Host–Mineral Trioxide Aggregate Inflammatory Molecular Signaling and Biomineralization Ability

Host–Mineral Trioxide Aggregate Inflammatory Molecular Signaling and Biomineralization Ability

Basic Research—Biology Host–Mineral Trioxide Aggregate Inflammatory Molecular Signaling and Biomineralization Ability Jessie F. Reyes-Carmona, DDS, M...

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Basic Research—Biology

Host–Mineral Trioxide Aggregate Inflammatory Molecular Signaling and Biomineralization Ability Jessie F. Reyes-Carmona, DDS, MS, PhD,*† Adair S. Santos, PhD,‡ Claudia P. Figueiredo, PhD,‡ Cristiane H. Baggio, PhD,‡ Mara C. S. Felippe, DDS, MS, PhD,* Wilson T. Felippe, DDS, MS, PhD,* and Mabel M. Cordeiro, DDS, PhD§ Abstract Introduction: The biological processes underlying the ability of mineral trioxide aggregate (MTA) to promote hard-tissue deposition and wound healing remain unclear. To further study these processes, specific signaling molecules related to the inflammatory response and the biomineralization process were analyzed to assess host-MTA interactions in vivo. Methods: For cytokine level quantification and immunohistochemical analysis, human dentin tubes were filled with ProRoot MTA (Dentsply, Tulsa Dental, OK) or kept empty and were implanted in subcutaneous tissues in the backs of mice. Dentin tubes were retrieved and subsequently observed using a scanning electron microscope. Results: MTA induced a time-dependent proinflammatory cytokine up-regulation up to 3 days. Immunohistochemical analyses showed an upregulated expression of myeloperoxidase, nuclear factor-kappa B, activating protein-1, cyclooxygenase-2, inducible nitric oxide synthase, and vascular endothelial growth factor on day 1. Scanning electron microscopic examination revealed the presence of apatite-like clusters on collagen fibrils over the surface of tubes containing MTA. With the increase in time after implantation, a more extensive mineralization showing a compact layer of apatite was observed. Conclusion: MTA induced a proinflammatory and pro–wound healing environment. The biomineralization process occurred simultaneously at the biomaterial-dentin-tissue interface, with the acute inflammatory response. This promoted the integration of the biomaterial into the environment. (J Endod 2010;36:1347–1353)

Key Words Apatite, bioactivity, biomineralization, inflammation, mineral trioxide aggregate, wound healing

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bioactive material should be capable of stimulating specific biological responses via biochemical and biophysical reactions that result in the formation of an apatite layer (1, 2). The ability to induce the formation of apatite allows the integration of the biomaterial into the environment (2). However, host responses to biomaterials are dependent on the innate and nonspecific immune responses that occur in the surrounding tissues (3). Biomaterials may elicit an inflammatory cascade comprising neutrophil and macrophage recruitment and adhesion, foreign body reaction, and fibrous encapsulation (4). Cytokines and growth factors secreted by inflammatory cells are the molecular messengers that promote inflammatory events and wound healing (5). Inflammatory cytokines such as, interleukin (IL)-1b, tumor necrosis factor a (TNF-a), and prostaglandins play an important role in the development of the inflammatory response. The expression of these proteins is controlled by some transcription factors, such as activating protein-1 (AP-1) and nuclear factor-kappa B (NF-kB) (6). NF-kB consists of a group of proteins, including p50, p65, and p105, which are sequestered in the cytoplasm in their resting state. When activated by agents such as cytokines, NF-kB undergoes phosphorylation, leading to its nuclear translocation and binding to specific sequences of DNA, which, in turn, results in gene transcription (7). At the onset of inflammation, a cytokine-mediated activation of NF-kB in macrophages results in nitric oxide production by the inducible nitric oxide synthase (iNOS) enzyme (8, 9). Experimental evidence suggests that a relationship exists between nitric oxide and prostaglandin E2 biosynthesis, the production of which is regulated by cyclooxygenase-2 (COX-2) (9, 10). Moreover, myeloperoxidase (MPO), a leukocytederived enzyme that catalyzes the formation of a number of reactive oxidant species, is linked to the acute phase of inflammation (11). Vascular endothelial growth factor (VEGF) is a glycoprotein with the ability to increase the permeability of blood vessels, an important vascular change observed during inflammation (12). Therefore, VEGF also plays a critical role in angiogenesis and neovascularization by participating in critical biological processes, such as tissue repair (13). Mineral trioxide aggregate (MTA) has been extensively used as a promising biomaterial for stimulating dentinogenesis and cementogenesis. Despite its widespread use in clinical practice, the mechanism by which it induces hard-tissue deposition remains unknown. Previous studies have shown that MTA releases calcium hydroxide, which interacts with a phosphate-containing fluid to produce calcium-deficient apatite via an amorphous calcium phosphate phase (14–16). A preliminary study provided compelling evidence of the biomineralization process promoted by the interaction of

