The results of this study indicate that the tested materials discolored tooth structure to various degrees, which was observed by the variation in the ∆Ε values in relation to the different material compositions. GMTA Angelus was intended as the positive control group for the materials tested, however, the difference between ProRoot WMTA and GMTA Angelus was not significant, having the most severe pattern in color change that was caused by both. Although the GMTA formula was modified by reducing the metal oxides such as iron, aluminum, and magnesium oxide which may take part in color change , the resulting formula which is the WMTA, is reported to cause same discoloration effect as the GMTA [8, 23], and as it was found in the present study.
The discoloration induced by MTA has been reported to be caused by bismuth oxide which is added as a radiopacifier. One of the possible mechanism of discoloration caused by bismuth oxide, is that oxidation of bismuth oxide destabilizes the oxygen in its formulation, and reacts with carbon dioxide to produce bismuth carbonate which is light sensitive that results in a black precipitate when exposed to light . Another theory is that bismuth oxide interacted with collagen and converted into a black precipitate .
In the other materials tested, the bismuth oxide was replaced by other constituents to serve as radiopacifier such as; zirconium oxide in Biodentine, tantalum and zirconium oxide in TotalFill, and barium zirconate in TheraCal. Thus, the discoloration in these three material groups was significantly less than WMTA and GMTA whether the materials were tested with saline or with blood. Furthermore, Biodentine in the saline group hardly caused any discoloration almost like the negative control, while the TotalFill and TheraCal have caused significantly more, but when the materials were tested with blood the difference between these three groups were not statistically significant but still significantly less than the MTA groups. These results agree with previous studies where the most color change was related to the MTA materials more than Biodentine or premixed bioceramic (EndoSequence RRM) [24,25,26,27]. Akbulut et al. (1917), reported that blood is the main causative factor in tooth color change rather than the type of calcium silicate cement used, they found no significant difference in tooth discoloration induce by MTA or Biodentine, but after bleaching, the teeth in the Biodentine group were found to be significantly more whitened than those teeth in the MTA groups .
The Biodentine groups also showed color stability over time particularly in the saline group. The color stability of Biodentine has been reported before [6, 29], and discoloration of the teeth with darkening of the coronal structure after one year was more evident for the MTA samples than with the Biodentine . In another study using bovine teeth, Marciano et al.  found that teeth filled with Portland cement that have different radiopacifiers other than bismuth oxide (zirconium oxide and calcium tungstate) have exhibited color stability, and no color change have occurred after zirconium oxide and calcium tungstate were placed in contact with collagen, but, discoloration was observed when the authors placed bismuth oxide in contact with the collagen . Similar findings have been reported more recently demonstrating again that, calcium silicate cement containing bismuth oxide was found to cause the highest color change compared to those with zirconium oxide or calcium tungstate as radiopacifiers when the cements were placed into extracted human premolars in contact with blood .
The discoloration caused by TheraCal and TotalFill was more than that caused by Biodentine in the saline test groups but when blood was added, the Biodentine became very similar to the TotalFill. The reason for this difference in discoloration potential is due to the differences in chemical structure of these three materials, some of which are affected by the blood more than the others. All three materials have zirconium compounds in their composition to act as radiopacifier instead of bismuth oxide, but Biodentine has some elements not present in others which include calcium carbonate, calcium oxide and iron oxide. On the other hand TotalFill differs in the presence of calcium phosphate in its composition, and other elements that are present only in the composition of TheraCal are barium sulfate, fumed silica, Sr glass, and resin. Further evaluations are required to investigate the chemical reactions involved in the color change of these materials.
This study revealed that after contamination of the specimens with blood, overall the estimated marginal means of ∆Ε were increased for the materials particularly; for the Biodentine (2.59) and TheraCal (1.36) and to a lesser extent for the TotalFill (0.79), but very little for the MTA groups (G: 0.44, W: 0.45). Apart from the hemostasis whether if it is fully archived or not, these materials come in contact with vital tissue, in vital pulp therapy procedures or in revascularization when applicable. Some of the components of these materials may have different reactions with blood and vital tissue. Thus, clinical studies should be performed investigating the influence of bleeding time of pulpal tissue and the thickness of remaining coronal tooth structure in tooth discoloration.
In the present study, comparing the ∆E values at 6 months for the experimental materials in both the saline and the blood groups, it could be noticed that the ∆E at 6 months for the groups of materials containing bismuth oxide (GMTA and WMTA) has not increased in greater amounts in the presence of blood relative to the other materials. This could be related to the high discoloration potential and high ∆E values caused by the material itself, irrespective to the presence of blood. While in the other groups of; TotalFill, Biodentine, and TheraCal, where the bismuth oxide were replaced with other radiopacifiers, the increase in the discoloration (∆E at 6 months) when tested with blood was almost double for Biodentine, lesser increase for the TheraCal and the least increase was for TotalFill. This could be related to the fact that both TotalFill and TheraCal are premixed materials with greater homogeneity compared to Biodentine which comes in Powder-liquid form that should be mixed in an amalgamator. This could make the mixture easy to entrap the blood molecules that could penetrate easily within the particles of the material, while this could be less possible in the premixed materials.
Previous studies reported that the porosities in the microstructure of calcium silicate-based materials may uptake blood components and cause discoloration . Also erythrocyte penetration into the tooth structure could intensify the discoloration as well . This was obvious in the positive control group where the pulp chamber cavity was filled with a cotton pellet moistened with 12 μL blood, which showed a very high value of color change (∆E) at 6 months (Fig. 4). It has been reported that sealing the dentinal tubules of the pulp chamber of bovine incisors with a dentin bonding agent, in a simulating regenerative endodontic treatment with the use of antibiotic and blood clot or platelet-rich fibrin, color change of coronal tooth structures filled with MTA or Biodentine was not different at 6 month follow-up .
Therefore, it is better for these materials to be used only after achieving complete hemostasis to decrease the potential of discoloration effect. Furthermore the presence of calcium carbonate in the Biodentine may also make the material more affected by the blood than the other materials tested. This needs more investigation as stated before. Indeed, Palma et al.  found that after 6 months, MTA with saline had higher ∆E value by 5.08 than samples of Biodentine with saline, while the MTA with blood had 3.65 higher ∆E value than the Biodentine with blood, thus the blood had more effect on the Biodentine samples than on the MTA samples as reported in their results.
It could be noticed that overall in both groups (saline and blood) there were no significant differences between the 3 months and 6 months times, thus, the discoloration effect of materials becomes less after 3 months, although this was not that clear for the TheraCal in particular as seen in Figs. 2 and 3.
Overall, and based on the result of this study, the calcium silicate-based materials that were tested in this study have caused tooth discoloration with large differences between them; those with bismuth oxide in their composition (GMTA, and WMTA) have caused the most severe discoloration. Therefore, MTA with bismuth oxides should be avoided in the anterior teeth. Contact with blood has increased the discoloration caused by the materials overall, therefore, in pulp capping procedures, hemostasis should be achieved to reduce the discoloration potential of the calcium silicate materials, even if they do not have bismuth oxide in their compositions.