## 4 Discussion
Previous ex vivo [11] and clinical [7] studies found greater color stability with Biodentine, which might be explained by the absence of bismuth oxide within its formulation and because of the faster setting time, which possibly limits blood sorption. More recently, two different bismuth-free biomaterials were presented (TotalFill and PCM), with a shorter setting time when compared to MTA, which might be predictive of a favorable result concerning its color stability. While a favorable cytocompatibility profile of both biomaterials was reported in previous studies [28; 29], there is no published data concerning color variation of PCM. TotalFill was the only biomaterial which did not suffer statistically significant differences regarding \(\Delta\)E over time, either in contact with saline solution or blood. Since TotalFill is a pre-mixed material (ready-to-use), not subject to powder/water ratio variations, it appears that this material has less interaction with the environment (saline/blood), thus presenting lower color change in regard to the baseline. Although a previous clinical study [30] reported no differences between MTA and TotalFill regarding consequent tooth discoloration, the use of a double antibiotic intracanal paste which induces discoloration per se, a small sample size and the selected tooth color assessment methods, might explain the disagreement of the obtained results. The importance of in vitro and/or ex vivo studies that allow the evaluation of specific variables and avoid confounding factors is therefore highlighted. Although PCM is also a pre-dosed material (self-mixing syringe), statistically significant differences were verified in the PCM/Blood group over time, which looks to be associated with the different adhesion ability of both TotalFill and PCM to the surrounding dentine. According to the results of the present study, TotalFill and PCM, like Biodentine, do not cause significant color change when compared with MTA. In fact, when individually analyzing the factor material, no statistically significant differences are found between Biodentine, TotalFill, and PCM. However, it is important to underline that, although TotalFill/Saline and TotalFill/Blood groups do not present statistically significant differences in terms of \(\Delta\)E over time, Biodentine presents absolute values of \(\Delta\)E consistently lower than those verified for TotalFill groups in both conditions (saline/blood), which might indicate that Biodentine leads to a lower clinical impact in terms of the perception of chromatic alterations. Moreover, group comparison of material/treatment interaction revealed that PCM contaminated by blood led to a statistically higher color change when compared to both Biodentine/Saline and Biodentine/Blood groups. Thus, considering the results of the present study, Biodentine might be the preferable material to use due to its lower esthetic impact. To summarize, the choice of biomaterial presents a critical factor for the achievement of a successful esthetic outcome. According to our results over a six-month period, Biodentine is the material presenting the lowest discoloration potential, followed by TotalFill and PCM. MTA is the material with lowest color stability. These considerations are verified either in the presence or absence of blood during regenerative procedures. Furthermore, in addition to showing the lowest discoloration potential, it is noteworthy that previous studies demonstrate that Biodentine might allow the execution of bonding procedures directly over it in an immediate timeframe (12 min after biomaterial placement), therefore potentially maximizing the procedure's long-term success [31]. Regarding the limitations of the present study, the absence of positive pulp pressure inherent to an ex vivo model might limit the blood flow towards the periphery. Therefore, in the future, the role played by blood exposure should be confirmed in vivo. Moreover, a longer follow-up period is desirable to confirm the obtained results over a long-term perspective.