Biocompatibility of Intracanal Medications Based on Calcium Hydroxide

Objective. The aim of this study was to evaluate the rat subcutaneous tissue reaction to calcium hydroxide-based intracanal medicaments, UltraCal XS (calcium hydroxide, barium sulphate, aqueous matrix), Hydropast (calcium hydroxide, barium sulphate, and propyleneglycol), and Calen (Calcium hydroxide, zinc oxide, colophony, and polyethyleneglycol), used as a control. Methods. Forty-eight rats (Rattus Norvegicus Holtzman) were distributed in three groups: Calen, UltraCal XS, and Hydropast. Polyethylene tubes filled with one of the medicaments were implanted in the dorsal subcutaneous. After 7 and 30 days, the implants were removed and the specimens were fixed and embedded in paraffin. Morphological and quantitative analyses were carried out in the HE-stained sections. The numerical density of inflammatory cells in the capsule was evaluated and statistical analyses were performed (P ≤ 0.05). Results. At 7 days, all materials induced an inflammatory reaction in the subcutaneous tissue adjacent to the implants. In all groups, a significant reduction in the number of inflammatory cells and giant cells was verified in the period of 30 days. Conclusion. These results indicate that the calcium hydroxide-based medicaments evaluated present biocompatibility similar to Calen.


Introduction
The success of endodontic treatment of teeth with periapical lesion depends on the reduction or elimination of the intraradicular infection [1,2]. The root canal mechanical preparation is not enough to eliminate this infection because many microorganisms are not only in the main root canal, but also disseminated throughout the root canal system. Therefore, the use of an intracanal dressing to eliminate the microorganisms is indicated [3][4][5][6].
Antimicrobial activity and biocompatibility are characteristics that an ideal intracanal dressing has to show [7]. Calcium hydroxide [Ca(OH) 2 ] has been widely used for its biological and antimicrobial activity [4,8], ability to dissolve organic tissue [9], and capacity to inactivate bacterial endotoxin [10,11]. Despite these properties, the Ca(OH) 2 has no satisfactory physical properties such as radiopacity to visualize on dental radiographs and flow capacity to facilitate its insertion in the root canal [12,13]. For this reason, it needs the incorporation of a radiopacifying agent and a vehicle to improve these characteristics [8,14].
Although the Ca(OH) 2 shows an excellent biocompatibility, the addition of other substances can affect its biological properties [8,12]. In the last years, it has been marketed the calcium hydroxide-based medicaments such as UltraCal XS (Ultradent Products, Inc.) and Hydropast (Biodinâmica Química e Farmacêutica Ltda., Brazil). Ultra-Cal XS is basically composed by 35% of calcium hydroxide, a radiopacifier, and a vehicle which does have not the proportions related by the manufacturer. Studies have 2 ISRN Dentistry demonstrated that UltraCal XS has a high pH value [15] and an effective antimicrobial activity against common endodontic bacteria of teeth with pulp necrosis [16]. On the other hand, Brazilian paste Hydropast, composed by 38% of calcium hydroxide, barium oxide, as a radiopacifying agent, and propilenoglycol as vehicle, is a recent material, and, therefore, until now, there are no studies of its biological properties.
Considering the recommendation of International Organization for Standardization [17], it is necessary in vitro and/or in vivo studies for evaluation of the biocompatibility of these new materials. Thus, the purpose of this study was to evaluate the tissue reaction of these calcium hydroxide-based medicaments in rat subcutaneous.

Animals and Experimental
Proceedings. This study was performed in accordance with the principles of animal care on animal experiments. The research protocol was authorized by the Ethical Committee for Animal Research of the São Paulo State University, Brazil (Dental School, UNESP, Araraquara).
The animals were anaesthetized with an intraperitoneal injection containing 80 mg/Kg of body weight of ketamine (União Química Farmacêutica Nacional S/A-Brazil) and 4 mg/Kg of body weight of xylazine (Virbac do Brasil Indústria e Comércio Ltda., Brazil). After shaved and disinfection with 5% iodine solution, a 20 mm-long incision in a head-to-tail orientation was made using a scalpel (no. 15, Fibra Cirúrgica, Joinvile, SC, Brazil) in the dorsal skin. Subsequently, the polyethylene tube containing Calen paste, UltraCal XS, or Hydropast was immediately implanted into the dorsal subcutaneous connective tissue. After implantation, the skin was closed with 4.0 silk suture (Vicryl; Johnson & Johnson: Ethicon Inc., New Brunswick, NJ, USA). One polyethylene tube filled with an intracanal paste was implanted in each animal and left for periods of 7 and 30 days. After experimental periods, the animals were killed by overdose of anesthetic solution, and the tubes were removed with surrounding connective tissue and prepared for paraffin embedding.