From the *Postgraduate Dentistry Program of the Federal University of Santa Catarina, Floriano´polis, SC, Brazil; †Department of Restorative Sciences, University of Costa Rica, San Jose, Costa Rica; ‡Department of Physiological Sciences, Federal University of Santa Catarina, Floriano´polis, SC, Brazil; and §Department of Morphological Sciences, Federal University of Santa Catarina, Floriano´polis, SC, 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 Reyes-Carmona, Department of Endodontics, School of Dentistry, University of Costa Rica, San Jose´, Costa Rica. E-mail address: [email protected] 0099-2399/$0 - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.04.029

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Basic Research—Biology MTA and dentin (15). The apatite formed by the MTA–phosphatebuffered saline (PBS) system was deposited among collagen fibrils. This promoted controlled mineral nucleation on dentin, which triggered the formation of an interfacial layer with an intratubular mineralization at the biomaterial-dentin interface (15, 16). Although MTA has been studied in numerous clinical and histological studies, there is little consensus regarding the mechanism involved during the inflammatory reaction and its correlation with the repair process and hard tissue formation. Therefore, in this study, we evaluated specific signaling molecules related to the inflammatory process and the biomineralization ability of MTA to assess host-biomaterial interactions in vivo.

Materials and Methods Ethical Concerns All experimental protocols used in this study were approved by the Animal Ethics Screening Committee and the Ethics Committee for Research with Human Beings of the Federal University of Santa Catarina, Santa Catarina, Brazil. Preparation of Specimens Eighty dentin tubes were prepared from extracted human tooth roots. The crowns and the apical thirds of the roots were removed using a low-speed water-cooled ISOMET diamond saw (Buehler, Lake Bluff, NY). Each root canal was enlarged to obtain 1.3-mm-diameter standardized cavities. Tube length was 5 mm, and their outer walls were abraded with a diamond bur to thin the walls to a 2-mm thickness. The dentin tubes were washed in distilled water and then autoclaved. Before implantation, the tubes were thoroughly irrigated with 17% EDTA followed by a 1% sodium hypochlorite solution, dried, and then filled with tooth-colored ProRoot MTA (Dentsply, Tulsa Dental, OK) or kept empty (negative control). Experimental Protocol Male Swiss mice (35-40 g) were anesthetized with ketamine hydrochloride (Dopalen; Division Vetbrands Animal Health, Jacareı´, SP, Brazil) and xylazine (Anasedan; Agribrands do Brasil Ltda, Paulı´nia, SP, Brazil). Four separate 1-cm incisions were made in the backs of mice at a 1-cm interval. The skin was deflected to create a pocket by a blunt dissection on one side of each incision. Each mouse received three dentin tubes: two filled with MTA and one empty; no specimen was inserted in the fourth pocket (sham) to ensure a rotation of sites. After 12 hours and 1, 3, and 7 days after implantation, the animals were euthanized, and the tubes together with surrounding tissues were removed. Half of the samples were fixed in 4% paraformaldehyde at 4 C for histological and immunohistochemical staining. To determine the protein level expression of IL-1ß, TNF-a and IL-10, the remaining half of the samples and surrounding tissues were excised and processed for tissue homogenate. Dentin tubes were retrieved and processed for scanning electron microscopic (SEM) analysis.

