Initial inflammatory response after the pulpotomy of rat molars with MTA or ferric sulfate

Abstract Purpose To compare, both qualitatively and quantitatively, the inflammatory cells, vascular density and IL-6 immunolabeled cells present in the pulp after pulpotomy with white MTA versus 15.5% ferric sulfate (FS). Methodology Forty-eight mandibular first molars from 24 Wistar rats were divided into MTA or FS groups and subdivided according to the period after pulpotomy procedure (24, 48 and 72 hours). Four teeth (sound and untreated) were used as controls. Histological sections were obtained and assessed through the descriptive analysis of morphological aspects of pulp tissue and the quantification of inflammatory cells, vascular density and interleukin-6 (IL-6) expression. Data were statistically analyzed (p<0.05). Results The number of inflammatory cells was similar in both groups, being predominantly localized at the cervical radicular third. In the MTA group, increased inflammation was observed at 48 hours. Vascular density was similar in both groups and over time, being predominant in the medium radicular third. No correlation was found between the number of inflammatory cells and the vascular density. Pulp tissue was more organized in MTA-treated teeth. In both groups, a weak to moderate IL-6 expression was detected in odontoblasts and inflammatory cells. Comparing both groups, there was a greater IL-6 expression in the cervical radicular third of teeth treated with MTA at 24 hours and in the medium and apical thirds at 72 hours, while in the FS group a greater IL-6 expression was found in the apical third at 24 hours. Conclusion The MTA group presented better histological features and greater IL-6 expression than the FS group. However, no difference was observed between the groups regarding the inflammatory status and vascularization, suggesting the usefulness of FS as a low-cost alternative to MTA.


Introduction
Pulpotomy consists of the coronal pulp amputation and treatment of remaining radicular tissue with capping agents that preserve its vitality and function.
To keep the vitality of pulp tissue and impair pathological alterations, exposed pulp stumps should be covered with a biocompatible material. 1 The growing interest in biocompatible materials promoted MTA as the capping material of choice for pulpotomies. In contact with periradicular or dental pulp tissues, MTA is able to maintain pulp vitality and to stimulate repair. 2 This material also enhances the deposition of mineralized tissue in root canals, thus causing radicular stenosis or pulp canal obliteration. 3,4 Despite the high clinical, radiographic and histological success rates of MTA, both tooth discoloration 5 and its high cost 6,7 are remarkable inconveniences. Ferric sulfate (FS) has been considered an appropriate and low-cost alternative capping material for primary teeth pulpotomy. 1 This is a coagulative and hemostatic agent that forms ferric ion-protein complex on contact with blood, thus reducing the inflammatory response. 8,9 The FS presents high clinical and radiographic success rates, comparable to that of formocresol. 10-13 However, studies assessing the histological condition of the remaining pulp are scarce. 4,14,15 The analysis of pulp response to capping materials is the best way to determine both their action mechanism as the pulp condition after pulpotomy. 16 Considering the pulp inflammatory events may favor the initial phases of pulp repair and that the exacerbation of inflammation often leads to necrosis of the entire tissue, 17 it is important to evaluate the acute pulp response after pulpotomy with different capping materials, to determine the most favorable one. Therefore, the purpose of this study was to compare, both qualitatively and quantitatively, the inflammatory cells, vascular density and IL-6 immunolabeled cells present in the pulp 24, 48 and 72 hours after pulpotomy with white MTA versus 15.5% FS.

