# Comparison of 2- and 3-Dimensional Cultured Periodontal Ligament Stem Cells; a Pilot Study
## 4 Discussion
In addition, studies about the differentiation potential of stem cells have shown that the intercellular interactions in embryonic stem cells are better expressed in a 3D environment [14; 34; 41]. For example, salivary-gland-derived progenitor cells can differentiate into hepatocytic and pancreatic islet cell lineages only when cultured in a 3D culture environment [42]. For this reason, 3D culture has attracted attention in stem cell biology and oncology, and in 3D-culture studies on dental stem cells [8; 14]. For example, 3D-cultured dental pulp stem cells or gingiva/papilla-derived stem cells show higher ALP activity than those grown in 2D culture [26; 27; 28]. In addition, the expression of genes associated with bone formation, such as BMP2, RUNX2, and dentine sialophosphoprotein (DSPP), are also highly expressed in these 3D-cultured cells [21; 32]. In this experiment, both 2D- and 3D-cultured PDLSCs showed osteo-differentiation. However, compared to the control group, ARS staining showed that the 3D-cultured PDLSCs had significantly higher absorbance than the 2D-cultured PDLSCs (20.8 and 1.6 times higher, respectively). This suggests that more mineralized tissue is produced by the 3D-cultured PDLSCs compared to their 2D counterparts [21]. RNA sequencing was used to analyze the gene expression profiles of PDSLCs grown in 2D and 3D culture environments. In previous studies, such gene expression profiles have been analyzed via a microarray or PCR, but both of these methods are limited by the fact that only selected genes can be identified. Since RNA sequencing can capture a wider range of active genes, it is possible to detect small changes in expression with a limited amount of transcripts [43]. In this study, RNA sequencing results showed that the expression of genes related to the ECM, angiogenesis, and inflammation, such as MMP1, SMOC1, MMP10, CXCL8, CXCL1, and EREG, was very high. Previously, 3D-cultured MSCs have been shown to display increased expression of genes related to angiogenesis, inflammation, and proliferation, such as TGS-6, IL24, VEGF, FGF-2, CXCR4, MCP3, RANTES, EGF, and SDF [36]. The increased expression of angiogenic genes is evidenced by the observation that 3D-cultured MSCs demonstrate more potent angiogenic effects than 2D-cultured MSCs, which allows them to restore more tissue than their 2D counterparts [44]. In addition, 3D-cultured MSCs show increased expression of osteogenic- and adipogenic-related genes, such as RUNX-2, OXC, OC, OPN, COLI, OCN, DDPP, DMP1, LPL, FABP4, PLN4, and PPAR [21; 32; 33; 34; 45]. These genes promote adipogenic and osteogenic differentiation by increasing the production of ECM proteins and endogenous growth factors [36]. The initial coalescence of the spheroids is caused by caspase-dependent IL1 autocrine signaling, which upregulates EGR2 [22]. It is noteworthy that this study did not show any significant difference in the expression of EGR2, however, the expression of EGR1 and EGR3 were increased 4.90 and 5.57 fold, respectively. Similarly, immunomodulatory mediators such as IL6 (2.07 fold) and TNFAIP6 (65.87 fold) and chemokine receptors such as CXCR (3.96 fold) were upregulated in response to IL1 autocrine signaling in the 3D spheroids [46; 47]. A previous study found that in the center of the spheroids, where the apoptosis zone is formed due to the restriction of nutrients and oxygen, hypoxia- and apoptosis-related genes such as VEGFA and hypoxia inducible factor (HIF) are also upregulated [14]. In this study, hypoxia-associated genes such as HIF1A and HIF3A and apoptosis-related genes such as IER3, COMP, and S100A8 were highly upregulated in the spheroids, while the expression of genes related to cell differentiation and proliferation such as RSPO2, DCLK1, and CXCL14 were highly downregulated. These results are consistent with the observation that over time, the apoptotic zone of the spheroid increases and the surface proliferation zone decreases. This study had several limitations.