DPSCs exhibit multidirectional differentiation potential, self-renewal replication and immunomodulation, and have promising applications in tissue engineering and cell therapy.[17] DPSCs, as a type of mesenchymal stem cell, have a higher proliferation rate, greater in vivo osteoinductive formation and multidirectional differentiation potential compared to bone marrow mesenchymal stem cells. [18] Furthermore, DPSCs can be obtained from wisdom teeth or teeth that require extraction for orthodontic purposes, which is a straightforward process, less traumatic to patients, and involves fewer ethical considerations.[19] A study by Nela et al. demonstrated that even after thawing after one year of cryopreservation at -80°C, the cell viability, proliferative capacity, and differentiation ability of DPSCs remained unaffected.[20] These advantages have expanded the clinical applications of DPSCs. Studies have demonstrated that DPSCs must be expanded and cultured in vitro to meet the needs of clinical treatment.[21] Nevertheless, prolonged in vitro expansion and culture will result in cellular senescence. The current research on cell senescence resulting from prolonged in vitro expansion and culture primarily focuses on other types of stem cells, with fewer studies on the senescence of DPSCs. Further research is required to elucidate the related aspects of DPSCs and the underlying mechanisms of senescence. This will facilitate the determination of the optimal number of in vitro expansion generations to ensure that the cell activity and number meet the clinical therapeutic application requirements.[21]
In this experiment, we successfully established a senescence model of DPSCs by extracting primary DPSCs and culturing them to 12 generations by in vitro expansion under standard culture conditions. DPSCs cultured to the third generation were selected and flow cytometry was performed to detect the expression of antigenic markers on their surfaces as well as for osteogenic-lipogenic induction. The results demonstrated that the negative markers CD34 and CD45 were expressed at low levels, while the positive markers CD73, CD90 and CD105 were highly expressed. Furthermore, the flow cytometry results were consistent with the surface immunostaining profiles of MSCs, as previously reported in the literature.[22] These findings indicated that the cells extracted exhibited the potential for osteoblastic and lipogenic differentiation, which confirmed that the cells were indeed MSCs. Following a prolonged period of in vitro passaging and expansion culture, it was observed that the proliferative capacity of the cells declined, accompanied by a gradual increase in the proportion of SA-β-gal-positive cells with each additional passage. This is a marker of cellular senescence, which suggests that the DPSCs exhibited a gradual process of cellular senescence with the increase in the number of passages. [23] Although DPSCs continued to exhibit low expression of CD34 and CD45, there was a tendency for CD73, CD90 and CD105 expression to decline with the number of passages, with the most significant decrease observed following in vitro expansion to 12 generations. The results of osteogenic-lipogenic induction experiments demonstrated that the osteogenic-lipogenic differentiation potential of DPSCs was reduced with the increase in the number of in vitro amplifications. These experimental results were consistent with those of previous studies.[24, 25] The above experimental results suggest that DPSCs with reduced polydifferentiation potential after aging may no longer be suitable for clinical treatment.
However, there is a divergence of opinion regarding the changes in stem cell surface markers with passaging. Kim et al. studied 55 generations of bone marrow MSCs cultured in vitro and found that the expression of stem cell surface markers tended to stabilise with increasing number of passages. [26] In contrast, Bakuplu et al.expanded DPSCs in vitro up to 12 generations and found that the expression of CD105, a surface marker of DPSCs, decreased with increasing number of passages, while the expression of CD73 and CD90 remained relatively stable.[27, 28] This is not exactly the same as our experimental results. The experimental results may be affected by various factors, including the source of pulp tissue, the stage of tooth development and the age of the patient. In addition, heterogeneity may increase during the culture process due to differences in culture time, medium used and number of passages. There are fewer studies on the expression of surface markers in DPSCs with different numbers of passages, which remains to be further explored. There are no specific surface markers for DPSCs. [28, 29] The typical markers for MSCs, including CD73, CD90 and CD105, do not appear to provide sufficient or specific indications to maintain the ‘stemness’ of DPSCs. Therefore, additional stem cell markers and other multiparametric immunophenotyping should be employed to validate DPSCs stemness.