Histological Procedures and Analysis.
The specimens containing the implanted polyethylene tubes were fixed in 4% formaldehyde (prepared from paraformaldehyde) buffered at pH 7.2 with 0.1 M sodium phosphate at room temperature for 48 hours. Subsequently, the specimens were dehydrated and embedded in paraffin. Serial 6 µm-thick sections were made parallel to the tube long axis and stained with hematoxylin and eosin (H&E) for morphological and morphometric analyses. The morphological analysis of the capsule in contact with the material on the opening of the tube was performed considering the following parameters: presence of inflammatory process, main cells (inflammatory cells or fibroblasts) present in the capsule, presence of multinucleated giant cells, and presence of collagen fibers.
The numerical density of inflammatory mononucleated cells and multinucleated giant cells was undertaken using a light microscope (BX51, Olympus, Tokyo, Japan) and an image analysis system (Image Pro-Express 6.0, Olympus). Three H&E-stained sections per animal were selected at intervals of at least 100 µm; in each section, a standardized field of 0.09 mm 2 of the connective tissue adjacent to the opening of the tube implanted was analyzed, totaling 0.27 mm 2 per animal. In each area, the total number of inflammatory cells was counted using the image analysis system at ×40 magnification; in each animal, the total number of inflammatory cells was divided by total area, and, then, the number of inflammatory cells/mm 2 was obtained. The differences between the groups were statistically analyzed using SigmaStat 2.0 software (Jandel Scientific, Sausalito, CA, USA); the data were submitted to ANOVA and Tukey test. The significance level accepted was P ≤ 0.05.

Morphological and Quantitative
Analyses. After 7 days of implantation, the capsule adjacent to the opening of the tubes filled with Calen, UltraCal XS, and Hydropast paste showed numerous inflammatory cells, mainly lymphocytes and macrophages. Usually, the inflammatory infiltration was evident in the innermost portion of the capsule, that is, in close juxtaposition to the materials (Figures 1(a), 2(a) and 3(a)). According to Table 2, no significant difference was verified in the numerical density of inflammatory cells among the groups. Multinucleated giant cells were also observed in the capsules formed in all groups ( Figures  1(a), 2(a), and 3(a)). However, in the Hydropast group the number of multinucleated giant cells was significantly higher in comparison to other groups; on the other hand, the capsule of the Calen group exhibited the lower number of multinucleated giant cells (Table 2). A significant decrease in the number of inflammatory cells and multinucleated giant cells was verified from 7 to 30 days, in all groups (Figures 1(b), 2(b), and 3(b); Table 2). In the period of 30 days, the capsule adjacent to Calen paste was formed by a dense connective tissue exhibiting typical  Values are expressed as mean ± standard deviation.
Equal letters indicate no statistically significant difference (P > 0.05) between the materials in the same experimental period. Different numbers indicate difference statistically significant (P ≤ 0.05) of each material in the different experimental periods.
bundles of collagen fibers between fibroblasts; lymphocytes and plasma cells were mainly present next to blood vessels (Figure 1(b)). In the UltraCal group, the capsule contained several fibroblasts among the inflammatory cells; usually, bundles of collagen fibers were only present in the outermost portion of the capsule (Figure 2(b)). The connective tissue of the capsule of the Hydropast exhibited several cells and scarce collagen fibers (Figure 3(b)). Although no significant difference was found in the number of inflammatory cells between the groups, the mean of the numerical density of inflammatory cells in the Hydropast group was around 1,400 cells/mm 2 , whereas in the other groups was around 965 cells/mm 2 . Moreover, in the Calen group the number of multinucleated giant cells was significantly lower in comparison to UltraCal and Hydropast groups ( Table 2).

