Systemic effect of mineral aggregate-based cements: histopathological analysis in rats

Abstract Objective: Several studies reported the local tissue reaction caused by mineral aggregate-based cements. However, few studies have investigated the systemic effects promoted by these cements on liver and kidney when directly applied to connective tissue. The purpose of this in vivo study was to investigate the systemic effect of mineral aggregate-based cements on the livers and kidneys of rats. Material and Methods: Samples of Mineral Trioxide Aggregate (MTA) and a calcium aluminate-based cement (EndoBinder) containing different radiopacifiers were implanted into the dorsum of 40 rats. After 7 and 30 d, samples of subcutaneous, liver and kidney tissues were submitted to histopathological analysis. A score (0-3) was used to grade the inflammatory reaction. Blood samples were collected to evaluate changes in hepatic and renal functions of animals. Results: The moderate inflammatory reaction (2) observed for 7 d in the subcutaneous tissue decreased with time for all cements. The thickness of inflammatory capsules also presented a significant decrease with time (P<.05). Systemically, all cements caused adverse inflammatory reactions in the liver and kidney, being more evident for MTA, persisting until the end of the analysis. Liver functions increased significantly for MTA during 30 d (P<.05). Conclusion: The different cements induced to a locally limited inflammatory reaction. However, from the systemic point of view, the cements promoted significant inflammatory reactions in the liver and kidney. For MTA, the reactions were more accentuated.


Introduction
Mineral Trioxide Aggregate (MTA) is one of the most used biomaterials in endodontics currently 3

. It is a
Portland cement (75.0%) (wt) added to dehydrated CaSO 4 (5.0%) and Bi 2 O 3 (20.0%), the latter being responsible for its radiopacity 2 . Because of its hydraulic nature, after the addition of water, the setting process of the cement begins by initially forming a hydrated silica gel 3 .
There is few information regarding the MTA cement manufacturing process. However, the companies the cement under conditions that guarantee its safe use. Portland cement, the main raw material of MTA, has several compounds in its formulation, the following being the mostly used ones: SiO 2 (21.2%); CaO (68.1%); Al 2 O 3 (4.7%); MgO (0.48%) and Fe 2 O 3 (1.89%) 3 process before being used, studies have proved the presence of heavy metals in the composition of MTA, among them As, Cr and Pb 11,12 . Nevertheless, it is 12 .
In spite of its proven biocompatibility and bioactivity 20,21 , undesirable effects of Bi 2 O 3 have been pointed out on the rate of Ca++ ions released from MTA, thus compromising its performance as a reparative material 3,25 . Furthermore, studies have reported that heavy metal salts, mainly Bi and Cd, are responsible for the expression of the heme oxygenase-1 enzyme (HO-1) in different cell lineages, compromising their regulatory functions and protective mechanisms 4,22 .
In a recent study, Khalil and Eid 18 (2013)  to address the deficiencies of the silicate-based cement 3,11,12,25 as ZnO, have been proposed as a feasible alternative to the compounds currently used for this purpose 26 . Therefore, the aim of this in vivo study was to investigate the local (subcutaneous) and systemic (liver and kidney) effects of MTA and a new reparative calcium aluminate-based cement (EndoBinder) hypothesis tested was that there would be no cements in the different evaluated tissues.

Animals
The entire study was developed according to the guidelines of the Research Ethics Committee on the Use of Animals (Process CEUA No. 3/2013), and the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). For this study, 40 male rats (Rattus novergicus, Wistar), weighing 300 g, were selected.
The number of specimens per group was determined based on other biological studies that used a similar quantity of animals 1,13,14 . In addition, sample size was calculated to set a number of needed specimens to among control and experimental groups. Animals were kept in plastic cages (40x32x17 cm) especial for this purpose, accommodated in an acclimatized bioterium (temperature: 21-23°C/relative humidity: 60±5%/12 h light-dark cycle), and received balanced rations (Nuvilab, Colombo, PR, Brazil)  After manipulating the cements, pre-sterilized polyethylene tubes measuring 1.5 mm of internal Systemic effect of mineral aggregate-based cements: histopathological analysis in rats g of cements using a sterile Lentulo spiral (Dentsply/ Maillefer, Ballaigues, Switzerland) 13 tubes with the tested cements, one of their extremities was heat-sealed to avoid cement extravasation 13 . After biopsies, the tissues and organs collected were immediately immersed in a 10% formalin solution (Merck, Darmstadt, Germany), where they remained for 24 hours at room temperature. After this period, all surgical parts were submitted to routine laboratory processing. Polyethylene tubes were removed before tissue sectioning, then the tissues were sectioned longitudinally, in 5 μm-thick semi-serial cuts, considering 10 cuts were discarded and 5 were stained with hematoxylin-eosin (Merck).

Histopathological examination
Histopathological examination was performed blindly and double-checked by two previously calibrated examiners with a concordance index of 94% between them, using an optical light microscope (Axio Star Plus, Carl Zeiss, Oberköchen, Germany).
The local (subcutaneous tissue) and systemic (liver and kidney) biocompatibility of the cements were evaluated considering the presence of the following histopathological events: inflammatory (fibroblasts and blood vessels) and macrophagic activity (mononuclear phagocytes and multinucleated giant cells) 5,13 . In addition to these tissue events, the presence of micro and macrovesicular steatosis, and the occurrence of apoptosis in hepatic cells were also the liver 5 , whereas, for the kidney, the presence of hypercellularity in the cortex was used 18 . According      However, at the period of 30 d, we noted a reduction in the extent of these events in the affected areas ( Figures 4A and B). At the end of the analysis, we index for the liver and kidney.

EBZnO group
The histopathological events observed in both time intervals of analysis were similar to those from the previous groups, both for livers and kidneys. We polymorphous and mononuclear cells of moderate extent in the initial period. Most blood vessels seemed congested, and had a ruptured endothelial layer, which the formation of various accessory vessels. In addition, we noted the presence of moderate microvesicular steatosis and several areas of intracellular edema. At the period of 30 d, we also noted a small quantity of hepatocytes in mitosis and apoptosis and a decrease the kidney, we observed vascular congestion and hypercellularity with an increase in the renal cortex density, followed by reduction of these features at the 1 (discrete).

WMTA group
At the period of 7 d, despite involving larger areas than the ones observed in the groups treated with different versions of EndoBinder, we noted the extent in the liver. Blood vessels seemed congested cells within and adjacently to them. In addition, we observed microvesicular steatosis and several areas of regional necrosis. In only one of the samples, we detected a foreign body in one of the tributary vessels of the hepatic portal system, associated with the dispersed residual material tested in this group. After and associated with moderate fibroangioblastic proliferation, with several hepatocytes that seemed to be undergoing mitosis and apoptosis. We also   it becomes methylated into its trivalent form, which is less toxic and more easily eliminated 8,11 . However, if the liver is not capable of metabolizing the arsenic in a reasonable time, it causes irreparable damage to the organ, even when the doses are not considered lethal 8,11 . The results observed in the histopathological analysis of the liver and kidney led us to believe that

Conclusion
In summary, the different mineral aggregatebased cements investigated in this study promoted tissue, which decreased with time. Thus, it could be stated that all cements were locally biocompatible.
Systemically, all cements caused adverse histological reactions on liver and kidney; there were more Systemic effect of mineral aggregate-based cements: histopathological analysis in rats accentuated reactions for MTA that persisted at the end of 30 d. In addition, it is worth to emphasize that these systemic reactions can not be considered irreversible, and further researches using longer periods of analysis must be conducted.