## 4 Discussion  
In the present study, we investigated the temporal changes in phenotypes and the expression of senescence-associated factors in rat DPSCs after 3P to 26P. As is consistent with the general characteristics of cellular senescence, cell shape and nuclear size increased with increasing passage number. Although the staining intensity of \(\gamma\)-H2A.X (indicative of DNA damage in cells) and 4-HNE (indicative of an increase in intracellular ROS) increased with increasing passage numbers, their onset timings were not concurrent. Complete cell growth arrest occurred after 26P. The proportion of SA-\(\beta\)-gal, p21, p16, and uPAR-positive cells also increased with increasing passage numbers, whereas the expression pattern of uPAR differed from that of the other three markers. The expression of SASP factors showed that _Tnf-_\(\alpha\) expression increased significantly earlier than _ll-6_ in replicative senescent DPSCs. CD90 has been established as a marker of DPSCs [14]. In this investigation, the prepared DPSCs at all passages exhibited CD90 expression, although a slight decrease was observed after 26P (Figure 2). Although the precise mechanism underlying this decrease is unknown, similar reductions in CD90 expression have been reported in replicative senescent adipose-derived stromal cells [41], bone-marrow-derived mesenchymal stem cells [8], and rat dedifferentiated fat (DFAT) cells [42]. It is possible that plasma membrane proteins undergo significant alterations as cellular senescence progresses. Indeed, the observed increase in uPAR expression, a surface antigen, supports this hypothesis. Figure 8: Gene expression of senescence-associated genes. * \(p\) < 0.05, ** \(p\) < 0.01, *** \(p\) < 0.001, and *** \(p\) < 0.0001; ns: no significant difference between any groups; one-way ANOVA with Tukey’s test; \(n\) = 3. In this study, DPSC proliferation significantly decreased at 15P, and low levels of cell growth, as indicated by poor Ki67 expression, were identified at 26P. p21 is involved in cell cycle arrest, and it mainly acts on CDK1/2 [43], whereas p16 acts on CDK4/6 [33]. Low p21 levels have limited effects on cell cycle arrest [34]. Indeed, in the present study, cell proliferation continued at 5P when only p21 was strongly expressed; conversely, pronounced growth arrest occurred after 26P when both p21 and p16 were strongly expressed. These results collectively indicate that enhanced p16 expression is the primary cause of cell cycle arrest in DPSCs. Our previous study demonstrated that 90% of rat DFAT cells (multipotent progenitor cells) undergoing replicative senescence at passage 60 exhibited p21 expression, while only 16% of rat DFAT cells exhibited p16 expression [44]. In the present study, only 50% of the DPSCs exhibited p21 expression, while 90% of the DPSCs exhibited p16 at 26P (Figure 7); the expression pattern of SA-\(\beta\)-gal, p21, p16, and uPAR of the DPSCs appears to be inconsistent with rat DFAT cells, even within the same rodent model. Furthermore, although the passage protocol may not be consistent with our protocol, [PERSON] et al. reported that 80% of human bone marrow-derived mesenchymal stem cells approximately at passage ten exhibited SA-\(\beta\)-Gal expression [45], whereas our DPSCs (5P and 15P) exhibited significantly lower positive cells (Figure 7). These findings may suggest that the expression timing and behavior of senescence markers are not consistent across all mesenchymal stem cell/progenitor cells, at least with respect to SA-\(\beta\)-gal, p21, p16, and uPAR. Further investigation is necessary to identify reliable and unified senescence markers for these cells. Our results showed that SA-\(\beta\)-gal and p16 exhibited similar expression patterns, whereas p21 and uPAR had distinct expression patterns (Figure 7). Previous studies have shown that p21, p16, and SA-\(\beta\)-gal have heterogeneous expression [46].