According to previous studies, we know that PDLSCs have the capability of self-renewal and can differentiate into various tissue-specific cells, such as osteoblasts, chondrocytes, and adipocytes (25). In recent years, based on the expanding understanding of the regeneration and potential mechanism of PDLSCs, we further found that PDLSCs expressed low immunogenicity and had immunoregulatory capability, and thus transplantation of allogeneic PDLSCs into the host would not result in immune rejection (18). Meanwhile, we have known that age-related loss of beneficial function of PDLSCs may be related to their pluripotency in periodontal tissue engineering (26). Therefore, this study compared and analyzed the age-related changes in the biological and immunological features of PDLSCs and their possible mechanisms.
Previous studies (8, 13) have shown that PDLSCs could be divided into two groups, namely, YPDLSCs and APDLSCs, based on a certain comparative age range. Our data showed that the positive expression rates of STRO-1 and CD146 in PDLSCs decreased with age, which was consistent with the findings of previous studies (13). Earlier investigations have shown that as the surface-specific markers of MSCs, namely, STRO-1 and CD146, can reflect changes in the number of PDLSCs (12). Moreover, STRO-1+ cells in MSCs have significantly enhanced inhibitory effects on lymphocyte proliferation compared with STRO-1− cells, i.e., STRO-1+ cells impart stronger immunoregulatory effects than STRO-1− cells, which supports our observations (27, 28). This suggests that the number and immunoregulatory characteristics of PDLSCs decrease with age, which coincides with the subsequent results of this study. Then, we found that with increasing donor age, the proliferation rate of PDLSCs decreased, whereas the level of apoptosis increased. These results agree with the findings of previous studies (26), indicating that there is a negative correlation between aging and the proliferation activity of PDLSCs. Active proliferation means that YPDLSCs have a greater self-renewal ability than APDLSCs. In addition to the ability of proliferation, we found that compared with YPDLSCs, the osteogenic and adipogenic differentiation potential of APDLSCs were significantly reduced, i.e., PDLSCs with greater osteogenic/adipogenic differentiation potential can be obtained within a short period of time when these are derived from young individuals. Some conflicting reports have been published. For example, Khan et al. (10) found that the adipogenic potential of BMSCs increased with age, whereas Shi et al. (29) reported no change in adipogenic potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs) from individuals of advanced age. These discrepancies may be due to variations in gene expression of different types of MSCs. Taken together, our study demonstrates that aging is associated with the deterioration of biological characteristics of PDLSCs.
In accordance with previous reports (20), YPDLSCs expressed HLA-І but not of HLA-II DR, CD80 and CD86. However, the present study did not observe any statistically significant difference between APDLSCs and YPDLSCs. As the secondary signaling molecules of T cell activation, these proteins are co-stimulatory molecules that play an important role in host immune responses and immunoregulation (30, 31). Therefore, the results of the present study show that both YPDLSCs and APDLSCs express low immunogenicity, i.e., donor age does not affect the immunogenicity of PDLSCs. To test the immunoregulatory functions of PDLSCs, we compared the effects of PDLSCs in different age groups using lymphocyte proliferation and mixed lymphocyte response (MLR) experiments. We conclude that PDLSCs from individuals of different age groups impart strong immunosuppressive effects on the proliferation of allogeneic PBMCs, whether in cell-cell contact culture or Transwell cultures, whereas the immunosuppressive effect of PDLSCs from elderly individuals was weaker than that of PDLSCs from young subjects. These findings agree with the results of previous reports using other kinds of MSCs, i.e., donor age influences immunoregulation in MSCs (14, 32, 33), although MSC inhibition of T cell proliferation is preserved in the elderly (34). Furthermore, we found that the difference in immunosuppressive ability between APDLSCs and YPDLSCs is not related to the apoptosis of PBMCs. These findings are similar to those of previous studies that observed that the anti-proliferative activity of MSCs is due to cell cycle arrest of active T cells at the G0/G1 phase and not by apoptosis (35). Therefore, our study demonstrates that aging can degrade the immunological characteristics of PDLSCs.
Previous studies have found that BMSCs from donors of different ages have significant differences in gene expression levels (36, 37). Hence, to explore the mechanisms that cause differences in the biological and immunological characteristics of PDLSCs from young and elderly individuals, we performed microarray analysis and used qRT-PCR and western blot analyses to verify our microarray results to identify genes that may play a key role in the age-related decline of PDLSCs. The results showed that the expression of genes related to osteogenic/adipogenic and immunosuppressive capacity in PDLSCs was regulated to varying degrees with increasing age. The expression of osteoblastic markers Runx2, ALP, and COL1A1 revealed age-related decline in both gene and protein expression levels, which supported the observed decline in osteogenic differentiation ability of PDLSCs due to aging. In accordance with previous reports, age is associated with changes in the differentiation ability of BMSCs, i.e., they become less bone and more fat, which is driven by the increase in the expression of PPARγ2, a specific transcription factor of adipocytes (38–40). However, in this study, the expression of adipogenic marker PPARγ2 showed age-related decline in both gene and protein expression levels. This may explain the difference in the effect of aging on the adipogenic differentiation between PDLSCs and BMSCs. Furthermore, in terms of immunosuppressive capacity, whether PDLSCs not co-cultured with PBMCs or PDLSCs co-cultured with PBMCs, compared with YPDLSCs, CCND3 and RC3H2 were up-regulated in APDLSCs, whereas CXCL12, FKBP1A, FKBP1B, NCSTN, P2RX7, PPP3CB, RIPK2, SLC11A1, and TP53 were down-regulated in APDLSCs. Among these differentially expressed genes, we found that compared with non-co-cultured PDLSCs, changes in the expression of CCND3 were the most significant in PDLSCs co-cultured with PBMSCs. Therefore, we again assessed CCND3 protein expression levels, which coincided with the gene expression levels. As indicated previously, CCND3 is known to be involved in regulating the cell cycle of activated T lymphocytes (41), and cyclin D3-/- animals cannot normally expand immature T-lymphocytes (42). Furthermore, in addition to its role in cell cycle progression, cyclin D3 imparts significant effects on PPARγ activity and subsequently on adipogenesis (43, 44). Hence, the gene expression patterns described in the current study may provide novel markers for aging in PDLSCs. In the future, we will further explore and clarify the mechanism underlying these differentially expressed genes.
Our comprehensive experimental study has confirmed the relationship between age and the biological and immunological characteristics of PDLSCs, thereby providing some valuable information for basic research and clinical applications. First of all, the proliferation, differentiation, and immunoregulatory ability of PDLSCs from young individuals are better than those of elderly PDLSCs, which means that young PDLSCs should be selected for the treatment of periodontitis or tooth regeneration. Second, published studies have shown that cryopreservation, as a feasible option for the long-term preservation of biomaterials such as stem cells (45, 46) and embryos (47), is an important method for maintaining cell activity and function, providing a pathway for the success of regenerative medicine and tissue engineering. Moreover, our previous studies have verified that cryopreservation does not change the structural integrity and functional viability of PDLSC sheets (48), allowing us to provide cellular products when patients need these. Therefore, we should pay attention to the storage of PDLSCs from young individuals for later use when the donor needs clinical treatment at a later age or for allotransplantation in other elderly people.