Discussion
Implantation in the subcutaneous connective tissues of experimental animals has been extensively used to evaluate the biocompatibility of endodontic materials [18,19]. Our findings indicate that UltraCal XS and Hydropast exhibit biological behavior similar to Calen (control group). At 7 days, an intense inflammatory reaction and foci of coagulative necrosis were seen in the adjacent capsule to the implanted materials. After 30 days, significant reduction in the inflammatory process was verified in all analyzed groups; usually, the capsule formed juxtaposed to the Calen paste exhibited inflammatory cells among typical bundles of collagen fibers and fibroblasts. The inflammatory reaction observed in the period of the 7 days may be attributed to the superficial necrosis promoted by calcium hydroxide-based materials [20]. Calcium hydroxide has an alkaline pH [21] and, when in contact with the connective tissue, induces the formation of a coagulative necrosis zone [22]. Coagulative necrosis refers to a spectrum of morphological changes in living tissue resulting from the action of enzymes on lethally injured cells. The mass of necrotic cells is characterized by preservation of the basic outline of the coagulated cells for a span of at least some days. As necrotic cells are unable to maintain membrane integrity, their contents leak out and elicit an inflammatory response that removes the cellular debris by phagocytosis, followed by healing [20]. The vehicles mixed with calcium hydroxide powder play an important role in the ionic dissociation process and so in the disinfection of the root canal and biocompatibility [8,23]. There are three main types of vehicle: water-soluble substances, viscous, and oil-based [8]. In the present study, the vehicles of the different medicaments did not interfere in their tissue reaction since the results were similar. In the periods of 7 and 30 days, the number of giant cells was significantly reduced in the Calen group in comparison to UltraCal XS and Hydropast. At 30 days, the capsule adjacent ISRN Dentistry 5 to Hydropast showed a significantly lower number of giant cells than the UltraCal XS. The giant cells are derived from the fusion of 20 or more monocytes/macrophages and are formed for removing exogenous agents [24]. In the period of 30 days, the high number of giant cells verified in the UltraCal XS group suggests that this material may release more irritant substances than the Hydropast and Calen.
In Calen paste, the calcium hydroxide is mixed to a viscous vehicle, polyethylene glycol 400, one of the most commonly used vehicles in root canal medicaments with low toxicity, high solubility in aqueous solutions, low immunogenicity and antigenicity [25], and antibacterial activity [5]. This vehicle releases calcium and hydroxyl ions more slowly and for longer periods than water-soluble and oil-based materials [26]. Because the releasing of H + , the polyethylene glycol 400 neutralizes the OH − released by calcium hydroxide and, thereby, reduces the superficial necrosis area [27].
The propylene glycol is used as vehicle in the Hydropast; the vehicle used in the Hydropast is classified as a viscous vehicle with high molecular weight and, as well as polyethylene glycol 400, prolong the action of calcium hydroxide in the root canal system [26]. Moreover, propylene glycol shows low toxicity and antimicrobial properties [28,29]. It was demonstrated that the addition of propylene glycol may not interfere in the biocompatibility of MTA in rat subcutaneous tissue [30].
Our results also suggest that the different radiopacifying agents of the pastes did not interfere in the tissue reaction. The endodontic materials should present sufficient radiopacity to be distinguished from adjacent anatomical structures, such as bone and teeth [31]. Zinc oxide, barium sulfate, bismuth oxide, and other components with iodine and bromine are some examples of radiopacifiers [12,14,27,32].
The zinc oxide of Calen paste does not affect the biological properties of calcium hydroxide [27,33]. Barium sulphate, radiopacifying agent of Hydropast, is also biocompatible because its cause no detrimental effect in rat subcutaneous tissue [34] or in periapical tissue in association with calcium hydroxide [14]; barium oxide in association with the Norian, an skeletal repair system (SRS), in tibiae defects of rats, maintains the properties of biocompatibility and osteoconductive materials of the SRS [35]. Although the manufacturer does not inform the radiopacifying agent of the UltraCal XS, our results demonstrated that this material has a good biological behavior.
Considering the methodology used in the present study, our findings indicate that UltraCal XS and Hydropast are biocompatible in subcutaneous tissue of rats.