## 4 Discussion  
In this study, similarities and differences were found between pPDLSCs and sPDLSCs grown in a 3D culture medium. In scaffold-free 3D media, pPDLSCs and sPDLSCs developed into spheroids. However, after one week, their proliferative ability was significantly decreased when compared to that observed after 24 h of culture. There was no significant difference between the two groups in terms of differentiation into bone tissue. pPDLSCs were more dominant in cartilage, while sPDLSCs were more dominant in fat. According to [PERSON] et al. [26], factors related to the properties of PDLSCs are typically determined by tissue origin, donor age, culture methods, and growth factors. The most commonly used tissue sources for PDLSCs include permanent teeth (3 rd molar, extracted orthodontic tooth) [27], deciduous teeth [28], granulation tissue [29], and ST [30]. Among these teeth, PDLSCs obtained from ST may be useful candidates for use in regenerative medicine for several reasons. First, STs are defined as those in addition to the normal series of deciduous or permanent dentition and are an ethical material for stem cells as they are tissues that must be removed [31]. Second, the recovered state is useful for cryopreservation without contamination, such as dental caries and periodontitis. Third, STs have rich sources of stem cells, such as the dental pulp, periodontal ligament, apical papilla, and dental cysts, enabling their greater utility for biological use and potential therapeutic applications. Figure 4: (**A**) Evaluation of osteogenesis, chondrogenesis and adipogenesis in 3D culture of pPDLSC and sPDLSC. BM: Basal media, OM: Osteogenic media, CM: Chondrogenic media, AM: Adipogenic media. Black scale bar: 200 \(\upmu\)m, Red scale bar: 40 \(\upmu\)m (**B**) No significant difference in osteogenic differentiation between pPDLSCs and sPDLSCs (\(p\) = 0.94). Significant differences in adipogenic (\(p\) = 0.003) and chondrogenic (\(p\) = 0.013) differentiation between pPDLSCs and sPDLSCs. * \(p\) < 0.05; Mann–Whitney U test was performed. Many studies have identified the characteristics of PDLSCs; however, it is important to note that culturing methods also play a crucial role. The culture environment influences the growth rate of cells, their differentiation characteristics, and the direction of cell size [32]. In culture, the important factors include the composition and form of the culture medium, concentration of oxygen [33], and number of cells during culture [34]; however, recent reports have highlighted the critical role of 3D culture [35]. In a 3D culture setting, cells aggregate to form spheroids, which offer advantages in terms of cell-cell interactions, better replication in the in vivo microenvironment, and improved cell signaling pathways [36]. In our study, pPDLSCs and sPDLSCs also formed spheroid-like structures in the initial stage; however, as the number of cells increased, the spheroid shape became more irregular. The shape of 3D cell aggregates can change over time owing to various factors, such as cell proliferation, cell death, mechanical stress, and oxygen and nutrient gradients. As cells grow, the diffusion of oxygen and nutrition are limited, affecting the apoptotic cell cycle. Moreover, degradation products may influence the shape of the spheroids. According to [PERSON] et al. [36], the growing mass increases the mechanical stress, which is also a factor of irregularity. The ULA plates used in this study showed different characteristics from those previously used in the 3D cell culture system, StemFit [37]. In our study, ULA plates served as a versatile tool for 3D cell culture by preventing cell attachment and facilitating spheroid formation. In contrast, StemFit, a tissue culture adhesive primarily designed for stem cell culture, which is currently not widely associated with dental research, could be relevant in dental stem cell applications or tissue engineering.