In Vivo Host Interactions with Mineral Trioxide Aggregate and Calcium Hydroxide: Inflammatory Molecular Signaling Assessment

In Vivo Host Interactions with Mineral Trioxide Aggregate and Calcium Hydroxide: Inflammatory Molecular Signaling Assessment

Basic Research—Biology In Vivo Host Interactions with Mineral Trioxide Aggregate and Calcium Hydroxide: Inflammatory Molecular Signaling Assessment J...

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Basic Research—Biology

In Vivo Host Interactions with Mineral Trioxide Aggregate and Calcium Hydroxide: Inflammatory Molecular Signaling Assessment Jessie F. Reyes-Carmona, DDS, MS, PhD,*† Adair R.S. Santos, MS, PhD,‡ Claudia P. Figueiredo, MS, PhD,‡ Mara S. Felippe, DDS, MS, PhD,* Wilson T. Felippe, DDS, MS, PhD,* and Mabel M. Cordeiro, DDS, PhD§ Abstract Introduction: Mineral trioxide aggregate (MTA) and calcium hydroxide [Ca(OH)2] are promising biomaterials for stimulating dentinogenesis and cementogenesis. This research was undertaken to understand how MTA and CA(OH)2 participate in the inflammatory, healing, and biomineralization processes. In this part of the study, we evaluated inflammatory signaling molecules promoted by in vivo host interaction with MTA and Ca(OH)2. Methods: Human dentin tubes were filled with ProRoot MTA (Dentsply Tulsa Dental, Tulsa, OK), Ca(OH)2, or kept empty. After 12 hours and 1, 3, 7, 15, 30, and 60 days of implantation in subcutaneous tissues in the backs of mice, the tubes and surrounding tissues were retrieved for cytokine level quantification and histological and immunohistochemical analysis. Results: MTA and Ca(OH)2 induced proinflammatory cytokine up-regulation for up to 3 days. Moreover, interleukin-10 overexpression was noted on the tissue in contact with the biomaterials during the acute phase of the inflammatory reaction. Immunohistochemical analyses showed an increased expression of myeloperoxidase, nuclear factor kappa B (NF-kB), cyclooxygenase-2, inducible nitric oxide synthase enzymes, and vascular endothelial growth factor on day 1 for all groups. Conclusions: MTA and Ca(OH)2 increased the activation of the NF-kB signaling system on day 1 for all groups. This finding can be associated with a proinflammatory and pro–wound healing environment, which was promoted earlier by MTA. (J Endod 2011;37:1225–1235)

Key Words Bioactivity, calcium hydroxide, cyclooxygenase-2, inducible nitric oxide synthase enzymes, inflammation,

mineral trioxide aggregate, nuclear factor kappa B, vascular endothelial growth factor, wound healing

M

ineral trioxide aggregate (MTA) and calcium hydroxide (Ca[OH]2) have been shown to be capable of inducing mineralized tissue formation at a variety of dental tissue sites and, subsequently, their potential applications within dentistry have expanded (1, 2). Studies have suggested that the similar clinical response of tooth tissues to both biomaterials is based on a comparable mechanism of action involving the release of calcium and hydroxyl ions (2–11). However, in vivo studies showed the benefits of MTA over Ca(OH)2, including thicker dentine bridge formation, decreased incidence of inflammation, and necrosis of pulp tissue (1, 5–8). The biological basis for the clinical benefits of MTA is yet to be completely elucidated although recent studies suggested that this bioactive cement may modulate tissue repair and hard-tissue deposition because of its biomineralization ability (12–14). The interaction of MTA with a phosphate-containing fluid produces calcium-deficient B-type carbonated apatites via an amorphous calcium phosphate phase (13, 14). The apatite formed by the MTA–phosphate-buffered saline (PBS) system is deposited among collagen fibrils, triggering the formation of an interfacial layer with intratubular mineralization at the MTA-dentin interface (13, 14). Furthermore, in a previous in vivo study, we showed that MTA induced a biomineralization process that occurred simultaneously with the acute inflammatory response (Reyes-Carmona et al, October 2009, unpublished data). Thus, we suggested that the precipitation of apatite by MTA during the acute phase of inflammation, together with its alkalinity, may induce changes in gene expression and signal several unrecognized pathways in different cell types, which are likely to contribute to repair and biomineralization. The mechanisms underlying host responses to biomaterials are dependent on their ability to induce the formation of apatite and on the innate and nonspecific immune response that occurs in the surrounding tissue (15, 16). However, biomaterials might evoke several signaling pathways to trigger the inflammatory cascade (16). Proinflammatory cytokines, such as interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), and prostaglandins (PGs), are known messenger molecules involved in the initiation of the inflammatory process and thus may contribute to

