (powder/distilled water) TSC Cement tricalcium silicate* with 20% ZrO2 † Tricalcium silicate, zirconium oxide 1g/340 mL MTAP White mineral trioxide aggregate‡ Tricalcium silicate, dicalcium silicate, calcium sulfate, silica, bismuth oxide 1g/340 mL MTAP, MTA Plus; TSC, tricalcium silicate cement. *Mineral Research Processing, Meyzieu, France. †Sigma-Aldrich, St Louis, MO. ‡MTA Plus; Avalon Biomed Inc, Bradenton, FL. Basic Research—Biology the immunohistochemical reaction for the detection of interleukin 6 (IL-6), a proinflammatory cytokine, were performed. The null hypoth- esis was that there would be no difference in tissue reactions caused by TSC and MTAP. Figure 1. Light micrographs of sections showing portions of capsules adjacent to th and (D–F) 15 days. (A–C) Several ICs (arrows) are observed, mainly in the portion vessels. (D–F) ICs (arrows) among blood vessels (Bv) and fibroblasts (Fb). Gc, g JOE — Volume 44, Number 3, March 2018 Material and Methods The present research protocol was analyzed and approved by the Ethical Committee for Animal Research of Araraquara Dental School, UNESP, Araraquara, S~ao Paulo, Brazil (Process CEUA no. 33/2014). e opening of the tubes implanted (T) in the subcutaneous tissue after (A–C) 7 of the capsule adjacent to the opening of the tubes (T). N, neutrophil; Bv, blood iant cells. Hematoxylin-eosin, 695�. Tissue Response and Immunoexpression of IL-6 459 Figure 2. Light micrographs of sections showing portions of capsules adjacent to the opening of the tubes implanted (T) in the subcutaneous tissue after (A–C) 30 and (D–F) 60 days. Typical fibroblasts (Fb) are observed among bundles of collagen fibers (Cf) in the capsules. Few ICs (arrows) are present, mainly in the capsules of MTAP and the CG. Mp, macrophages; Bv, blood vessels. Hematoxylin-eosin, 695�. Basic Research—Biology In this study, 32 rats were used and distributed into 3 groups: TSC (TSC [Mineral Research Processing, Meyzieu, France] and 20% ZrO2 [Sigma-Aldrich, St Louis, MO), MTAP, and a control group (CG, empty polyethylene tubes). The composition and proportion of the materials evaluated are described in Table 1. After anesthesia with 80 mg/kg ketamine chloride 10% (Virbac do Brasil Ind�ustria e Com�ercio Ltda, S~ao Paulo, S~ao Paulo, Brazil) and 8 mg/kg xylazine chloride 2% (Uni~ao Qu�ımica Farm- acêutica Nacional S/A, S~ao Paulo, S~ao Paulo, Brazil), administered by the intraperitoneal route, the dorsal skin was shaved and disin- fected with 5% iodine solution. A 2-cm incision was made with a no. 15 scalpel blade (Fibra Cir�urgica, Joinville, Santa Catarina, Brazil). Afterward, polyethylene tubes (Embramed Ind�ustria e Com�ercio Ltda, S~ao Paulo, S~ao Paulo, Brazil), measuring 10 mm long � 1.5 mm in diameter, were filled with TSC or MTAP and immediately implanted into the subcutaneous tissue. The site of 460 Andrade et al. the incised skin was sutured with simple stitches using 4-0 silk thread (Ethicon Inc, S~ao Jos�e dos Campos, S~ao Paulo, Brazil). Two polythene tubes were used in each animal; 5 animals per group were used in each time point. After 7, 15, 30, and 60 days of implantation, the animals were euthanized with anesthetic overdose, and the implants with the sur- rounding tissues were removed. The specimens were immediately immersed in a formaldehyde 4% solution (prepared from paraformal- dehyde) buffered with sodium phosphate 0.1 mol/L (pH = 7.2) for 48 hours. After fixation, the specimens were dehydrated, treated with the xylene, and embedded in paraffin. Longitudinal sections (6-mm thick) were obtained and stained with hematoxylin-eosin for morpho- logic analysis and quantification of inflammatory cells (ICs) in the capsules. Other sections were adhered to slides previously treated with silane 4% (Sigma-Aldrich) to perform the immunohistochemical reaction for IL-6 detection. JOE — Volume 44, Number 3, March 2018 TABLE 2. Numeric Density of Inflammatory Cells (ICs) and Interleukin 6 (IL-6)-immunolabeled Cells in the Capsules of the Tricalcium Silicate Cement (TSC), MTA Plus (MTAP), and Control Groups at 7, 15, 30, and 60 Days TSC MTAP Control 7 days IC 