## 4 Discussion
This is because p21 is regulated by p53 transcription factors [43], whereas p16 is regulated by transcription factors such as Est1 and Et2 [47], indicating that p21 and p16 are activated through different mechanisms. Cells exhibiting promoter activation abilities for SA-\(\beta\)-gal and p16 INK4a showed increased SA-\(\beta\)-gal activity [48], whereas other studies using electron radiation (not replicative senescence) reported that the expression of SA-\(\beta\)-gal and p16 was not correlated in cells such as aging fibroblasts [49]. Furthermore, in chondrocytes, SA-\(\beta\)-gal exhibits different expression of uPAR, a novel senescence-related marker [50]. Thus, we sought to confirm the senescent markers after different passages in the DPSCs. Considering that more than 90% of the cells were positive for SA-\(\beta\)-gal and p16 after 26P in this study, it is probable that uPAR is expressed in most cells concurrently with p16 or SA-\(\beta\)-gal. Conversely, 20% of the cells expressed uPAR after 3P, whereas p21, p16, and SA-\(\beta\)-gal positive cells were undetectable. Although uPAR is considered a promising new surface antigen for senescent cells, these results indicate that the relationship between uPAR expression and senescent DPSCs may require careful interpretation. The contemporary theory of cellular senescence is recognized as dynamic and multi-step. The secretion patterns of SASP factors vary according to the stage [27; 51] and type of cellular senescence (replicative senescence vs. SIPS) [52]. [PERSON] et al. reported that various SASP factors, including cytokines, alter the initiation of their expression along the early and full senescence [27]. IFN-\(\gamma\) is predominantly secreted by T cells and natural killer cells (NK cells) rather than MSCs or multipotent progenitor cells, while we have demonstrated that DFAT cells after 60 passages exhibited increased IFN-\(\gamma\) expression compared to cells at passage 3 [42]. In the present study, after 26 passages, cells express _Tnf-\(\alpha\)_ and _Il-6_, but there is no significant enhancement of _Ift-\(\gamma\)_ expression. Although there is the difference between DFAT cells and DPSCs, these findings may suggest that the prepared cells from 3 to 26 passages have undergone pre-, early-, and full (mid)-states of senescence and have not progressed to the late (deep) state of senescence. The number of 4-HNE-positive cells increased with increasing passage numbers in this study (Figure 5). Previous research has demonstrated that long-term culture induces an increase in ROS production within human umbilical cord MSCs [53]; ROS is a significant factor that accelerates senescence [40]. More recently, _Cmpk2_ gene expression has garneredconsiderable attention in stress-induced premature senescence (SIPS); senescent cells induced by glucocorticoids exhibit strong _Cmpk2_ expression and increased ROS activity [54]. Moreover, _Cmpk2_ expression is known to increase through stimulation via lipopolysaccharide-charide (for macrophages) and damage-associated molecular patterns via the toll-like receptor [55; 56]. Meanwhile, in our study, negligible differences in _Cmpk2_ expression were observed between 26P cells and other passaged cells. Unlike SIPS, replicative senescence may have a minimal effect on increasing _Cmpk2_ expression and subsequent ROS production in DPSCs. Nevertheless, it would be premature to conclude that _Cmpk2_ expression has no relevance to the replicative senescence of rat DPSCs. The DPSCs prepared in the present study are likely to be early senescent cells. Late (deep) senescence cells may exhibit different _Cmpk2_ expression patterns. Further rigorous investigation is necessary to elucidate the relationship between replicative senescence in rat DPSCs and _Cmpk2_ expression. In the present study, _Tnf-\(\alpha\)_ and _Il-6_ (SASP factors) expression significantly increased at 15P and 26P. [PERSON] et al. reported that TNF-\(\alpha\) stimulation induces premature senescence of rat nucleus pulposus cells [57].