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This study aims to compare the biological characteristics of human dental pulp stem cells (hDPSCs) isolated from different-aged populations and examine the effects of Biodentine on proliferation and odonto/osteogenic differentiation of hDPSCs isolated from the elderly in vitro. hDPSCs were isolated from three different-aged populations: group A (≤18 years old), group B (19–59 years old), and group C (≥60 years old). The adhesion, proliferation, odonto/osteogenesis, and senescence were compared. The optimal concentration of aqueous Biodentine extract was determined by CCK-8 assay, alkaline phosphatase (ALP), and alizarin red staining (ARS). The effect of Biodentine on odonto/osteogenic gene and protein expression of hDPSCs in each group was evaluated by quantitative real-time PCR (QRT-PCR) and Western blot. hDPSCs were successfully isolated from three different-aged populations. Flow cytometry revealed that all isolated hDPSCs were positive for CD73 (>90%), CD90 (>90%), CD146 (<30%), and negative for CD45 (<1%). There existed an age-related decline in proliferation, odonto/osteogenic gene expression, and S-phase fraction (p < 0.05), an increase in senescence genes and p21 and p16 expression, and time needed for cell adhesion. Biodentine promoted hDPSC proliferation and mineralization in each group, particularly at a concentration of 0.2 mg/mL. Biodentine markedly enhanced odonto/osteogenesis-related gene and protein expression in each group (p < 0.05). hDPSCs can be obtained from populations of all ages. Though there is an age-related decline in their biological properties, hDPSCs from the elderly still maintain certain proliferation and multidirectional differentiation abilities. Biodentine can significantly promote the proliferation and odonto/osteogenic differentiation of hDPSCs isolated from the elderly over 60 years old, which could be considered a pulp capping material for vital pulp therapy in the elderly. Nevertheless, the efficacy of Biodentine in clinical application has to be further studied. [ABSTRACT FROM AUTHOR]