Methodology Experimental design
The Animal Ethics Committee approved the protocol of this study (570/2014). Considering a prior study, 18 the sample size of 7 teeth per group achieved 95% power and 1% significance level. We added 10% to the final sample size to compensate for possible losses, leading to a final sample size of 8

Results
The sound teeth presented a normal pulp tissue, characterized by a loose connective tissue containing several types of cells and thin-walled blood vessels in the central area, as well as an odontoblast layer in the periphery. No inflammation was observed in these teeth. In untreated teeth, a mild inflammation, with a small number of neutrophils, was detected near the coronal opening, while the remaining tissue exhibited an aspect of normality.
In the tested groups and during the three periods of evaluation, the inflammatory response was greater in the cervical third (Figures 1 and 2), gradually decreasing through the medium and apical third. The inflammatory infiltrate, mainly composed of neutrophils, was more intense at 48 hours. Vascularization was predominant in the medium third, with inflammatory cells inside and around the vessels.
In the MTA group, inflammatory cells were grouped into the tissue surrounding the material, with superficial necrosis in some histological sections, while the deeper tissue was well organized and exhibited several fibroblasts among the bands of collagen fibers, which are features of normality ( Figure 3). In the FS group, the tissue adjacent to the material was acellular, followed by a layer of disperse inflammatory cells, sometimes presenting necrotic areas. In some sections, the deeper tissue was scarcely organized (Figure 4). There was no significant correlation between Occasionally, a diffuse immunostaining was found in the extracellular matrix of the connective tissue ( Figure   5). In the control teeth (sound and untreated ones), no immunostaining was detected.  In the quantification of IL-6 immunostaining, a triple significant interaction between the studied factors was detected in the analysis of variance (F test, p=0.0039); thus, interactions were decomposed.
One should consider that high values represent the low IL-6 expression, whereas low values represent the high IL-6 expression.
When comparing the immunostaining caused by either MTA or FS in the same radicular third and at the same experimental period, greatest IL-6 expression was found in the MTA group, mainly in the cervical third at 24 hours (p=0.0074), in the medium third at 72 hours (p=0.0250), and in the apical third at 72 hours (p=0.0331) ─ assessed by Tukey test. Higher immunostaining was found in the apical third at 24 hours, particularly in teeth from the FS group in comparison with the MTA group (Tukey test, p=0.0091, Table 3).
In the comparison between different periods for the same material and the same radicular third, by Tukey test, a statistically significant difference was found in the cervical third of teeth treated with

Discussion
The initial inflammatory reaction after pulpotomy is not completely elucidated, and it is believed that pulpotomy failures may be related to undiagnosed, subclinical inflamed pulp. 20 Therefore, it is important to conduct analyses that seek to understand the pulp repair mechanism after the application of a capping material since an exacerbated initial inflammatory response may cause deeper tissue necrosis. 17 Considering that inflammation is an early event, we studied the pulp response 24, 48 and 72 hours after the execution of the pulpotomies.
We chose the animal model of the rat mandibular molar free of caries 4,18,21,22 due to ethical aspects, as one cannot extract a human primary tooth right after pulpotomy to evaluate the initial pulp responses to different materials. According to Dammaschke 23 (2010), rat molars are appropriate to test capping materials. Although this model is not completely clinically relevant, it provides a way to study the cellular and molecular mechanisms that occur during dental pulp repair.
MTA was chosen for this study since it is the gold standard capping material for pulpotomy of primary teeth, due to its high clinical, radiographic and histological success rates. 3,24,25 However, important limitations of MTA include: tooth discoloration, high cost, considering the survival time of the primary teeth in the oral cavity 5 and difficulty with handling and insertion due to its grainy consistency and the possibility of it breaking down due to a long setting time and prolonged maturation phase. 26 The FS is a low-cost alternative to MTA and, according to a systematic review, the clinical success rates after pulpotomy with FS ranges from 78 to 100%, while the radiographic ones stem from 42 to 97%. 27 Although clinical and radiographic evaluation of pulpotomized teeth using FS has been reported in several studies 1,12,13,15    treatment. The presence of necrotic tissue suggests that MTA, similarly to calcium hydroxide, initially causes a coagulation necrosis when in contact with the pulp connective tissue due to its high alkalinity. 29 The alkaline pH and the release of calcium ions evoke the inflammatory reaction. However, calcium ions react with carbon dioxide present in the tissue and originate calcite crystals, thus reducing the inflammatory process. Therefore, the use of MTA as a Further studies should be conducted to confirm the histological success of FS after pulpotomy of primary teeth.