**3. Results**

In the cell viability assay, no significant differences were shown among the ProRoot MTA, Biodentine, and positive control groups during the test period (*p* > 0.05). The Endocem Zr and RetroMTA groups differed significantly from the control group on days 5 and 7 (*p* < 0.05). Out of all groups, the IRM group showed the lowest viable cell level after 24 h (*p* < 0.05) (Figure 1).

**Figure 1.** Relative cell viability based on methyl-thiazoldiphenyl-tetrazolium (MTT) assay. Asterisks represent statistically significant differences between the positive control and experimental groups.

In the cell migration assay, no significant differences were found among the ProRoot MTA, Biodentine, Endocem Zr, and control groups during the test period (*p* > 0.05). The RetroMTA group had lower cell migratory ability than the control group on days 3 and 4 (*p* < 0.05). Cell migration was not shown in the IRM group, and significant differences were observed between the IRM and positive control groups at days 1–4 (*p* < 0.05; Figure 2). Representative images of the cell migration in all groups are shown in Figure 3.

**Figure 2.** Wound healing rate of all tested calcium silicate-based cements. Asterisks represent statistically significant differences between the positive control and experimental groups.

**Figure 3.** Representative images of cell migration based on wound healing assay (scale bar = 250 μm).

In the live/dead staining assay, GDSCs in contact with IRM extract showed low viable cell density, whereas GDSCs in contact with the other experimental cements showed favorable cell growth relative to the control group (Figure 4).

**Figure 4.** Results of live/dead staining assay of all tested calcium silicate-based cements (scale bar = 200 μm).

In the ARS assay, GDSCs exposed to ProRoot MTA, Biodentine, and RetroMTA eluates resulted in a meaningful increase in the formation of calcium (Ca) compared to the Endocem Zr and control groups on day 21 (*p* < 0.05; Figure 5).

In the pH measurement, the pH of all experimental calcium silicate-based cements in deionized water was higher (pH > 10.0) than the pH of deionized water without experimental powder (Table 2).

**Figure 5.** Relative rate of mineralized nodule formation based on Alizarin red staining assay. Different superscript letters indicate statistically significant differences (scale bar = 500 μm).


**Table 2.** The pH of each experimental calcium silicate-based cement.

#### **4. Discussion**

In some clinical conditions such as the repair of root resorption, a fast initial setting time is required to prevent the dissolution of materials into blood and oral fluids. Less tooth discoloration is also an important factor esthetically. Some alternative calcium silicate-based cements, such as Biodentine, Endocem Zr, and RetroMTA, were introduced for these reasons to replace ProRoot MTA. Furthermore, biocompatible and bioactive calcium silicate-based cements can promote rapid healing of adjacent periodontal tissue. Therefore, in the present study we evaluated the cytotoxicity and mineralization potential of four calcium silicate-based cements on GDSCs.

In this study, we analyzed the biocompatibility of ProRoot MTA, Biodentine, Endocem Zr, RetroMTA, and IRM using MTT and wound healing assays. The ProRoot MTA, Biodentine, and control groups showed higher cell viability and migratory ability compared to the IRM group. The Endocem

Zr and RetroMTA groups showed lower cell viability compared to the control group (Figure 1), and the RetroMTA group also had a slower cell migration rate than the control group (Figure 2).

Biodentine is a novel bioceramic calcium silicate-based cement that possesses biocompatible and noncytotoxic properties [22,27]. In one study, the highest migration rate of dental pulp stem cells was found in the Biodentine group, and scanning electron microscopy revealed superior cell adhesion on disks of Biodentine [22]. In another study, cell viability was highest in the Biodentine group followed by the ProRoot MTA group, although viability on the glass ionomer cement (Ketac Molar Aplicap; 3M ESPE, Seefeld, Germany) was significantly lower [27]. Other published studies showed that Biodentine eluates resulted in low-to-moderate negative consequence on cell viability and on cell migratory ability [28]. One possible explanation is that high density level of Biodentine in the growth medium seriously lower stem cell proliferation [29].

Endocem Zr is a pozzolan-based, white calcium silicate cement developed to improve shortcomings such as a long setting time and tooth discoloration [30]. If bismuth oxide, which is used as a radiopacifier in ProRoot MTA, interacts with the collagen fibrils in dentin, it can lead to tooth discoloration. Bismuth oxide is substituted by zirconium oxide in the Endocem Zr formulation [31]. Lee et al. reported that Endocem Zr showed a similar inflammatory response to dental pulp tissue as did ProRoot MTA; however, its formation of a calcific barrier was inferior to that by ProRoot MTA [30].