From the *Postgraduate Dentistry Program of the Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil; †Department of Restorative Sciences, University of Costa Rica, San Jose, Costa Rica; ‡Department of Physiological Sciences, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil; and § Department of Morphological Sciences, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil. Dr Reyes-Carmona is a fellow of University of Costa Rica. Supported in part by Grants in Aid for Scientific Research from the University of Costa Rica and Federal University of Santa Catarina. Address requests for reprints to Dr Jessie F. Reyes-Carmona, Department of Restorative Sciences, University of Costa Rica, San Jose, Costa Rica. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2011.05.031

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Basic Research—Biology a sequence of healing events (17). Moreover, myeloperoxidase (MPO), a polymorphonuclear leukocyte indicator, has been widely used as an inflammatory marker of both acute and chronic conditions (17). Cytokine secretion has also been associated with activation of nuclear factor kappa B (NF-kB) (15). NF-kB is an inducible transcription factor that consists of heterodimers of RelA (p65), c-Rel, RelB, p50, and p52 and acts as a central coordinator of innate and adaptive immune responses. While being activated, NF-kB undergoes phosphorylation and translocates into the nucleus, where it binds to a unique decameric DNA nucleotide sequence, which promotes gene transcription (18). A cytokine-mediated activation of NF-kB in macrophages induced an upregulation in the inducible nitric oxide synthase enzymes (iNOS) at the early stages of the inflammatory cascade (19–21). Experimental data suggest that NF-kB activation results in the induction of gene expression of iNOS and cyclooxygenase-2 (COX-2) (21, 22). COX-2 was originally identified as an inducible enzyme expressed at the site of inflammation (13). However, recent evidence shows that COX-2 promotes the expression of vascular endothelial growth factor (VEGF) and subsequent angiogenesis (23–25). Although the overall results of clinical and histological studies involving MTA and Ca(OH)2 materials are very positive, further investigation is needed to provide detailed information regarding cellular and molecular events involved in the inflammatory reaction and its correlation with the repair process and hard tissue formation. A more complete understanding of the mechanisms of action of MTA and Ca(OH)2 is critical to understanding how oral and dental tissues may be repaired or regenerated during preventive and restorative procedures (2). This study was undertaken to understand how MTA and Ca(OH)2 actually participate in inflammatory, healing, and biomineralization processes. In this part of the study, we evaluated some specific signaling molecules related to the inflammatory process promoted by in vivo host interaction with MTA and Ca(OH)2.

Materials and Methods Animals All animal care and experimental protocols used in this study were approved by the Animal Ethics Screening Committee of the Federal University of Santa Catarina (Florianopolis, Santa Catarina, Brazil). The experiments were conducted using 55 male Swiss mice aged 5 to 7 weeks old (35–40 g) housed in polycarbonate cages placed in a ventilated, temperature-controlled room. Animals were kept in a 12-hour light/dark cycle, with controlled humidity (60%  5%) and temperature (25 C  1 C). The commercial pellet diet and distilled water were available ad libitum. Experiments were performed during the light phase of the cycle. The animals were acclimatized to this environment for 5 days before testing. Preparation of Specimens The research protocol was approved by the Ethics Committee for Research with Human Beings of the Federal University of Santa Catarina. In total, 165 dentin tubes were prepared from extracted human tooth roots. The crowns and the apical third of the roots were removed using a water-cooled low-speed ISOMET diamond saw (Buehler, Lake Bluff, NY). In each root, the space of the canal was enlarged with a #5 Gates-Glidden bur to obtain 1.3-mm-diameter standardized cavities. The length of the tubes was 5 mm, and their outer walls were abraded with a diamond bur to thin the walls to 2-mm thickness. The dentin tubes were washed in distilled water and then autoclaved. Before the implantation, the tubes were thoroughly irrigated with 17% EDTA and 1% sodium hypochlorite, dried, and then filled with tooth-colored 1226

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ProRoot MTA (Dentsply Tulsa Dental) or CA(OH)2 (Merck, Frankfurt, Germany) or kept empty to be used as controls.