The mRNA expression profiles reflect the biological behaviour and function of dental pulp stem cells (DPSCs) cultured in vitro over a long period of time. Changes in these mRNA expression profiles are closely related to functional changes during in vitro ageing. This experimental study identified a series of mRNAs associated with the senescence of DPSCs and their associated molecular mechanisms, which may be potential mediators of changes in biological properties such as reduced proliferation and differentiation after long-term in vitro expansion and culture. A total of 1159 differentially expressed (DE) mRNAs were identified between P12 and P3. Further analysis of these DE mRNAs revealed that changes in CFH, Wnt16, IDI1, COL5A3, and HSD17B2 mRNAs were closely associated with cellular senescence. CFH is a complement inhibitor that plays a key role in complement homeostasis.[30] The sequencing results demonstrated that the CFH gene exhibited elevated expression in senescent DPSCs subjected to long-term expansion and in vitro culture. Furthermore, mutations and variants of the CFH gene have been demonstrated to be significantly associated with a number of human age-related diseases, including cancer and age-related macular degeneration.[30–32] Wnt16, a member of the Wnt family, has been shown to be closely associated with osteogenic differentiation, cellular senescence and tumourigenesis.[33–35] Additionally, it has been implicated in the proliferation and differentiation of stem cells.[36] The results of RNA-seq sequencing demonstrated that: Wnt16 gene expression was observed to be upregulated, while the results of the osteogenic induction assay indicated that the osteogenic capacity of stem cells exhibited a gradual decline with increasing passages. This suggests that the elevated expression of the Wnt16 gene may potentially inhibit osteogenic differentiation. Jiang et al.demonstrated that the Wnt/β-catenin protein signalling pathway inhibits the osteogenic differentiation of human mesenchymal stem cells and MC3T3-E1 cells (mouse embryonic osteoblasts).[37–39] This assertion is corroborated by the findings of our own experiments. However, Carolyn et al.have proposed that the Wnt16 gene stimulates the osteogenic differentiation of perivascular stem cells (PSC).[40] These disparate findings indicate that the Wnt signalling pathway plays a pivotal role in the regulatory network of endosteal homeostasis. Both overactivation and inactivation of Wnt signalling can result in skeletal deformities, bone diseases and cartilage loss.[41] The number of studies investigating the Wnt16 gene and osteogenic differentiation of stem cells is limited, and the role of this gene in osteogenesis in DPSCs cells remains to be further elucidated. Isopentenyl diphosphate isomerase 1 (IDI1) is an enzyme that encodes a peroxisomal localization, which removes toxic hydrogen peroxide produced by different oxidative enzymes in the peroxisomal respiratory pathway. [42] It is involved in processes such as cell division and proliferation and is associated with age-related diseases.[43] The experimental results demonstrated that the expression of IDI1 was reduced in senescent DPSCs. This reduction in the synthesis of peroxisome-localised enzymes and the catabolism of hydrogen peroxide leads to an exacerbation of cellular senescence. The collagen type V alpha 3 chain (COL5A3) is a member of the collagen family that is closely associated with osteogenesis and tumourigenesis. The experimental results indicate that cellular senescence is associated with a reduction in COL5A3 expression. Pavitra K et al.demonstrated that hypoxia stimulates the expression of COL1A1, COL5A1 and COL5A3 in osteoblasts, which plays a role in maintaining bone volume by promoting collagen production.[44] Chen et al.observed that collagen inhibits immune signalling in the tumour microenvironment (chemokines) production, thereby inhibiting anti-tumour immune responses.[45] The loss of collagen results in an increase in chemokine levels, which in turn facilitates the proliferation of cancer cells. 17-β-hydroxysteroid dehydrogenase type 2 (HSD17B2) is a protein associated with estrogen synthesis and regulates estradiol (E2).[46] The experimental results indicated that HSD17B2 expression was upregulated, suggesting that it may be associated with cellular senescence. Lu et al.demonstrated that the inhibition of HSD17B2 activity suppressed E2 inactivation, increased endogenous estrogen levels, and improved bone metabolism-related indicators.[46] In conclusion, these DE mRNA changes are closely associated with cellular senescence, disease development, and osteogenic differentiation, among other factors. One limitation of this experiment is that we did not perform gene knockdown. To study the effect of gene knockdown on dental pulp stem cell senescence, further studies are needed in subsequent experiments.
