Effects of D-galactose Induction on Aging Characteristics of the Human Dental Pulp Cell Culture Model: An In Vitro Study

Objective: This study aimed to investigate the effects of D-galactose (D-gal) on cellular senescence induction, cell proliferation, mineralization production, and odontogenic gene expression of isolated human dental pulp cells (HDPCs).
Methods: Isolated HDPCs were cultured and assigned to 4 groups: 1) control, 2) 1 g/L D-gal, 3) 10 g/L D-gal, and 4) 10 g/L D-gal with Biodentine (BD). Cell proliferation was evaluated at 24, 48, and 72 hours using Alamar Blue® assay. To evaluate cellular senescence at 48 hours, senescence-associated beta-galactosidase (SA-β-gal) activity and senescence-related genes (p16 and p21) were assessed with SA-β-gal staining assay and quantitative reverse-transcription polymerase chain reaction (qRT-PCR), respectively. To examine the mineralization potential under differentiating conditions, quantitative staining with Alizarin Red S and mineralization-related gene expression (dentin sialophosphoprotein, DSPP) were investigated at 14 days. One-way ANOVA was used for statistical analysis. The statistical significance level was set at 0.05.
Results: One and 10 g/L of D-gal significantly decreased cell proliferation at 72 hours (p<0.05). SA-β-gal-positive cells were significantly observed in cells treated with both concentrations of D-gal (p<0.05). The expressions of genes p16 and p21 were markedly increased in cells treated with 10 g/L D-gal (p<0.05). The addition of BD did not promote cell proliferation but significantly improved cellular senescence by reducing SA-β-gal activity, p16, and p21 expression (p<0.05). For mineralization potential, the amount of mineralization was similar among groups under differentiating conditions. The reduction of DSPP gene expression was obvious only in the 10 g/L D-gal group (p<0.05). The addition of BD did not show a significant effect on mineralization.
Conclusion: Ten g/L of D-gal can effectively induce aging phenotypes and reduce DSPP gene expression in HDPCs. Co-incubation with BD extract reduced the expression of these aging phenotypes. Mineralization production was not altered in the presence of D-gal. The data support the development of in vitro model for aging dental pulp. (EEJ-2024-07-108)