The current study exposed cements for two periods of time, 7 days and one month, in order to assess the progress of the hydration process and microstructure of the materials exposed to various media.
To simulate the intracanal coronal barrier used in RET, HCSCs were placed in polythene cylindrical molds and transferred to Eppendorf tubes so that their lower surface was in contact with fresh human whole blood or PRF, which is used as a natural scaffold in regenerative treatments.
Hydraulic calcium silicate-based cements need moisture to set, which they acquire from physiological tissue fluids and blood. The term bioactivity usually refers to the release of OH− and Ca2+ ions, which interact with the mineral components of dentine to form a mineral bond . Calcium hydroxide, as a hydration by-product of HCSCs, combines with phosphate in the environment and forms hydroxyapatite as the key element in inducing hard tissue formation . PBS is a simulated tissue fluid containing phosphate that mimics clinical conditions in laboratory studies and was considered an ageing medium in the control group of the present study [20, 24].
The present results revealed that in contrast to PBS and PRF, blood contamination significantly increased chromatic alteration associated with all materials. This finding is in accordance with the results of studies that reported increased discoloration following blood contamination of HCSCs [20, 21, 25–27].
Fe3+, a dark brown by-product of natural oxidation and reduction of erythrocyte ferrous (Fe2+) may cause chromatic alteration of materials [26, 27]. Also, it has been demonstrated that diffusion of blood components into the porosities of partially hydrated cements may exacerbate chromatic alteration . The partial absence of erythrocytes in platelet derivatives such as PRF might be the cause for less color change compared to whole human blood .
The chromatic alteration of OrthoMTA specimens exposed to blood and PRF was significantly more pronounced than TotalFill and RetroMTA. This finding may be linked to the content of tetracalcium aluminofrrite and bismuth oxide in OrthoMTA, which is not present in other test cements. Presence of metal components such as iron and bismuth oxide is one of the important factors affecting the color of endodontic cements and subsequently tooth crown discoloration. The oxidation of residual iron components, in the set material, relating to the calcium aluminoferrite phase of the powder, has been considered as a possible mechanism for tooth discoloration by tooth-colored MTAs . Moreover, relating to the mechanism of discoloration caused by bismuth oxide, the theory of bismuth oxidation has been proposed. This reaction results in the creation of unstable oxygen, the reaction of oxygen with carbon dioxide, and then the production of bismuth carbonate as a discoloration agent [28, 29]. In this regard, some studies suggest that MTA chromatic alteration may be related to bismuth oxide, which has been added to both white and grey MTA as a radiopacifier . While several studies have shown less tooth discoloration in the cement containing zirconium oxide or tantalum oxide than bismuth oxide containing cements [16, 21, 28]. It should be noted that the final discoloration of the tooth can be due to the discoloration of the cement itself, the interaction of bismuth oxide in some types of cement with dentin, or the penetration of blood and its components into dentinal tubule. Therefore, discoloration caused by blood may mask the effects of cements with or without bismuth oxide in tooth discoloration and prevent an accurate study of the color stability of HCSCs.
