## 4 Discussion  
In regenerative endodontics, biocompatible materials come into direct contact with blood clips, which act as natural scaffolds that contain DPSCs, and they regenerate dentin-pul complexes through an appropriate biological response. Thus, the requisites for biocompatible materials in regenerative endodontics are that they be non-toxic to dental pulp tissues and capable of inducing dentin repair or regeneration. MTAs are commonly used as biocompatible materials. In this study, DPSCs were exposed to the eluates of MTA cements to simulate the conditions of regenerative endodontics, and cell viability was measured using the CCK-8 assay for the 2D group and the CellTiter-Glo assay for the ULA group with observations of the morphology of the attached DPSCs or DPSC microspheres. Almost all of the groups of cells exhibited normal morphology and structure (Figure 1). In the 2D culture, the DPSCs exhibited a fibrobl morphology, as has been observed in a previous study [12]. In contrast, when DPSCs were cultured in ULA, one large spheroid was observed. The diameter of this spheroid of DPSCs showed a tendency to decrease because the cells clustered together over time. In the IRM group, DPSCs were not attached, but remained suspended when cultured in 2D; when cultured in ULA, they did not aggregate with each other and instead spread apart. This is consistent with the low cell viability observed in the treatment with the IRM eluate. The CCK-8 assay measures the reduction of tetrazolium, and the change from WST-8 to formazan, which occurs through enzyme activity in viable cells and has a specific color [17]. However, in the ULA group, where cells are organized as microspheres, it is difficult to accurately measure the change in color when conducting tetrazolium reduction analysis. This is because the tight cell-cell interactions in compact spheroids hamper the subsequent reduction to formazan, leading to an under-representation of the actual number of healthy cells [18]. CellTiter-Glo is a luminescence assay that determines the number of viable cells in culture by quantifying the total ATP content. In the results of our study, cells treated with IRM eluate exhibited significantly lower absorbance in CCK-8 and CellTiter-Glo assays; the CCK-8 assay showed moderate absorbance values in the presence of all other MTA test groups. This difference was probably a result of the differences in the composition of  
Figure 3: Scanning electron microscopic images of the DPSCs that were on the surface of different MTA materials. the materials. A previous study reported that zinc oxide eugenol, a major component of IRM, has lower biocompatibility than calcium silicate, a major component of MTAs [19]. We performed the cell viability test, taking measurements on days 1, 3 and 7. According to the previous investigation of ours [12] and the preliminary pilot study, day 3 and day 5 showed a similar level of cell viability regardless of the experimental groups. Thus, we decided to investigate the immediate, early (day 1 and 3), and mature (day 7) stages of cell viability without providing the day 5 information. In this study, the odontoblastic differentiation and mineralization effect of the experimental groups were investigated using ALP activity test and qualitative alizarin red staining assay. ALP activity is a biochemical marker for osteoblasts and the formation of new bone. In regenerative endodontic procedures, DPSCs differentiate into osteoblasts and form mineralized tissues. ALP releases free phosphate ions, which react with calcium ions to form a calcium phosphate precipitate, the molecular unit of hydroxyapatite. A previous study reported that ALP exhibited a pronounced expression one week after its induction, and it can be considered to be an early marker of osteo/odontogenic differentiation [20]. In this study, IRM was excluded because it had little cell viability, and basal media and osteogenic media were set as negative and positive control groups, respectively.