Experimental Protocol The animals were divided into seven groups, with n = 10 for the 12 hours and 1, 3, and 7-day experimental periods and n = 5 for the 15-, 30-, and 60-day time points. Mice were anesthetized with 80 mg/kg of ketamine hydrochloride (Dopalen; Division Vetbrands Animal Health, Jacareı, SP, Brazil) and 10 mg/kg of xylazine (Anasedan; Agribrands do Brasil Ltda, Paulınia, SP, Brazil). Then, four separate 1-cm incisions were made in the backs of mice at 1-cm intervals. The skin was deflected to create four subcutaneous pockets by a blunt dissection on one side of each incision, two in the cranial portion and two in the caudal portion. Each mouse received three dentin tubes, two filled with each material and one empty, whereas no specimen was inserted in the fourth pocket (sham). After 12 hours and 1, 3, 7, 15, 30, and 60 days after implantation, the animals were euthanized, the tubes with surrounding tissues were removed, and the surrounding tissues were collected. Half the samples (n = 5) from the 12-hour to the 7-day time points were fixed in 4% paraformaldehyde, at 4 C, for histological and immunohistochemical staining. To determine the protein level expression of IL-1b, TNF-a, and IL-10 in the remaining half of the samples (n = 5), the dentin tubes were retrieved for scanning electron microscopy analysis (presented in part 2 of this study), and the surrounding tissues were excised using a rounding cutting tool to obtain the same amount of tissue from all of the groups; this tissue was processed for tissue homogenate. The sham group was identified following the four-quadrant design of the 1-cm incisions made in the backs of the mice at 1-cm intervals from each other. Histological Analysis All tissues were processed using conventional histochemical techniques, embedded in paraffin, sectioned at 3-mm thickness, mounted on glass slides, and deparaffinized. For general histology analysis, slices were stained with hematoxylin and eosin by standard techniques. The connective tissues in contact with the material on the tube openings or on an empty opening were photographed (5), and then 4 consecutive images per sample at 40 magnification were captured. Reactions in the tissues in contact with the material on the tube openings were analyzed by a previously calibrated examiner for the presence and localization of inflammatory cells and angiogenic sprouting. Immunohistochemical Analysis Tissue sections were deparaffinized, and immunohistochemistry analysis was performed using the following antimouse primary antibodies and respective dilutions: rabbit polyclonal antimyeloperoxidase (MPO, 1:300; Dako Cytomation, Carpinteria, CA), mouse monoclonal anti-VEGF (1:200; C-1, Santa Cruz Biotechnology Inc, Santa Cruz, CA); rabbit polyclonal anti–COX-2 (1:200); rabbit polyclonal anti-phospho-p65 NF-kB (1:50); and rabbit polyclonal anti-iNOS (1:100, all from Cell Signaling Technology, Beverly, MA). High-temperature antigen retrieval was applied by immersion of the slides in a water bath at 95 C to 98 C in 10 mmol/L trisodium citrate buffer at a pH of 6.0 for 45 minutes. The nonspecific binding was blocked by incubating sections for 1 hour with goat normal serum diluted in PBS. After overnight incubation at 4 C with primary antibodies, the slides were washed with PBS and incubated with the conjugated secondary antibody Envision plus, JOE — Volume 37, Number 9, September 2011

Basic Research—Biology ready-to-use (EnVision Doublestain System, Dako Cytomation) for 1 hour at room temperature. The sections were washed in PBS, and the visualization was completed using 3,30 - diaminobenzidine (Dako Cytomation) and counterstained lightly with Harris hematoxylin solution. Control and experimental tissues were placed on the same glass slide and processed under the same conditions. A negative control for each reaction was performed by abolishing the primary antibody. One image of hematoxylin and eosin and of each immunohistochemical stained tissue sections per sample and time point for all mice were acquired using a digital camera (A620; Canon, Lake Success, NY) connected to a light microscope (Axiostar Plus; Carl Zeiss, Oberkochen, Germany). Settings for image acquisition were identical for control and experimental tissues. In total, four consecutive images per sample of the tissue in contact with the material on the tube openings or with an empty opening were captured at 40 magnification. The threshold optical density was obtained using the NIH ImageJ 1.36b imaging software (National Institutes of Health, Bethesda, MD). The total pixel intensity was determined, and data were expressed as optical density.

