To establish an acidic environment, this study used butyric acid, which is one of the by-products of the metabolism of anaerobic bacteria, the dominant bacteria in endodontic infections. Therefore, to simulate infectious conditions in laboratory studies, the use of butyric acid has been recommended [12, 18, 19]. PBS is a simulated tissue fluid containing phosphate that can be used to mimic normal in vivo conditions in laboratory studies [20].
Among the three materials used in the current study, Biodentine had the largest significant difference in volume ratio based on the setting condition. Therefore, this material is most affected by the acidic environment during setting process. The relatively rapid setting time of Biodentine (9–12 min) is attributed to calcium chloride, the accelerator in the liquid [21]. Initial contraction occurs at the beginning of setting. However, delayed expansion occurs after. The set material releases calcium ions into the aqueous solution. Setting continues for at least 14 days with the exchange of ions in the surrounding environment [22, 23]. In this study, the materials were immersed in acidic solution (butyric acid, pH = 5.4) for 5 days. The acidic environment might have influenced the initial contraction or affected the setting process with ion exchange in the surrounding environment, which lasted for 14 days. Aksel et al. have reported that the different effects of acidic and neutral pH levels on the properties of the materials might be correlated to the inhibition of the setting reaction [15], which may lead to the fast dissolution of the materials in an acidic environment. In relation to this reason, the solubility of the material may impair the dimensional expansion by preventing the accumulation of hydroxyapatite on the material surface [24]. Similarly, Grench et al. have reported that Biodentine has a higher wash-out tendency, with the loss of materials upon contact with blood and other fluids [9]. Moreover, Agrafioti et al. have shown that ProRoot MTA had hexagonal crystal in scanning electron microscope (SEM) after it was immersed in citric acid for 3 months, whereas Biodentine had smooth spheroidal crystal [25]. They have concluded that these structural changes in hydroxyapatite in an acidic condition may affect the solubility and porosity formation of Biodentine. In addition, Namazikhah et al. have reported that when the environment is more acidic, the setting MTA was more porous [14]. Considering the result of this study, it should be considered clinically that the volume ratio of the materials immersed in acidic solution was significantly lower than that immersed in normal saline, and it was most remarkable in Biodentine.
When comparing the Vf (%) values of the three materials, all materials had similar volume values. By conducting experiments on in-vitro settings, the handling of related variables was reduced. During the experiment, Biodentine had a similar operability to that of packable composite resin, and it was easy to pack. Endocem MTA is less viscous and easier to pack, and the texture is extremely fine and has a mud-like consistency [11]. ProRoot MTA had high adhesion as it adhered well to the instrument, and it came out even after it was packed into the cavity. Thus, the material will be more difficult to operate than the other two materials in high-level procedures, including periapical surgery.
Kim et al. have reported that ProRoot MTA had a higher gap formation than Endocem MTA when it was used as a retrofilling material in vitro [11]. Moreover, the superior consistency of Endocem MTA established a lower gap formation than ProRoot MTA. Choi et al. have found that Endocem MTA set significantly faster and was more resistant to washout than ProRoot MTA [7]. In our study, Endocem MTA had a higher Vf ratio than ProRoot MTA. However, the difference was not statistically significant. Although all the materials formed 3-mm cavities and operated according to the manufacturer’s instructions in vitro, the results of previous studies were different from ours. The difference may be attributed to the technique sensitivity during MTA manipulation. Thus, differences can be observed based on who conducted the experiments.
The images of all the setting materials in acidic conditions had a noticeable radiolucency in the area in contact with the acidic solution (Fig. 2). When calcium silicate cements set, they undergo ion exchange with the environment. Tian et al. have reported that exposure to an acidic environment enhanced the release of Si and Ca ions from ProRoot MTA and reduced the apatite formation capacities of this material [17]. Moreover, they used butyric acid with pH 5.4 in this study. Loranzo et al. have reported that Endocem MTA had increased calcium ion release after it was immersed in acid solution for 7 days [26]. Moreover, they have found prismatic crystalline structures on the surfaces of Endocem MTA after it was immersed in acidic solution. These findings are consistent with those of previous studies that reported about the presence of cubic, prismatic, and needle-like crystalline structures in bioactive cements exposed to blood, PBS, and butyric acid, respectively [16, 27, 28]. Thus, during these radiolucent phases, it is supposed that a higher ionic releases might occur in calcium silicate cements when set in an acidic environment than in saline, and these ions may contain radiopaque ions.
This experiment had some limitations. First, the size of the cavities formed were not exactly similar. However, the difference was not significant. We attempted to simulate a clinical situation using real human teeth. However, there was a difference in size, and when the cavity was larger, it was easier to fill the materials. Second, some surface areas where the radiolucency was observed were not assessed. Thus, further studies that analyze the surface areas using EDX, SEM, or micro-hardness test for the evaluation of actual leakage must be conducted.
