After root canal preparation, it is important to completely fill the root canal system to ensure clinical success over time. The choice of endodontic sealers depends on their capacity to provide an adequate sealing and stimulate periodontium tissue formation. Thus, the ideal endodontic sealer should exhibit adequate physicochemical and biological properties and should be easy to manipulate. Apexit Plus was launched in an attempt to provide a flawless seal at the apical foramen as well as to allow periodontal tissue repair. In the present study, some physical properties of Apexit Plus were evaluated; we also evaluated whether this endodontic sealer is biocompatible and potentially bioactive.
4.1. Physical Properties
An adequate endodontic sealer must present radiopacity to be distinguished from bone and teeth and to facilitate the evaluation of the root filling’s quality [
28]. Here, the radiopacity of Apexit Plus and Sealapex was greater than 3 mm of aluminum as recommended by ANSI/ADA Specification 57 [
20]. In its composition, Apexit Plus contains bismuth-rich particles as radiopacifier [
29], resulting in a radiopacity medium value above 3.44 mmAl. Similar values were also found by other studies [
6,
29]. However, Sealapex containing lanthanum dioxide (20%) in addition to bismuth trioxide [
30] exhibited a radiopacity value of about 6.82 mm Al. In fact, Sealapex has demonstrated greater radiopacity than Apexit Plus [
6].
The setting time of the sealers evaluated according the ANSI/ADA Specification 57 [
20] revealed that setting time of Apexit Plus was reduced in comparison with that of Sealapex. The greatest difference was observed at initial setting time, since the initial setting time of Apexit Plus occurred after around 5 h, whereas the initial setting time of Sealapex was only observed after 19 h. The final setting of Apexit Plus occurred almost around 18 h and, for Sealapex, it occurred after 21 h. Although there is no definitive standard for the setting time of endodontic sealers, it has been suggested that a prolonged setting time may be responsible for the increase solubility. In fact, Sealapex exhibited greater solubility than Apexit Plus, reinforcing the concept that the extended setting time leads to an increase in solubility. The delay in the setting time of Sealapex has been associated with calcium oxide in its composition; the calcium oxide in contact with water gives rise to calcium hydroxide, which delays the setting time process of the sealer [
6]. Moreover, the high solubility of Sealapex has been attributed to the non-homogeneous setting reaction of this sealer, forming a fragile matrix [
7,
8] with low dimensional stability [
31] due to high water absorption [
32].
According to the ISO 6876/2012 [
19], the endodontic sealers must exhibit the minimum value for flow of 17 mm. Our findings revealed that both sealers showed values for flow within the specification of the ISO 6876/2012 [
19]. However, Apexit Plus had a higher flow value (around 25 mm) than that of Sealapex (around 20 mm). It is important to emphasize that a greater flow rate allows an adequate filling of the root canal; however, an excessive flow is not ideal, as it increases the risk of apical extrusion of the sealer [
33].
4.2. Biological Properties
Our findings showed that Apexit Plus had an effective antibacterial action against
Enterococcus faecalis, a facultative anaerobic Gram-positive coccus responsible for root canal treatment failures [
34]. Here, the findings were obtained using the direct contact test for 60 minutes, a widely used methodology performed to assess the antimicrobial activity of materials [
35,
36]. The antimicrobial properties of calcium hydroxide-based sealers has been related to hydroxyl ion release and, consequently, to an increased alkaline pH in the environment promoted by fresh sealers [
4]. Some studies have reported that, in the first hours, Apexit Plus provides a highly alkaline pH (about 12) to the medium [
12]. Considering that Sealapex initially provides a pH around 7.18 to the medium [
30], it is possible that the high effectiveness of Apexit Plus against
Enterococcus faecalis may be due to the elevated pH initially provided by this endodontic sealer. However, the antimicrobial effectiveness of Apexit Plus may decrease over time, since the pH decreases when the hydroxide calcium-based sealers take hold, culminating in a significant loss of antimicrobial efficacy of these sealers.