Determination of Cytokine Levels In brief, full-thickness tissue samples were homogenized in phosphate buffer containing 0.05% Tween 20 (Merck AG, Darmstadt, Germany), 0.1 mmo/L phenylmethylsulfonyl fluoride, 0.1 mmol/L benzethonium Ca chloride, 10 mmol/L EDTA, and 20 KIU aprotinin A. The homogenate was centrifuged at 3,000 rpm for 10 minutes, and the supernatants were stored at 80 C until further analysis. Before specific cytokine analyses, the total amount of proteins was determined using the Protein Assay–Bradford Method Biotechnology Grade kit (E535; Amresco Inc, Cleveland, OH). IL-1b, TNF-a, and IL-10 levels were evaluated using DuoSet enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The results were expressed as a pg/mg tissue protein concentration. Statistical Analysis For statistical analysis, all data were expressed as mean  standard error of the mean. For parametric data, statistical significance of differences between the groups was determined by two-way analysis of variance followed by the Bonferroni post-test. Statistical analyses were performed using GraphPad Prism 4 software (GraphPad Software Inc, San Diego, CA). A P value of less than .05 was considered to be statistically significant.

Results Inflammatory Cytokine Expression Profile The expression levels of cytokine genes are shown in Figure 1. In all experimental groups, the total amount of cytokine expression decreased after day 3. Although MTA and Ca(OH)2 induced an overall proinflammatory cytokine up-regulation during the first 3 days, there was no apparent material-dependent effect on the classes of cytokines produced, whereas a time-dependent manner was shown. TNF-a and IL-1b expression peaked at 12 and 24 hours, respectively, in all experimental groups. IL-1b and TNF-a expression, at 12 hours and at days 1 and 3, was significantly up-regulated in the MTA and Ca(OH)2 groups when compared with empty tubes and sham (P < .05). By day 7, no differences were found. Our findings reveal that the inflammatory response induced by MTA and Ca(OH)2 peaks during the acute response and its resolution can be seen starting on day 7. The cytokine measurement JOE — Volume 37, Number 9, September 2011

Figure 1. Cytokine levels (pg/mg protein) of (A) TNF-a, (B) IL-1b, and (C) IL-10 on tissue homogenates. Each column represents the mean  standard error of the mean. *P < .05 versus the empty tube group. #P < .05 versus sham. **P < .05 versus CA(OH)2.

assay showed that MTA stimulated the up-regulation of TNF-a expression at 12 hours when compared with Ca(OH)2 (P < .05). However, at the other experimental times, no statistical difference was found. However, IL-10 expression was up-regulated on days 1 and 3 and peaked on day 1 in all experimental groups (Fig. 1C). MTA and Ca(OH)2 displayed a significant up-regulation of IL-10 expression when compared with the empty tube and sham conditions at 12 hours and on days 1 (P < .05) and 3 (P < .01). On day 7, no significant statistical difference was observed between the experimental groups.

Inflammatory Response Assessment Histomorphological Findings. At 12 hours, the tissue surrounding all experimental groups contained primarily neutrophils (Fig. 2). A mild acute inflammatory reaction, consisting of neutrophils, lymphocytes, macrophages, and few young fibroblasts, was observed in the sham condition. The transition from an acute phase to a moderate chronic response was denoted by a decrease in neutrophil recruitment between days 1 and 3 in all experimental groups. Host-MTA and Ca(OH)2 Inflammatory Molecular Interactions

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Figure 2. Representative photomicrographs of hematoxylin and eosin staining of different groups and time periods (40).

On day 3, a mild inflammatory cell infiltration consisting mainly of lymphocytes and macrophages was present in a fibrous capsule surrounding the opening of the tubes containing the biomaterials as well as the empty tube. Fibroblasts were the predominant cell population in the sham condition, with few macrophages and lymphocytes found within the tissue. Moreover, edema was decreased. On days 7 and 15, a chronic inflammatory cell infiltration, consisting primarily of macrophages, fibroblasts, lymphocytes, and few giant cells, was present in a thinner fibrous capsule (Fig. 2). The intensity of inflammation was reduced at days 30 and 60, when the inflammatory cell population was diminished and the fibrous capsule near the tube was thinner (Fig. 2). Despite the similarity in the responses of MTA and Ca(OH)2, the greatest areas of necrosis by coagulation occurred in tissues surrounding Ca(OH)2. Immunohistochemical Analyses. The immunoreactivity analyses for NF-kB, iNOS, COX-2, VEGF, and MPO are shown in Figures 3 through 7. The different proteins expressed in a time-dependent manner. 1228