Technology, Beverly, MA), rabbit polyclonal antiphospho-p65 NF-kB (1:50, Cell Signaling Technology), rabbit monoclonal antiphospho-cjun AP-1 (1:50, Cell Signaling Technology), and rabbit polyclonal anti-iNOS (1:100, Cell Signaling Technology). High-temperature antigen retrieval was applied by immersing the slides in a water bath at 95 C to 98 C in 10 mmol/L trisodium citrate buffer (pH = 6.0) for 45 minutes. The nonspecific binding was blocked by incubating sections for 1 hour with goat normal serum diluted with PBS. After overnight incubation with primary antibodies at 4 C, the slides were washed with PBS and incubated with the ready-to-use secondary antibody EnVision Plus (DakoCytomation EnVision Doublestain System, Carpinteria, CA) for 1 hour at room temperature. The sections were washed again in PBS, and visualization was completed using 3,30 -diaminobenzidine (DAB) (DakoCytomation) and counterstained lightly with Harris’ hematoxylin solution. Both control and experimental samples were placed on the same glass slide and processed under the same conditions. Images of the stained tissue sections were acquired using a digital camera (Canon A620, Lake Success, NY) connected to a light microscope (Axiostar Plus; Carl Zeiss, Oberkochen, Germany). Settings for image acquisition were identical for both control and experimental tissues. Four consecutive images per sample, captured at 40 magnification, were taken of the tissues in contact with the material on the tube’s opening or on an empty opening. The threshold optical density was obtained using 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 Briefly, full-thickness tissue samples were homogenized in phosphate buffer containing 0.05% Tween 20, 0.1 mmol/L phenylmethylsulphonyl fluoride, 0.1 mmol/L benzethonium chloride, 10 mmol/L EDTA, and 20 KIU aprotinin A. The levels of IL-1b, TNF-a, and IL-10 were evaluated using DuoSet ELISA kits according to the manufacturer’s recommendations (R&D Systems, Minneapolis, MN). The results were expressed as picogram per milligram (pg/mg) of tissue protein concentration. SEM Analysis After the experimental periods, the retrieved dentin tubes were briefly washed in distilled water and sputter coated with gold for SEM observation (Philips SEM XL 30; Philips, Eindhoven, The Netherlands) at an accelerating voltage of 10 kV. Statistical Analysis Data of cytokine measurement (pg/mg) and optical densities of immunohistochemistry staining were expressed as mean  standard error of the mean. Two-way analysis of variance followed by a Bonferroni posttest was performed to analyze differences between the groups (p< 0.05).

Results Histological and Immunohistochemical Analyses For hematoxylin-eosin and immunohistochemistry staining, tissues were embedded in paraffin, sectioned at a 3-mm thickness, and prepared on conventional glass slides. Tissue sections were deparaffinized, and immunohistochemistry was performed using the following primary antibodies and respective dilution ratios: 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; Cell Signaling 1348

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Inflammatory Cytokine Expression Profile Cytokine expression profile is shown in Figure 1. For all groups, the total amount of cytokines expressed decreased after day 3. Although MTA induced a proinflammatory cytokine up-regulation during the first 3 days, there was no apparent material-dependent effect on the classes of cytokines produced. However, a time-dependent manner was observed. In all the experimental groups, the expression of TNFa and IL-1b peaked at 12 and 24 hours, respectively. The expression levels of IL-1b and TNF-a in the MTA group at 12 hours, 1 day, and JOE — Volume 36, Number 8, August 2010

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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 < 0.05 versus the empty tube group. #p < 0.05 versus the sham group.

3 days were significantly up-regulated when compared with those in the empty tubes and sham (p < 0.05). By day 7, no significant differences were found in any group. Meanwhile, IL-10 expression was upregulated between days 1 and 3, and the expression peaked on day 1 in all the experimental groups (Fig. 1C). The MTA group showed a significant increase in IL-10 expression compared with the empty tube and sham groups at 12 hours, 1 day (p < 0.05), and 3 days (p < 0.01). On day 7, the experimental groups continued to exhibit IL-10 expression. However, the magnitude of IL-10 expression decreased, and no statistically significant difference was observed between the groups.

Inflammatory Response Assessment: Histomorphological and Immunohistochemical Findings After 12 hours, the tissue surrounding all experimental groups contained primarily neutrophils. In all groups, neutrophil recruitment decreased between days 1 and 3, and cellular populations of macrophages and lymphocytes increased and remained elevated from days 3 to 7. On day 7, the inflammation intensity had diminished, and a chronic inflammatory cell infiltration consisting primarily of macrophages, fibroblasts, lymphocytes, and few giant cells was present in a thin fibrous capsule. These findings show the transition from an acute phase to a moderate chronic response. Immunoreactivity analyses for MPO, AP-1, NF-kB, iNOS, COX-2, and VEGF are shown in Figure 2. These analyses revealed the expression of the different proteins in a time-dependent manner. MPO expression peaked at 24 hours in all experimental groups, whereas a significant increase was observed in tissues in contact with MTA between 12 hours and day 1 when compared with the control groups (p < 0.001). AP-1 expression was slightly elevated in all groups. However, MTA showed a significant up-regulation of the transcriptional factor on day 1 JOE — Volume 36, Number 8, August 2010