482.5 (45.9)b,1 448.8 (34.8)b,1 324.0 (29.9)a,1 IL-6 475.5 (16.4)a,1 480.0 (14.4)a,1 464.4 (19.8)a,1 15 days IC 185.1 (28.2)ab,2 214.8 (43.6)b,2 154.0 (8.1)a,2 IL-6 185.1 (28.2)ab,2 195.5 (12.6)a,2 182.2 (12.6)a,2 30 days IC 160.0 (30.3)a,2 135.5 (29.0)a,3 148.8 (8.0)a,2 IL-6 148.8 (18.5)a,3 151.1 (14.9)a,3 144.4 (17.5)a,3 60 days IC 71.1 (9.5)a,3 70.3 (16.1)a,4 69.0 (6.5)a,3 IL-6 75.9 (8.9)a,4 75.5 (9.3)a,4 71.1 (9.9)a,4 Values are mean (standard deviation). The comparison among periods (P < .05) is indicated by different superscript numbers in the various columns. The comparison among groups (P < .05) is indicated by different superscript letters (a, b, c, and d) in the various lines. Basic Research—Biology Numeric Density of ICs The images were obtained with a digital camera (DP-71; Olympus, Tokyo, Japan) attached to a light microscope (BX-51, Olympus). The number of ICs was quantified using an image analysis system (Image Pro-Express 6.0, Olympus), as previously described (12). Quantitative analysis was performed in all samples or implants; in each implant, 3 hematoxylin-eosin–stained sections of the capsule were used at inter- vals of at least 100 mm. In each section, a standardized field of 0.09 mm2 of the capsule in close juxtaposition to the opening of the im- planted tube was captured, totaling 0.27mm2 per implant. In each field, the number of ICs (neutrophils, lymphocytes, plasma cells, and macro- phages) was computed. In this way, in each implant, the number of IC/mm2 was obtained. The numeric density of ICs was measured by 2 calibrated and blinded examiners. Immunohistochemical Reaction for the Detection of IL-6 For IL-6 detection, the primary goat antibody anti–IL-6 (Santa Cruz Biotechnology Inc, Santa Cruz, CA) was used. Deparaffinized sec- tions were immersed in 0.001 mol/L sodium citrate buffer (pH = 6.0) and submitted to microwave oven cycles for 20 minutes at 90�C–94�C for antigen retrieval. After cooling, the slides were washed in 0.05 mol/L Tris-HCl buffer (pH = 7.4) and subsequently immersed in 5% hydrogen peroxide for 20 minutes for blocking endogenous peroxidase. After washing, the sections were incubated with 2% bovine serum albumin (Sigma-Aldrich) for 20 minutes. Sections were incubated overnight in a humid chamber at 4�C with anti–IL-6 antibody diluted 1:100. Subsequent to washing in Tris-HCl buffer, the sections were incu- bated for 40 minutes at room temperature with a multilink solution con- taining biotinylated rabbit/mouse/goat antibodies (LSAB; Dako Inc, Carpinteria, CA). After washing in Tris-HCl buffer, the sections were incu- bated with horseradish peroxidase complex (System-HRP, Dako Inc) for 40minutes at room temperature. Subsequent to washing in buffer, perox- idase activity was revealed by chromogen 3.3-diaminobenzidine-HCl (DAB, Dako Inc) for 3 minutes. The sections were counterstained with Carazzi’s hematoxylin. As a negative control, some sections were submit- ted to all the stages, except incubation with primary antibody; in this step, the sections were incubated in nonimmune serum. Numeric Density of Cells Positive for IL-6 With the purpose of verifying whether there were differences among the groups, the number of IL-6–immunolabeled cells was JOE — Volume 44, Number 3, March 2018 estimated in the capsules of the 5 implants of each group/time point. As previously described, Image Pro-Express 6.0 Olympus software was used. The numeric density of IL-6–immunolabeled cells was obtained by 2 calibrated and blinded examiners. For each animal, the number of immunolabeled cells was quantified at �695 in a stan- dardized field (0.09 mm2) of the capsule in close juxtaposition to the opening of the implanted tube. Thus, the number of IL-6–immunola- beled cells/mm2 per animal was obtained (8, 12). Statistical Analysis The quantitative data were submitted to statistical analysis using the Sigma Stat 2.0 program (Jandel Scientific, Sausalito, CA). After normality was confirmed, 2-way analysis of variance and the Tukey test were used at a level of significance of 5%. Results Morphologic and Numeric Density Analysis of ICs At 7 days, the