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MPO expression reached its peak at 24 hours in all experimental groups (Fig. 3). The immunoreactivity analysis for MPO showed a significant increase in protein expression between 12 hours and day 1 in tissues in contact with MTA as well as in the empty tube and sham (P < .001). At 12 hours and day 1, in the tissues in contact with Ca(OH)2, the expression of MPO was higher than that with the empty tube (P < .001). At 12 hours and days 1 and 3, the immunohistochemical analysis showed a significant increase in the expression of MPO in the tissue in contact with Ca(OH)2 when compared with the sham condition (P < .001). To further define some signaling pathways activated by inflammatory stimuli, we studied the transcriptional factor NF-kB, which is able to modulate the expression of COX-2. All experimental groups induced NFkB phosphorylation and their subsequent translocation to the nucleus of inflammatory cells (Fig. 4). This transcriptional factor presented a peak of phosphorylation at 12 hours and, as expected, MTA and Ca(OH)2 significantly up-regulated NF-kB expression when compared with empty tubes and sham (P < .01). JOE — Volume 37, Number 9, September 2011

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Figure 3. (A) Representative images for MPO immunohistochemical analysis. (B) Negative control of the immunohistochemical reaction. (C) Staining intensity and stained area of MPO immunoreaction are expressed as optical density. Each column represents the mean  standard error of the mean. *P < .05 versus the empty tube group. #P < .05 versus sham. **P < .05 versus CA(OH)2.

COX-2 and iNOS expression reached their peak at 12 and 24 hours, respectively, in all groups (Figs. 5 and 6). No significant difference was found between MTA and Ca(OH)2 in the expression of these proteins (P > .05); however, up-regulated expression was shown when compared with the empty tube and sham (P < .01). MTA produced a significant increase in the expression of COX-2 when compared with Ca(OH)2 (P < .01). VEGF expression was up-regulated at all time periods (Fig. 7). At the acute phase of inflammation, VEGF was mainly expressed in the presence of inflammatory cells, such as neutrophils and macrophages. Therefore, on day 7, VEGF expression by fibroblasts was also shown.

Discussion MTA and Ca(OH)2 have been used extensively as promising biomaterials for stimulating dentinogenesis and cementogenesis. However, there is little consensus on whether these biomaterials induce inflammation, wound healing and hard-tissue deposition. To date, few JOE — Volume 37, Number 9, September 2011

studies analyzing specific inflammatory signaling molecules have been performed in vivo. Thus, our study focused on a possible signaling pathway that can trigger healing and repair events. Ideally, the cellular and molecular reactions of dental materials would be evaluated in human dental tissues. However, bioethical concerns and the technical demands of applying these materials in animal teeth, as well as the lack of an ideal animal model, have prompted researchers to look for alternative models. To date, several studies have used mouse skin models to assess inflammatory reactions (25) and even tissue regeneration and engineering (25). The development and characterization of preclinical experimental models are important before clinical applications and to aid in the understanding of tissue reactions. The inflammatory response induced during host-biomaterial interaction is traditionally assessed by histological or immunohistochemical analysis (19). Until recently, in vivo studies investigating the link between cell interrogation of materials and intercellular signaling relied on polymerase chain reaction (26–30), protein array, or ELISA (17, 19, 26–29). In this study, we used ELISA to

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Figure 4. (A) Representative images for NF-kB immunohistochemical analysis. (B) Negative control of the immunohistochemical reaction. (C) Staining intensity and stained area of NF-kB immunoreaction are expressed as optical density. Each column represents the mean  standard error of the mean. *P < .05 versus the empty tube group. #P < .05 versus sham. **P < .05 versus CA(OH)2.

assay the expression levels of the cytokines IL-1b, TNF-a, and IL-10 in the implanted region. The types and levels of cytokines surrounding a biomaterial may initially drive acute and chronic inflammatory reactions and later initiate the wound-healing response while inflammation resolves (30). Our results showed that MTA and Ca(OH)2 induced a time-dependent proinflammatory cytokine up-regulation during the acute phase of the inflammatory response. In the MTA and Ca(OH)2 groups, IL-1b and TNF-a 1230