Although Apexit Plus recruited a higher number of inflammatory cells when compared with Sealapex, inflammatory reaction decreased over time. It is conceivable to suggest that the high pH of Apexit Plus may induce a more accentuated inflammatory reaction when compared with Sealapex. Moreover, it is possible that irritant substances, such as disalicylate and alkyl ester of phosphoric acid, may be released by Apexit Plus, promoting an inflammatory reaction more accentuated than Sealapex over the short term (7 days). At 7 days, the capsules of Apexit Plus exhibited cells with pyknotic nucleus (i.e., condensed chromatin), indicating that this sealer may initially exert cell damage to the subcutaneous connective tissue. It is important to emphasize that, in the Sealapex specimens, acidophilic areas (typical of tissue degeneration) were also observed. Macrophages were also found in these areas of capsules, indicating that the cellular debris and remnants of extracellular matrix may be internalized by phagocytic cells. However, a reduction in the numerical density of the inflammatory infiltrate was verified in the capsules over time, suggesting that the damage promoted by Apexit Plus is transitional. In fact, differences in the inflammatory infiltrate were not seen between Apexit Plus and Sealapex at 15, 30, and 60 days. From 7 to 15 days, the inflammatory infiltrate reduced significantly in Apexit Plus, whereas in the Sealapex specimens this reduction was not significant. It is possible that the maintenance of the inflammatory reaction in the Sealapex specimens at 15 days occurred due to its high solubility [
8]. The cytotoxicity of Sealapex freshly prepared on MG-63 cells and human gingival fibroblasts is maintained for a longer period than AH Plus and EndoSeal [
37].
Apexit Plus shows low or no cytotoxicity effect on L929 mouse fibroblasts [
10] and human periodontal ligament fibroblasts [
9]. Moreover, no genotoxicity was induced by Apexit Plus on an immortalized cell line derived from human pulp [
38]. Our morphological analysis showed that inflammatory cells were gradually replaced by fibroblasts distributed among bundles of collagen fibers in the capsules of all groups over time. This idea is supported since, from 7 to 60 days, the amount of birefringent collagen increased significantly in the Apexit Plus and Sealapex specimens, similarly to that observed in the control group. Thus, Apexit Plus and Sealapex allowed the replacement of the inflammatory reaction by connective tissue, indicating that these sealers are biocompatible [
25,
26,
39].
Our histochemical findings showed von Kossa-positive structures in the Apexit Plus and Sealapex specimens indicating, therefore, the calcium precipitation in the capsules [
15,
16,
25,
27]. Furthermore, the examination under polarized light revealed birefringent structures in the capsules from unstained sections next to those submitted to the von Kossa method. It is well established that calcium ions bond with the carbon dioxide from tissue fluid, generating calcium carbonate crystals, which exhibit birefringence [
27]. Calcium ions are essential for the formation of hydroxyapatite [
40] and exert an important role in biomineralization [
5]. Moreover, calcium ions are also essential for proliferation and differentiation of mineralized tissue-producing cells [
41]. Here, strong ALP immunolabelling was predominantly observed in the cells close to the von Kossa-positive structures. Since ALP at alkaline pH releases inorganic phosphate [
17,
41], researchers have suggested a pivotal role played by this enzyme in the early stages of hard tissue formation. Considering that Apexit Plus and Sealapex release hydroxyl and provide an alkaline pH to the medium [
11,
31], it is not surprising that these sealers stimulate the ALP expression by cells of the subcutaneous connective tissue, as observed in the present study. This evidence is confirmed by the fact that immunostained cells were not seen in the connective tissue of the control group, except in vascular cells. Endothelial cells can, occasionally, exhibit ALP immunoexpression as reported in the literature [
41,
42]. In the control group, the ALP-immunolabelled cells may have a participation in the regression of the inflammatory infiltrate and tissue remodeling, since alkaline phosphatase secreted by endothelial cells acts as an anti-inflammatory agent stimulating wound healing [
42]. In conclusion, our findings indicate that Apexit Plus is biocompatible, favors calcium precipitation, and may stimulate the osteogenic potential by subcutaneous connective tissue cells in rats.