## 4 Discussion
There were no differences between the 2D and ULA groups, and the ALP activity in the cells that were grown with all types of MTA was comparable to that of the control group. In particular, PRM stimulated the highest levels of ALP in DPSCs, which was consistent with a previous report that found that PRM exhibited a high level of ALP activity [21]. We utilized qualitative alizarin red staining to evaluate the mineralization of the extracellular matrix for the 2D- and ULA-cultured DPSCs (Figure 2). For the 2D groups, the PRM and BIO groups showed a greater increase in staining compared to control group, which differed to the WRP group. PRM and BIO are currently referred to as hydraulic calcium silicate cements (CSCs), and a recently released systematic review reported that the application of these MTA-type CSCs led to no major differences in biocompatibility and odontogenic effect when applied to human pulp-derived cells [22; 23]. These reports were coincident with our study regardless of the culture condition, though our study examined the effect of various CSCs on the two- and three-dimensionally cultured DPSCs. To the best of our knowledge, the three-dimensionally cultured microsphere-forming DPSCs have not been investigated very much, so our findings might provide the potential evidence that supports the utilization of the differently cultured DPSCs in endodontic regenerative research or their future application in clinical procedures. Generally, the alizarin red staining assay is used to quantitatively analyze the amount of odontogenic differentiation. However, due to the different characteristics of the three-dimensionally cultured microspheres of DPSCs, the quantitative comparison between 2D and 3D culture methods could not be conducted. To negotiate the effect of the culture methods and the different type of the MTA materials, RNA- or DNA-level laboratory examination, such as RNA sequencing, is required due to the methodological differences in the process of analysing the three-dimensionally cultured cells. Although our study did not analyze the degree of the quantitative mineralization staining, WRP exhibited much less mineralization staining after undergoing 21 days in the odontogenic differentiation-conditioned medium compared to PRM and BIO. As mentioned previously, WRP is the newly formulated premixed CSC, which is a ready-to-use bioceramic paste. According to the manufacturer's information, the materials are composed of two major groups of chemical components. The first are tricalcium silicate, dicalcium silicate, and zirconium oxide, which are most similar to that of other traditional MTA-type CSCs; and the second are polyethylene glycol, polypropylene glycol, and calcium sulfate dehydrate, which are different from conventional materials. Although our study has not explored the physicochemical and compositional properties of the experimental materials, the premixed-type CSCs might not have the chemical composition necessary to induce the mineralization or odontogenic differentiation. The biomaterials that are used in endodontics make direct contact with pulp and periodontal tissues. Cell attachment to a material surface is a good indication that the material is biocompatible. Furthermore, if the material stimulates cell proliferation or mineralization, then it is likely to promote the healing process. Observing the material surface with SEM (Figure 3) can confirm whether the DPSCs adhere to it. In the PRM and BIO groups, DPSCs adhered to the surface well, whereas in the WRP group, only a cloud of dead cells was observed, and almost no cells with fibrobl morphology were observed. Further examinations, such as RNA extraction and PCR assay and Western blot analysis with the specific markers (DSPP, DMP-1, Runx2, FGF-1 and VEGF), scratch wound healing assay, and RNA sequencing, might provide more detailed scientific evidence, which could contribute to the utilization of the DPSC microspheres in laboratory and clinical settings.