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expression, at 12 hours and on days 1 and 3, were significantly upregulated compared with empty tubes and sham conditions. By day 7, no significant differences were found. These data suggest that the presence of these biomaterials in the dentin tubes had an impact on the intercellular signaling that occurs at the implantation site. Nonetheless, an overexpression of proinflammatory cytokines occurred during the acute phase. IL-10 overexpression, mainly in tissues surrounding dentin tubes filled with MTA and Ca(OH)2, occurred at JOE — Volume 37, Number 9, September 2011

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Figure 5. (A) Representative images for COX-2 immunohistochemical analysis. (B) Negative control of the immunohistochemical reaction. (C) Staining intensity and stained area of COX-2 immunoreaction are expressed as optical density. Each column represents the mean  standard error of the mean. *P < .05 versus the empty tube group. #P < .05 versus sham. **P < .05 versus CA(OH)2.

the same time as this acute phase. The high concentration of IL-10 after implantation may have signaled the decrease in proinflammatory cytokine production observed at days 3 and 7 because IL-10 has strong anti-inflammatory properties. IL-10 overexpression in tissue surrounding MTA and Ca(OH)2 during the acute phase of inflammation supports the idea that these biomaterials promote an antiinflammatory effect (27, 31). Neutrophils have a short lifespan, in the order of hours to days, and disappear from the injured tissue more rapidly than do macroJOE — Volume 37, Number 9, September 2011

phages, which have a lifetime of days to weeks or months (16). Eventually, macrophages become the predominant cell type, resulting in a chronic inflammatory response (16, 32). MPO analyses showed that neutrophils were the predominant cells at the implantation sites during the first 24 hours in all experimental groups. Between days 1 and 3, mostly macrophages and lymphocytes migrated into the tissue. On days 7 and 15, a mild chronic inflammatory cell infiltration was present in a thicker fibrous capsule. At days 30 and 60, inflammatory cell numbers were diminished, and the fibrous capsule near the tube

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Figure 6. (A) Representative images for iNOS immunohistochemical analysis. (B) Negative control of the immunohistochemical reaction. (C) Staining intensity and stained area of iNOS immunoreaction are expressed as optical density. Each column represents the mean  standard error of the mean. *P < .05 versus the empty tube group. #P < .05 versus sham.

was thinner. These data show the expected transition from an acute proinflammatory phase to an anti-inflammatory and pro–wound-healing chronic environment. Macrophages may be the most important cell in chronic inflammation because of the large number of biologically active compounds they produce, including neutral proteases, chemotactic factors, arachidonic acid metabolites, reactive oxygen metabolites, complement components, coagulation factors, growth-promoting factors, and cytokines 1232

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(16). It is widely accepted that macrophages play a critical role in mediating the host response to biomaterials, and that is why perhaps in our study, macrophage recruitment was more prominent on day 1 than on day 3. This finding suggests that macrophages could be the cells responsible for inducing the host inflammatory response through the release of inflammatory molecules such as IL-1 and VEGF. Our previous in vivo study showed that MTA induced the activation of NF-kB at the early stage of inflammation (33). As expected, JOE — Volume 37, Number 9, September 2011

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Figure 7. (A) Representative images for VEGF immunohistochemical analysis. (B) Negative control of the immunohistochemical reaction. (C) Staining intensity and stained area of VEGF immunoreaction are expressed as optical density. Each column represents the mean  standard error of the mean. *P < .05 versus the empty tube group. #P < .05 versus sham.

the current study showed that NF-kB is involved in Ca(OH)2-stimulated signal transduction. NF-kB has been found to induce activation in many cell types in response to a broad range of stimuli, which typically include proinflammatory cytokine secretion (22). It is widely known that the activation of NF-kB is implicated in the induction of gene expression of COX-2 and iNOS (22, 34). COX-2 has been known as the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandin in the acute and chronic inflammatory states (18). iNOS synthesizes nitric oxide, which induces dilatation of blood vessels JOE — Volume 37, Number 9, September 2011

and enhances COX-2 catalytic activity (22). As shown, the expression of NF-kB peaked on day 1. Moreover, COX-2 and iNOS expression also was increased on day 1; although levels began to decrease on day 3, the expression of both genes remained detectable until day 7. Our findings are in agreement with those of Minamikawa et al (22), who suggested that MTA induces phosphorylation of NF-kB inhibitor alpha (IkBa), which frees NF-kB complexes. Activated NF-kB complexes translocate to the nucleus and stimulate the expression of COX-2. Released prostaglandin E2 (PGE2) induces transcription of iNOS in an autocrine Host-MTA and Ca(OH)2 Inflammatory Molecular Interactions

