## 4 Discussion
Therefore, sPDLSCs can differentiate in 2D and 3D cultures, but their osteogenic differentiation is lower than that of cells extracted from permanent teeth. RNA sequencing was conducted to analyze the gene expression profiles of sPDLSCs grown in 2D and 3D cultures. Analyses of gene expression profiles by microarray or PCRare limited by the fact that only selected genes can be identified. Because RNA sequencing captures a wider range of active genes, it is possible to detect small changes in expression with a limited amount of transcript [27]. The RNA sequencing results showed that the expression of genes related to the ECM, angiogenesis, cell proliferation, and the inflammatory response (such as AREG, SMOC1, MMP1, STC1, MMP10, PGF, and PTGS2) was very high. Indeed, 3D-cultured MSCs reportedly exhibit increased expression of genes related to angiogenesis, inflammation, and proliferation, such as TGS-6, IL24, VEGF, FGF-2, CXCR4, MCP3, RANTES, EGF, and SDF [21]. The increased expression of angiogenic genes is confirmed by the observation that 3D-cultured MSCs had greater angiogenic effects than 2D-cultured MSCs, enhancing tissue regeneration [28]. In addition, 3D-cultured MSCs show increased expression of genes related to osteogenesis and adipogenesis, such as RUNX-2, BMP2, OXC, OC, OPN, COLI, OCN, DDPP, DMP1, LPL, FABP4, PLN4, and PPAR [15; 18; 19]. These genes promote adipogenic and osteogenic differentiation by increasing the production of ECM proteins and endogenous growth factors. The initial coalescence of spheroids is caused by caspase-dependent IL-1 autocrine signaling, which upregulates EGR2 [16]. In this study, the 3D-cultured sPDLSCs showed upregulated expression of EGR2 (2.3-fold). In addition, expression of EGR1 and EGR3 increased 5.4- and 9.7-fold, respectively. Similarly, immunomodulators, such as TN-FAIP6 (24.5-fold), are reportedly upregulated in response to IL1 autocrine signaling in 3D spheroids [29; 30]. In the center of spheroids, where the apoptosis zone is formed as a result of nutrient and oxygen limitation, hypoxia- and apoptosis-related genes, such as VEGFA and hypoxia-inducible factor (HIF), are upregulated. In this study, hypoxia-associated genes, such as VEGFA (6.1-fold) and HIF3A (2.0-fold), and apoptosis-related genes, such as IL24 (156-fold) and COMP (SFRP2), were highly upregulated in the spheroids. In addition, the expression of genes related to angiogenesis, such as PGF (341-fold) and PTGS2 (195.6-fold), was highly upregulated. These results are consistent with the observation that over time, the apoptotic zone of the spheroid increased and the surface proliferation zone decreased. This study has several limitations. First, the decreased diameter and reduced proliferative ability of the 3D-cultured sPDLSCs may have been a result of their inherent characteristics or the morphologic characteristics of the spheroid, which hamper the supply of nutrients and oxygen, and the discharge of waste products. Our results are compatible with those of other studies that used 3D pallet cultures, low-adhesive plates, or scaffolds. In this regard, it may be necessary to compare other 3D methods, such as a bioreactor. Second, it may not have been optimal to use assays optimized for a 2D environment to evaluate 3D-cultured cells. For example, the CCK-8 assay involves measuring the color change caused by the dehydrogenation of water-soluble tetrazolium salts by living cells. Therefore, it is not ideal for comparing the absorbance of 2D and 3D cultures quantitatively, because the number of cells contacting the solution differs. It is difficult, or impossible, to find protocols and assays optimized for 3D-cultured sPDLSCs, so further research is needed to establish optimized methods for analyzing 3D-cultured cells. Third, this was a pilot study of 2D- and 3D-cultured sPDLSCs, and so the sample was small. Fourth, several prior studies on spheroid morphology involved observations over only 5-7 days.