RetroMTA is another fast-setting calcium silicate cement thanks to its zirconium component, which shortens the setting time by increasing the hydration rate of Portland cement [32]. In a study by Chung et al., RetroMTA showed similar biocompatibility and angiogenic effects on human dental pulp cells as ProRoot MTA; therefore, it is an effective pulp capping material [33]. In comparison, Endocem Zr showed irregular cytotoxic effects and derived less vascular endothelial growth factor and angiogenin expression [33]. In a previous study, both ProRoot MTA and RetroMTA resulted in significantly higher cell viability compared to the positive control, whereas ProRoot MTA had a higher radiopacity than RetroMTA [23]. Another study found that set RetroMTA showed better biological responses compared to a set calcium-enriched mixture (BioniqueDent, Tehran, Iran) and Angelus MTA (Angelus MTA, Londrina, Paraná, Brazil) in a mouse L929 fibroblast cell line [19].

In this study, we evaluated the calcium nodule formation ability associated with ProRoot MTA, Biodentine, Endocem Zr, and RetroMTA using an ARS assay. We found that ProRoot MTA resulted in more mineralization potential than Biodentine and RetroMTA, which is in accordance with a previous study in which ProRoot MTA showed better osteogenic potential than Biodentine based on real-time polymerase chain reaction expression analysis, alkaline phosphatase activity, and calcium nodule formation data [34]. In another study, Biodentine showed significantly decreased alkaline phosphatase activity compared to ProRoot MTA [35]. Its differences in composition and the rate of dissolution in culture medium may be one reason for the lower mineralization activity of Biodentine [34]. Differences in types of cells, culture condition, and time of culturing may have affected the results. In both studies, alveolar bone marrow stem cells rather than dental pulp stem cells were used [34,35].

Meanwhile, a different study showed that Biodentine has a comparable efficacy to ProRoot MTA in the clinical setting and may be considered as an interesting substitute for ProRoot MTA in pulp capping procedures [36]. In that study, well-arranged odontoblast layers and odontoblast-like cells formed tubular dentin under the osteodentin [36]. Furthermore, Wongwatanasanti et al. reported that only Biodentine showed a positive ARS compared to ProRoot MTA and RetroMTA groups [37]. They concluded that Biodentine, ProRoot MTA, and RetroMTA all induce stem cell apical papilla (SCAP) proliferation; however, only Biodentine induces significant SCAP differentiation [37]. Another study also showed that SCAP mineralization was greater in the Biodentine group than the ProRoot MTA group [38]. These studies used SCAP for the ARS assay [37,38], unlike our study, which used GDSCs. The differences in osteogenic gene expression can be explained by differences in cell origin and developmental status at the time of incubation. Therefore, further investigation is required to clarify the different results.

A previous study reported that Endocem MTA and Endocem Zr are related with remarkably less Ca ion release compared to ProRoot MTA [31]. When the three cements were immersed in PBS for 2 weeks, these cements created Ca- and phosphorous (P)-incorporating apatite-like materials. ProRoot MTA showed precipitates which has a higher Ca/P ratio compared to Endocem Zr [31]. Unlike ProRoot MTA, in which calcium and silicon are the predominant compositions, Endocem Zr is largely composed of zirconia with a small quantity of calcium and silicon. In the present study, Endocem Zr showed the lowest calcium nodule formation ability among the experimental calcium silicate-based cements, in accordance with a previous study [30]. Ca ions contribute to the formation and mineralization of hard tissue. Therefore, extended Ca release from calcium silicate-based cements can influence the osteogenic potential of bone marrow stem cells and osteoblast progenitors [39,40]. In the present study, all tested calcium silicate-based cements were associated with an alkaline pH (Table 2), consistent with the findings of previous studies [23,41,42]. The high alkalinity of the materials contributes to their osteogenic potential as a suitable condition for matrix formation and antimicrobial ability is created.

Unfortunately, the reason why various calcium silicate-based cements elicit different biological responses was not thoroughly investigated in this study. Further study on the association between chemical components of the calcium silicate-based cements and biological responses of cells is necessary. Furthermore, proper characterization of each calcium silicate-based cement is required.