capsules in all the groups exhibited several ICs, mainly lymphocytes and macrophages among the blood vessels (Fig. 1A–C). At 15 days, the capsules contained fibroblasts and collagen fibers, particularly in the TSC group and the CG. However, ICs and blood vessels were still clearly observed in the capsules (Fig. 1D–F). Occasionally, some multinucleated giant cells were also observed, mainly in the internal portion of the capsules, adjacent to the materials (Fig. 1D). After 30 and 60 days, the connective tissue of the capsules contained several fibroblasts between the collagen fiber bundles; the scarce ICs were represented by plasma cells, macrophages, and mast cells (Fig. 2A–F). Quantitative analysis revealed that in all the groups the number of ICs was significantly higher at 7 days. After this period, the statistical analysis of each group revealed a gradual and significant reduction in the number of ICs in the capsules of all groups (P < .001) over time. At 7 days, no significant differences were verified between TSC and MTAP (P = .592); however, these values were statistically higher in comparison with those of the CG (P < .001). In turn, in the periods of 30 and 60 days, no significant differences were observed among TSC, MTAP, and the CG (P = .955) (Table 2). Immunohistochemical Detection of IL-6 and Numeric Density of Immunopositive Cells The sections of capsules submitted to the immunohistochemical reaction for IL-6 showed ICs (lymphocytes, plasma cells, macrophages, and mast cells) and some fibroblasts exhibiting positivity to immunore- action. Morphologic analysis revealed immunolabeled cells in the cap- sules of all the groups; however, an accentuated immunopositivity was observed in the capsules at 7 days in comparison with the other periods (Fig. 3A–L). On the other hand, sections of capsules incubated without the primary antibody (negative control) exhibited no immunopositive cells (data not shown). According to Table 2, the number of IL-6–immunolabeled cells was significantly higher at 7 days than at 15, 30, and 60 days. In all the groups, the number of immunolabeled cells was significantly reduced from 7 to 60 days. However, no statistically significant differ- ences were detected among TSC,MTAP, and the CG in any of the periods. Discussion Histopathological analysis of the subcutaneous response after different experimental periods evaluates not only the tissue character- istics to irritant potential but also the duration of this effect on tissues (5, 20, 21). The present study showed that TSC and MTAP induced Tissue Response and Immunoexpression of IL-6 461 Figure 3. Light micrographs of sections showing portions of capsules adjacent to the opening of the tubes (T) implanted in the subcutaneous tissue after (A–C) 7, (D–F) 15, (G–I) 30, and (J–L) 60 days of implantation. Sections were submitted to immunohistochemistry reaction for IL-6 detection and counterstained with hematoxylin-eosin. Several immunolabeled cells (arrows, brown/yellow color), especially plasma cells, mast cells, and some fibroblasts (Fb), are observed in the capsules at 7 and 15 days. At 60 days, scarce cells exhibit immunolabeling for IL-6. Bv, blood vessels; Mp, macrophage. 500�. Basic Research—Biology an inflammatory reaction in the subcutaneous tissues. However, this reaction was gradually reduced, leading to the formation of dense connective tissue in the capsules. The reduction in the inflammatory reaction occurred concomitantly with the reduction in the immunoexpression of IL-6 in the capsules adjacent to the materials, indicating that the experimental TSC and MTAP are biocompatible ma- terials. The inflammatory reaction initially caused by TSC andMTAPmay be related to the hydroxyls (OH�) released after hydration of the ma- terials, providing an environment with an alkaline pH (3, 14, 18). The alkaline pH stimulates recruitment of the leukocytes of the blood vessels (8, 12, 22, 23) and, therefore, may explain the higher values of ICs verified in the initial time point. The tissue reaction observed is a consequence of the host response to substances released by the materials. Therefore, the