## 4 Discussion
Also, it was previously suggested that calcium silicate-based sealers, such as Bio C sealer, have more pronounced biological properties than resin-based sealers, such as AH Plus [28]. Although some primary studies have been made to investigate the effect of the mineral trioxide aggregate (MTA) based bioceramic material revealing an expected high bioactivity and biocompatibility [29; 30], as far as we know, no cytotoxicity studies have been performed until now using Bio MTA+. On the other hand, AH Plus did not seem to cause any cytotoxic effect on cells when applied in the material-conditioned medium, while Bio MTA+ and Bio C sealers revealed growing cytotoxicity with increasing compound concentration, reaching a decrease in the metabolic activity of HGF-1 cells of 50% with 100 mg/mL. The material-conditioned medium with Bio C sealer revealed a significantly higher pH than the control medium. This indicates that Bio C sealer has a strong capacity to release hydroxyl ions and the alkaline pH is a possible cause of high cytotoxicity, which could explain the results shown by this material [31]. In addition, previous studies have shown that AH Plus has low solubility while Bio C sealer has high solubility [32]. This high solubility of Bio C sealer may suggest an increased solute degradation in the culture medium, possibly responsible for the cytotoxic effects demonstrated by Bio C sealer conditioned-medium tests. Despite the fact that, as mentioned above, some studies have revealed a cytotoxic effect of AH Plus, some other studies reported similar results to those that we obtained in the material-conditioned medium, where no cytotoxic effect of AH Plus was detected [1; 5; 33; 34]. These discrepant results may be due to the lack of standardized methodologies. Thus, further studies are needed to clarify this effect of AH Plus. In general, it has been reported that there is a possible association between the antimicrobial properties of the endodontic sealers and their cytotoxic effects, regarded as the lack of selective toxicity towards the microorganisms [35; 36]. The antimicrobial properties of the endodontic sealers are an important aspect of the success of the endodontic treatment since an efficient antimicrobial behaviour from the sealers will reduce the residual amount of microorganisms left by the root canal's cleaning procedures. Regarding the sealers' antimicrobial behaviour, an increase in the pH of the environment may be translated into higher antimicrobial efficiency, namely against _Enterococcus faecalis_, the most prevalent bacteria in (re)infected root canals. [37]. The pH increase of the medium during the Bio C sealer incubation will most likely result in an efficient antimicrobial behaviour by this sealer. Previous studies have demonstrated good antimicrobial activity (against both Gram-positive and Gram-negative bacteria) by calcium silicate-based sealers also containing oxide compounds [38]. Additionally, [PERSON] et al. have also demonstrated that the antimicrobial activity of the endodontic sealers is higher for the freshly mixed sealers, and tends to decrease after sealers setting [38]. Related to AH Plus, its antimicrobial activity was previously demonstrated [39], and more recently [PERSON] et al. have shown that although this sealer has a strong antimicrobialactivity when freshly prepared (from the formaldehyde released during the polymerization process), it decreases after time [40], which could be related with the low solubility of this sealer. Although no antimicrobial studies have been made specifically using Bio MTA+ sealer, studies have been made using other Mineral Trioxide Aggregate (MTA) based sealers, demonstrating that the high amounts of oxide compounds, such as iron oxide, aluminum oxide, sodium oxide, and magnesium oxide, are capable of cell wall's damaging [38]. Beyond the study of cytotoxicity, we also performed the analysis of the cell morphology after incubation with the material-conditioned media.