## 4 Discussion  
Although endodontic sealers have a significant impact on endodontic treatment resolution, no extensive comparative studies have been made regarding the cytotoxicity and biocompatibility of the newly developed sealers [20]. The study of cytotoxicity and biocompatibility of the root canal sealers is crucial considering that they are applied in direct contact with periapical tissue [5; 21]. Therefore, the selection of the best sealer to be used in clinical practice--particularly in a minimal invasive endodontic approach since the goal is to preserve more of the natural tooth structure--must be taken into account. The chemical and physical properties, as well as the sealing ability of endodontic sealers, should ensure the effective sealing of the remaining root canal space to prevent reinfections or further damages. Also, their biocompatibility determines the interaction with the surrounding tissues, by releasing antimicrobial substances and tissue regeneration molecules that promote the healing process [5; 7; 8; 21]. In this context, a cell line of immortalized human gingival fibroblast (HGF-1) was considered as a cellular model since fibroblasts are predominant cells in connective tissue, being highly predominant in pediodontal and perimplant connective tissues, responsible for the maintenance and production of the extracellular matrix [22]. Several methods have been used to evaluate the toxicity of root canal sealers in vitro [3; 4]. Two different methods were applied: incubation in real-time (using a cell culture system with inserts) and material-conditioned media. Thereby, we were able to evaluate both the effect of the sealers through incubation in real-time with the cells and the effect of the root canal sealers diffusing substances on the culture medium. Regarding the incubation in real-time exposure, both MTT and SRB assays revealed a cytotoxic effect of AH Plus at 100 mg/mL  
Figure 4: Morphology study after 24 h of HGF-1 fibroblasts incubation with each sealer with material-conditioned media with 100 mg/mL (500\(\times\)). (**A**) Control group: cells with normal extensions and intact membranes; (**B**) AH Plus group: cells with irregular morphology, with compromised membranes and loss of cell content; (**C**) Bio MTA+ group: cells with some morphological alterations namely rounded cells and compromised cell membranes with loss of cell content; (**D**) Bio C group: several morphological changes including rounded cell shape, loss of membrane integrity and leakage of cellular content. after 24 h (\(p\)-value \(<\) 0.001), making this effect even more pronounced after 72 h of incubation. No cytotoxic effects were detected by using Bio MTA+ and Bio C sealer for 24 h, while slight cytotoxicity was observed by testing these compounds for 72 h. Previous studies revealed similar results regarding AH Plus toxicity [3; 11; 23; 24]. This cytotoxicity has been attributed to the release of formaldehyde from amines added to accelerate the polymerization of epoxy resin, as well as to the presence of bisphenol-A (BPA) in its composition that had been identified as a mutagenic component [11; 25; 26]. BPA, commercially used in the manufacture of epoxy resins, was identified as one of the first produced synthetic estrogens, and has endorsed the recent discussion about endocrine-disrupting chemicals regarding their presence and potential leakage from the products whose composition they integrate, as dental sealants. Although the data concerning the effective leakage of BPA from resin-based dental sealants has shown to be contradictory, this possibility must remain a concern [27]. Moreover, other studies also demonstrated that Bio C sealer, along with another bioceramic premixed root canal sealer, showed higher cell viability and biocompatibility than AH Plus [11; 14], also attributed to the calcium ions, present in their composition, elution into the medium [11].