(p < 0.001) (Figs. 2 and 3). All experimental groups induced pronounced phosphorylation of NF-kB, which peaked at 12 hours. As expected, MTA significantly up-regulated NF-kB expression compared with empty tubes and shams (p < 0.01). In all groups, the expression of COX-2 and iNOS peaked at 12 and 24 hours, respectively (Fig. 2). However, MTA caused a significant increase in the expression of COX-2 and iNOS when compared with the empty tubes and sham (p < 0.01). The expression of VEGF was increased at all time periods (Fig. 2). During the acute phase of inflammation, VEGF was mainly expressed in the presence of neutrophils and macrophages (Fig. 3). On day 7, VEGF expression by fibroblasts was also observed.

In Vivo Biomineralization Ability of MTA SEM examination of dentin tubes showed the presence of apatitelike clusters (Fig. 4). SEM-EDAX indicated that the precipitates mainly contained calcium and phosphorus with Ca/P molar ratios of 1.60 to 1.64 (Fig. 4). It was possible to observe numerous apatite-like clusters deposited on collagen fibrils all over the surface of dentin tubes containing MTA in as early as 12 hours from implantation. With the increase in the implantation time, a more extensive mineralization was observed; many of these precipitates formed agglomerates. After 7 days, a compact apatite layer was observed all over the surface of the dentin tubes (Fig. 4).

Discussion Despite the progress made in understanding the molecular biology that controls the mechanism of action of MTA (17–19), the exact mechanism of wound healing and the nature of hard-tissue formation remain unclear and, consequently, a matter of extensive research. Therefore, our study focused on the inflammatory reaction and its

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Figure 2. Immunoreactivity analysis for MPO, AP-1, NF-kB, iNOS, COX-2, and VEGF. Staining intensity and stained area of antibodies immunoreaction are expressed as optical density. Each column represents the mean  standard error of the mean. )p < 0.05 versus the empty tube group. #p < 0.05 versus sham.

correlation with the biomineralization process to better understand the mechanisms underlying host responses to MTA. The inflammatory reaction is closely related to the healing process (20, 21). The body’s defense reactions involve several regulatory functions and numerous molecular mediators (20). Because repair begins at the onset of inflammation, it is necessary to further understand the inflammatory process. Initially, during the inflammatory response, IL-1b and TNF-a have a proinflammatory effect followed by a regulatory effect in the later stages of inflammation, reducing immune activity (22, 23). This fact may explain the findings of our study in which overexpressed cytokine levels in the acute phase tended to decrease over time. Probably, the regulatory effect of these cytokines signaled the resolution of the inflammatory reaction during the chronic phase. Additionally, our data suggest that the presence of MTA in the dentin tubes had an effect on the intercellular signaling that occurs at the implantation site. It is interesting to note that there was a simultaneous overexpression of IL-10 in MTA specimens. Because IL-10 has been shown to down-regulate cytokine production, IL-10 up-regulation may have signaled the decrease in cytokine production observed at later time points (20). As suggested in previous studies, our data support the idea that MTA has an anti-inflammatory effect (17, 22). MPO immunostaining showed that neutrophils were the predominant cells at the implantation site during the first day. Neutrophil recruitment decreased from day 1 to day 3; after which, mostly macrophages and lymphocytes migrated into the tissue. As a result, by day 3, the number of inflammatory cell numbers was diminished, indicating the beginning of the resolution phase in the inflammatory process. This histomorphological change may explain the expected transition 1350