host 462 Andrade et al. cells (inflammatory and resident) produce and release several cyto- kines and growth factors that play a specific role in the complex cascade of cellular events involved in the tissue response. Among the different cytokines, IL-6 is considered a powerful mediator of the inflammatory process (24). An association has been observed between the reduction in immu- noexpression of IL-6 and a reduction in the inflammatory process in the capsules adjacent to the calcium silicate–based cements implanted in the rat subcutaneous tissues (8, 12), indicating that these materials do not have an irritating effect on connective tissue during a prolonged period. The gradual and significant reduction in the number of immu- nolabeled cells for IL-6 suggests that the capsules adjacent to the ma- terials implanted undergo an intense remodeling process from 7 to JOE — Volume 44, Number 3, March 2018 Basic Research—Biology 60 days. Moreover, our results point out that this cytokine may be involved in the response promoted by tricalcium silicate–based ma- terials because a reduction in both immunolabeled cells for IL-6 and in the number of ICs was observed. Considering that no significant differences in the IL-6 immunoexpression were observed for TSC and MTAP compared with the CG, it is possible to suggest a low irri- tant potential of the materials to the tissues. Despite this, the mate- rials promoted a greater recruitment of ICs than the empty tube. Therefore, these materials may release some substances that stimu- late the migration and differentiation of cells, such as macrophages and plasma cells, frequently observed in the capsules at 7 days. How- ever, the inflammatory process was replaced by a capsule containing well-developed bundles of collagen fibers between fibroblasts at 30 and 60 days after implantation. This tissue response indicates that the materials are biocompatible. It has been shown that ZrO2 added to calcium silicate cement promotes adequate physicochemical properties and a better biologic response than Bi2O3 (12). Nevertheless, in the present study, MTAP, which presents Bi2O3, promoted a subcutaneous tissue response similar to that of TSC. The hypothesis for this finding may be related to the fact that MTAP does not have Portland cement in its compo- sition, but instead it has tricalcium silicate (approximately 50%), di- calcium silicate, calcium sulfate, and silicon dioxide in addition to Bi2O3 (approximately 30%). The substitution of Portland cement by tricalcium silicate and dicalcium silicate allows better control of heavy metals that are found in MTA (18), favoring the hydration reaction (3). Conclusion TSC and MTAP caused a similar subcutaneous implantation reac- tion in rats, indicating that both present a similar immune response. The significant reduction in the inflammatory reaction accompanied by the decrease in IL-6 immunoexpression in the capsules over time indicates that these tricalcium silicate–based materials may have potential as root repair material. Acknowledgments The authors thankMr Pedro S�ergio Sim~oes andMr Luis Antonio Potenza for their kind help and technical assistance. Supported by the S~ao Paulo Research Foundation (FAPESP nos. 2015/03437-6 and 2014/13353-1). The authors deny any conflicts of interest related to this study. References 1. Camilleri J, Montesin FE, Brady K, et al. The constitution of mineral trioxide aggregate. Dent Mater 2005;21:297–303. JOE — Volume 44, Number 3, March 2018 2. Camilleri J, Sorrentino F, Damidot D. Investigation of the hydration and bioactivity of radiopacified tricalcium silicate cement, Biodentine and MTA Angelus. Dent Mater 2013;29:580–93. 3. Grech L, Mallia B, Camilleri J. 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Material and Methods Numeric Density of ICs Immunohistochemical Reaction for the Detection of IL-6 Numeric Density of Cells Positive for IL-6 Statistical Analysis Results Morphologic and Numeric Density Analysis of ICs Immunohistochemical Detection of IL-6 and Numeric Density of Immunopositive Cells Discussion Conclusion Acknowledgments References