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 four groups: control, 1 g/L D-gal, 10 g/L D-gal, and 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 (dentine 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: 1 g/L D-gal and 10 g/L of D-gal significantly decreased cell proliferation at 72 hours compared to the control group (p<0.05). SA-β-gal-positive cells were significantly more prevalent in both D-gal-treated groups than in the control group (p<0.05). The expressions of genes p16 and p21 were markedly increased in cells treated with 10 g/L D-gal compared to the control group (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) compared to the group without BD. 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)