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from an acute proinflammatory phase to an anti-inflammatory and pro–wound healing chronic environment. Biomaterials might elicit several signaling pathways to trigger the inflammatory cascade (3, 24, 25). Therefore, we analyzed the expression of selected NF-kBregulated gene products (iNOS and COX-2), AP-1 and VEGF by immunohistochemical staining. Our results showed that NF-kB was involved in MTA-stimulated signal transduction in the early stages of inflammation. Therefore, we suggest that MTA induces phosphorylation of IkBa, which leads to the freeing of NF-kB complexes. Activated NF-kB complexes translocate to the nucleus and stimulate the expression of COX-2. Released prostaglandins, in turn, induce the transcription of iNOS in an autocrine manner (26). Recent evidence shows that the production of prostanoids by COX2 promotes the expression of VEGF and subsequent angiogenesis (11). Moreover, AP-1 may induce VEGF expression. However, we observed that VEGF overexpression remained stable during the various time periods. During the acute phase, VEGF upregulation was attributed to its ability to increase the permeability of blood vessels - an important vascular change observed during the early stages of inflammation. On day 7, VEGF was mainly expressed by fibroblasts, suggesting sustained angiogenesis for the induction of a repair process. Our study provides compelling evidence of the in vivo biomineralization process promoted by MTA. SEM analysis showed the presence of the deposition of apatite-like clusters on collagen fibrils as early as 12 hours after implantation. SEM-EDAX indicated that the precipitates were mainly composed of calcium and phosphorus. Previously, we showed that the interaction of MTA with dentin in a phosphate-containing fluid produces an amorphous calcium phosphate phase, which acts as a precursor during the formation of carbonated apatite (15, 16). It is

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Figure 3. Representative images for tissue in contact with MTA at day 1 (40).Scale in 50 mm. (A) Hematoxylin and eosin staining. Immunohistochemical reaction for (B) MPO, (C) AP-1, (D) NF-kB, (E) iNOS, (F) COX-2, and (G) VEGF. (H) Negative control of the immunohistochemical reaction. (This figure is available in color online at www.aae.org/joe/.)

important to highlight that SEM-EDAX analysis showed similar results for precipitates formed by MTA after subcutaneous implantation. Our findings corroborate those of previous in vitro studies that suggest that calcium ions released by MTA react with phosphate, yielding carbonate apatite precipitates (14, 27). It is well known that the organic matrix possesses properties that can initiate and regulate the formation of mineral crystals. Thus, crystal nucleation and controlled growth are considered to be matrix-mediated or matrix-regulated processes (27–29). Type I collagen is the template for the controlled deposition of calcium phosphate, but by itself it does not have the capacity to induce matrix-specific mineralization (27, 30). Noncollagenous proteins present in the mineralized dentin matrix, such

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as dentin matrix protein 1, have been implicated as having a regulatory function in dentin formation (27). Recombinant dentin matrix protein 1 molecules have been thought to perform specific molecular recognition in conjunction with the apatite surface in order to guide calcium phosphate clusters through the collagen matrix during recruitment (15, 27). This process has been described as controlled biomineralization (15). To our knowledge, our study is the first to provide evidence that the biomineralization process occurs simultaneously with the initial acute inflammatory response. Therefore, we hypothesize that together with the alkalinity of the material, the precipitation of apatite by MTA during the acute phase of inflammation may contribute to the signaling of several unrecognized pathways in different cell types. Apatites may

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Figure 4. SEM photomicrographs of the surface of dentin tubes filled with MTA after subcutaneous implantation. (A) Formation of several apatite-like precipitates at 12 hours (3,000). (C) Energy Dispersive X-Ray Analysis (EDAX) spectrum revealing the chemical composition and the Ca/P ratio of the precipitate shown in B (8,000). Observe the deposition of precipitate on the collagen fibers (D, 2,500) (E and F, 4,000). Increased deposition of precipitates at day 1 (G, 2,500) (H and I, 5,000), and in areas of higher mineralization, collagen fibers exhibited a typical ‘‘corn-on-the-cob’’ appearance (arrows). Mineralization areas were more extensive as the implantation time increased (J and K, 3,000), forming a compact layer at 7 days after implantation (L, 100). (This figure is available in color online at www.aae.org/joe/.)

induce changes in gene expression and subsequently in cell functional activity. These changes are likely to contribute to repair and biomineralization process. Recent studies showed that MTA induced mineralization and mineralized tissue proteins messenger RNA expression of cementoblasts and bone cells, which play a crucial role in cemental and osseous repair and regeneration (31, 32). Therefore, we suggest that several biological mechanisms in combination with the bioactivity of MTA may explain its ability to induce mineralized tissue deposition. Our data provide scientific background to develop novel biomaterials aimed at exploiting the natural regenerative potential of pulp and bone tissues. In summary, we showed that MTA induces a proinflammatory and pro–wound healing environment. The biomineralization process 1352

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occurs simultaneously with the acute inflammatory response. We suggest that when MTA is implanted, a series of biochemical and biophysical reactions occurs at the MTA-dentin-tissue interface. Subsequently, this activates cellular and tissue events in the inflammatory and biomineralization processes and culminates in the formation of an apatite-like layer that allows the integration of the biomaterial into the environment.

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