KEGG analysis has identified numerous signalling pathways associated with human diseases, including viral myocarditis, pertussis, coronavirus disease (COVID-19) and basal cell carcinoma. These pathways play a pivotal role in the functional changes induced by ageing, thereby underscoring the significance of DPSCs ageing in the pathogenesis and clinical treatment of cancer, cancer and metabolic diseases.
Among the interactions between DE-encoded proteins, BMP4, ISG20, BST2 and Wnt5a were identified as key genes interacting with many other DE mRNAs in this network.BMP4, a member of the TGF-β superfamily, is involved in a variety of biologically regulated processes such as cell proliferation and cellular differentiation.[47] BMP4 plays a key role in bone formation, and the gene may promote alveolar bone development by increasing the expression levels of Runx2, BSP and OCN.[48] ISG20 has been associated with certain RNA virus-induced diseases, and a study of ISG20 elucidated its involvement in antiviral mechanisms, which found that overexpression of recombinant ISG20 in cultured cells increased cellular resistance to infection with certain RNA genomes of viruses.[49, 50] BST2, also known as CD317, is involved in a variety of physiological and pathological processes, including inflammation, immune regulation and tumourigenesis, and BST2 is overexpressed in various malignant tumours, suggesting that BST2 may be associated with certain tumour disorders induced by cellular senescence.[36, 51–53] Wnt5a is a representative Wnt protein of the non-classical Wnt signalling pathway, which plays an important role in the development and maturation of various tissues and organs, and is closely related to a variety of diseases such as infectious diseases, cancer and metabolic disorders.[54] Studies on Wnt5a and related aspects of its signalling pathway may provide new ideas for the diagnosis and treatment of human diseases.[55] In summary, these proteins are closely related to the biological changes of DPSCs after long-term in vitro amplification and culture. However, whether these proteins can be used as markers for clinical detection remains to be further investigated.
In addition, DPSCs are derived from neural crest cells and have unique advantages in nerve repair, cartilage formation and corneal reconstruction.[56–59] Furthermore, in addition to its use in repairing teeth and maxillofacial bone tissue, it can also be used to repair tissues outside the oral cavity, such as nerves, cartilage and other systems. Dental pulp stem cells are also receiving increasing attention in the field of regenerative medicine. Autologous stem cell therapy for diabetes and myocardial infarction have likewise shown promising applications.[60.61]
Dental pulp stem cells extracted by the pulp tissue block method exhibited low CD34 and CD45 expression, high CD73, CD90 and CD105 expression, and demonstrated osteogenic and lipogenic differentiation potential. The number of in vitro expansions was found to induce cellular senescence of pulp stem cells, which resulted in a reduction in proliferative capacity and osteogenic and lipogenic differentiation potential. It is possible that changes in mRNA induced by prolonged expansion in vitro may be a potential mechanism for the senescence of dental pulp stem cells. The differential expression of genes, including CFH, WNT16, HSD17B2, IDI and COL5A3, may be of significant importance in the context of pulp stem cell senescence.It is crucial to explore the molecular mechanism behind the senescence of DPSCs in order to establish a more robust theoretical basis for the clinical application of DPSCs.