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Basic Research—Biology manner. As in our study, CA(OH)2 showed a similar expression; we suggest that both biomaterials have a similar mechanism of action. Evidence suggests that PGE2 enhances alkaline phosphatase activity and bone sialoprotein activation; both genes are involved in mineralization (22). Thus, MTA and Ca(OH)2 stimulate the expression of COX-2, which produces PGE2 in an autocrine manner via NF-kB activation (22). Thus, it can be hypothesized that the induction of PGE2 by MTA and Ca(OH)2 might play an important role in hard tissue deposition. VEGF immunostaining revealed that the overexpression of this glycoprotein remained stable at the various time periods. At the acute phase, the up-regulation of VEGF was attributed to its ability to increase the permeability of blood vessels, an important vascular change observed during the early stages of inflammation (23). On day 7, VEGF expression mainly occurred in fibroblasts, suggesting the induction of angiogenesis and neovascularization (23). Host responses to biomaterials are complex processes involving several mediators. Therefore, we decided to analyze the integrated responses of the classes of genes examined in this study based on their respective roles. According to Brodbeck et al (30), the genes detected in this study may be classified as proinflammatory (IL-1b, VEGF, and TNF-a), anti-inflammatory (IL-10), pro–wound healing (IL-1b and VEGF), and anti–wound healing (TNF-a and IL-10). Based on this classification, we can draw out some conclusions from our findings. Our study showed that TNF-a expression peaked at 12 hours, and that on days 1 and 3, IL-1b expression was higher than that of TNF-a. Also, it was possible to note a decrease over time on IL-10 expression. As known, IL-1b has a proinflammatory/pro–wound-healing cytokine role, activating both inflammatory cells (lymphocytes and monocytes) and wound-healing cells (fibroblasts). However, IL-10 acts in the opposite fashion; it down-regulates the activity of these cell types and suppresses further cytokine production, leading to an anti-inflammatory/anti–wound-healing effect. Thus, TNF-a, a proinflammatory/ anti–wound-healing gene, may trigger the inflammatory cascade. The up-regulation of IL-1b up to day 3 activates inflammatory cells and fibroblasts and consequently stimulates repair, whereas IL-10 up-regulation at the acute phase promotes the resolution of the inflammatory process. Therefore, at later stages, IL-10 down-regulation is necessary to orchestrate wound healing and repair, as seen on day 7. MTA and Ca(OH)2 have been shown to be capable of creating a favorable environment for wound healing and repair. Our findings indicate that both biomaterials have a similar mechanism of action. However, it is pertinent to note that MTA triggers a proinflammatory and prorepair response much earlier than that observed in Ca(OH)2. A possible explanation is the presence of larger areas of coagulation necrosis in the tissues in contact with Ca(OH)2, leading to a decrease in cell viability and in local microvascularization; this is reflected in the immediate inflammatory response. In fact, previous data have shown that MTA provided a decreased inflammatory reaction and pulp necrosis when compared with Ca(OH)2 (5–8, 34). However, these findings may reflect the experimental period of time because these studies examined later stages, beyond the acute inflammatory response and during chronic inflammatory events. In summary, we showed that MTA and Ca(OH)2 increased the activation of the NF-kB signaling system on day 1 for all groups. This finding can be associated with a proinflammatory and pro–wound-healing environment, which was promoted earlier by MTA.

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

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31. Silva MJ, Vieira LQ, Sobrinho AR. The effects of mineral trioxide aggregate on cytokine production by mouse pulp tissue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:e70–6. 32. Williams DF. On the mechanisms of biocompatibility. Biomaterials 2008;29: 2941–53. 33. Reyes-Carmona JF, Santos AS, Figueiredo CP, Baggio CH, Felippe MC, Felippe WT, Cordeiro MM. Host–mineral trioxide aggregate inflammatory molecular signaling and biomineralization ability. J Endod 2010;36:1347–53. 34. Parirokh M, Torabinejad M. Mineral trioxide aggregate: a comprehensive literature review—part III: clinical applications, drawbacks, and mechanism of action. J Endod 2010;36:400–13.

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