Bioactive glass 45S5 promotes odontogenic differentiation of apical papilla cells through autophagy

The mechanism of the odontogenic differentiation of apical papillary cells (APCs) stimulated by bioactive glass 45S5 is still unclear. This study aims to investigate the effect of autophagy on the odontogenic differentiation of APCs stimulated by bioactive glass 45S5.

APCs were isolated and cultured in vitro, and the cell origin was identified by flow cytometry. The culture medium was prepared with 1 mg/mL 45S5, and its pH and ion concentration were determined. The experiments were divided into control, 45S5, and 3-methyladenine (3-MA) 45S5 groups. In the 45S5 group, APCs were induced to culture with 1 mg/mL 45S5. In the 3-MA 45S5 group, the autophagy inhibitor 3-MA was added to 1 mg/mL 45S5. Protein immunoblotting assay (Western blot) was used to detect the expression of autophagy-associated proteins of microtubule-associated protein 1 light-chain 3β (LC3B) and P62 after 24 h of induction culture in each group. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of bone sialoprotein (BSP), Runt-related transcription factor 2 (Runx2), dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) after 7 d of induction culture. Cellular alkaline phosphatase (ALP) staining analyzed cellular ALP activity at 7 d of induction, and alizarin red staining evaluated the formation of mineralized nodules at 21 d of induction.

The pH of the 45S5 extract culture medium was 8.65±0.01, which was not significantly different from that of the control group (P>0.05). The silicon ion concentration of the 45S5 induction culture medium was (1.56±0.07) mmol/L, which was higher than that of the control group (0.08±0.01) mmol/L (P<0.05). The calcium ion concentration of the 45S5 induction culture was (1.57±0.15) mmol/L, which was not significantly different from that of the control group (P>0.05). Western blot results showed that LC3B-Ⅱ/Ⅰ ratio increased and P62 expression decreased in the 45S5 group compared with those in the control group (P<0.05). By contrast, the ratio decreased and the expression increased in the 3-MA 45S5 group compared with those in the 45S5 group (P<0.05). RT-qPCR results showed that the expression of BSP, Runx2, DMP-1, and DSPP enhanced in the 45S5 group compared with that in the control group (P<0.05), but the expression decreased in the 3-MA 45S5 group compared with that in the 45S5 group (P<0.05). Semi-quantitative analysis of ALP staining and alizarin red staining showed that the ALP activity was enhanced, and the formation mineralized nodule increased in the 45S5 group compared with those in the control group. The ALP activity weakened, and the formation mineralized nodules were reduced in the 3-MA 45S5 group compared with that those in the 45S5 group.

Cell autophagy participates in the odontogenic differentiation of APCs induced by 1 mg/mL 45S5 in vitro.