Preprints

Background/purpose: The utilisation of human dental pulp stem cells (hDPSCs) as autologous stem cells for tissue repair and regenerative techniques represents a significant area of research globally. The objective of this experiment was to investigate the effect of long-term in vitro culture on the multidifferentiation potential of human dental pulp stem cells and the potential molecular mechanisms involved. Materials and methods: The tissue block method was employed to extract hDPSCs from orthodontic minus extraction patients, which were then expanded and cultured in vitro for 12 generations. Stem cells from passages 3, 6, 9 and 12 were selected. Flow cytometry was employed to detect the expression of stem cell surface markers, while CCK-8 was used to assess cell proliferation ability. β-galactosidase staining was employed to detect Cellular senescence, alizarin red S staining was employed to assess osteogenic potential, while Oil Red O staining was used to evaluate lipogenic capacity. RNA sequencing analysis was conducted to identify differentially expressed genes in DPSCs and to investigate potential mechanisms. Results: With increasing passage number, pulp stem cells showed an increase in senescent cells and a decrease in proliferative capacity and osteogenic-lipogenic multidifferentiation potential. The expression of the stem cell surface markers CD34 and CD45 was stable, whereas the expression of CD73, CD90 and CD105 decreased with increasing passages. According to RNA-seq analysis, The differentially expressed genes CFH, WNT16, HSD17B2, IDI and COL5A3 may be associated with stem cell senescence. Conclusion: An increase in in vitro expansion has been observed to induce a state of cellular senescence in pulp stem cells, which in turn results in a reduction in their proliferative capacity and osteogenic-lipogenic differentiation potential. Differential expression of genes such as CFH, Wnt16, HSD17B2, IDI, and COL5A3 may represent a potential mechanism for the induction of cellular senescence in pulp stem cells.