In the current study, XRD analysis of the three cements revealed high peaks of calcium hydroxide, after 1-week exposure to PBS, PRF and blood. However, the analysis of OrthoMTA and TotalFill exposed to blood and PRF after 1 month confirmed that the intensity of calcium hydroxide peaks reduced qualitatively. Like the present study, it has been reported that no peaks of calcium hydroxide formed on ERRM cements, which is similar to TotalFill exposed to blood, after 28 days, but contrary to cements in contact with water and HBSS . Regarding the reduction of calcium hydroxide in the specimens of OrthoMTA and TotalFill exposed to PRF and blood for 1-month, it seems that the hydration process of calcium silicate cements arrested, and dissolution of calcium hydroxide commenced. Nekoofar et al.  found no peaks of calcium hydroxide in specimens mixed entirely with blood, which is considered to be due to the inhibition of calcium hydroxide formation and/or its dissolution. In addition, the formation of amorphous calcium silicate hydrate (CSH) following to the hydration of HCSC should be considered, because only crystalline compounds are traceable in the XRD patterns . Also, the lack of this amorphous content as well as absence of ettringite, as hydration reaction indicators, could be due to the short time of incubation and incomplete hydration [19, 31]. On the other hand, the effects of blood and PRF on the amounts of calcium hydroxide at different time intervals, may be related to differences in the chemical composition of HCSCs. In assessing the hydration behavior of bismuth contained HCSCs, bismuth remains an unreacted powder in the hydrated composition of these cements, affecting the MTA hydration mechanism. This element enters the structure of hydrated calcium silicate and forms calcium silicate hydrate-bismuth (CSH-Bi), which can affect the formation and dissolution rate of calcium hydroxide and, consequently, the bioactivity of the hydrated material , thus preventing complete hydration [19, 33].
All cements tested in the study had a different surface microstructure after 1 week and 1 month of exposure to PBS, which may be due to differences in chemical composition. This finding might be related to the bioactivity of HCSCs and the deposition of apatite crystals in the presence of fluid containing phosphate [20, 24]; as seen in the present study where RetroMTA cement was exposed to PBS for one month. Indeed, over time, the size of the precipitated crystals and surface microstructure as well as the chemical composition of the PBS- exposed specimens changed.
Also, all cements exposed to PBS had different surface microstructures compared to cements exposed to blood and PRF, indicating that the cements were affected by blood and PRF. The crystalline microstructure was seen in all cements exposed to PBS while it was not observed in the cements exposed to blood and PRF. This result is in accordance with Nekoofar et al.  who revealed the unfavorable effects of blood contamination on the microstructure and hydration behavior of MTA. Furthermore, the surface microstructure of specimens exposed to PRF and blood for 1 month varied compared to the 1-week samples. This indicates changes in the hydration process, over time, which resulted in different surface microstructures [24, 31].
EDS analysis of the material revealed that the surface precipitations in all groups, mainly contained high peaks of carbon (C), oxygen (O) and calcium (Ca), which reflect the nature of HCSCs, as reported in similar studies [18, 24, 34]. It has also been reported that high peaks of calcium, oxygen, and silicon occur during EDS analysis of pure Portland cement exposed to PBS and PRF , which might be due to the release of hydrated calcium silicate and calcium hydroxide following hydration reaction of the HCSCs . In the present study, the peaks of bismuth (Bi) were observed specifically in OrthoMTA specimens, while the peaks of zirconium (Zr) in RetroMTA, TotalFill, and the peaks of tantalum (Ta) was detected only in TotalFill specimens, which can be justified by the radiopacifier content of these cement [18, 24, 34].
Aluminum (Al) was seen in all specimens, which can play a role in the formation of the ettringite crystalline microstructure . Aluminum contributes to the formation of tetra-calcium aluminoferrite and tricalcium aluminate in the OrthoMTA cement , this may be considered a reason for greater amounts of Al in the EDX analysis. However, Camilleri stated that the presence of aluminum in HCSCs is generally rare .
In the present study, phosphorus was observed in the EDS analysis of all specimens, which could be due to the presence of phosphates in PBS, blood, and PRF. Previous studies have also shown the presence of phosphorus in blood and PRF [18, 20, 27]. Furthermore, TotalFill BC RRM contains phosphorus as monobasic calcium phosphate [18, 24, 34]. High peaks of oxygen can be attributed to the presence of oxygen (O) in the chemical composition of all calcium silicate materials . The presence of carbon (C) may also be related to carbon dioxide and carbon in blood and its derivatives, and subsequently the presence of calcium carbonate deposits, which are formed by the reaction of calcium and carbonate ions in the environment . In addition, carbon is related to use of the carbon grid before SEM-EDS analysis.