Abstract:

Objective ·To explore the effect of all-trans retinoic acid (ATRA) of different concentrations on osteogenic differentiation of jaw bone mesenchymal stem cells (jBMSCs) in rats. Methods ·jBMSCs from 4-week-old Sprague-Dawley (SD) rats were isolated and cultured with whole bone marrow adherence method. The surface antigens were identified by using flow cytometry. Alkaline phosphatase (ALP) staining/alizarin red staining, oil red O staining and alcian blue staining were used to prove the multilineage differentiation potential of jBMSCs after osteogenic, adipogenic and chondrogenic induction respectively. jBMSCs were induced in osteogenic medium with ATRA of concentration of 0.01, 0.1, 1, 5, 10, 20 μmol/L in vitro, and dimethyl sulfoxide (DMSO) was used as control group. Cell viability of jBMSCs in different groups were determined by CCK8. ALP staining and alizarin red staining were used to investigate the osteogenic ability of jBMSCs in each group and screened the concentrations for subsequent experiments. Quantitative real-time polymerase chain reaction (qPCR) and immunofluorescence staining were used to analyze the expressions of osteogenesis-related genes and proteins in jBMSCs of different concentrations. Results ·The flow cytometry analysis showed that more than 98% of P1 jBMSCs were positive for CD29⁺CD90⁺CD31^-CD45^-, which was congruent with the characteristics of bone mesenchymal stem cells. The results of ALP staining/alizarin red staining, oil red O staining and alcian blue staining indicated that the P1 jBMSCs had the multilineage differentiation potential of osteogenesis, adipogenesis and chondrogenesis. The results of ALP staining/alizarin red staining showed that the osteogenic activity and mineralization ability of jBMSCs in 0.01, 0.1 and 1 μmol/L ATRA groups were increased compared with those in the control group, while the osteogenic activity and mineralization ability were decreased when the concentration of ATRA increased, especially higher than 5 μmol/L (all P<0.05). qPCR analysis showed that the mRNA expression levels of osteogenesis-related genes such as Alp, bone sialoprotein (Bsp), collagen type Ⅰ α1 (Col1a1) and osteocalcin (Ocn) were higher in the 0.1 and 1 μmol/L ATRA groups compared to the control group. However, further increasing the concentration of ATRA led to a decrease in gene expression levels, and when the concentration exceeded 5 μmol/L, it began to be lower than the control group level (all P<0.05). The immunofluorescence staining showed that the expression of osteogenic related proteins SP7, ALP and OCN in the 0.1 and 1 μmol/L ATRA groups were increased compared to the control group, while further increasing the concentration of ATRA led to a decrease in protein expression. When the concentration was higher than 5 μmol/L, it began to be lower than the control group level (all P<0.05). Conclusion ·Lower concentrations (0.1, 1 μmol/L) of ATRA can promote the osteogenic differentiation of rat jBMSCs, and the promoting effect reaches its peak at 0.1 μmol/L, while the effect can be weakened by further increasing the concentration. Higher concentrations (5, 10, 20 μmol/L) of ATRA could inhibit the osteogenic differentiation of rat jBMSCs, showing an inhibitory effect. In this study, the dual-directional effect of retinoic acid on osteogenic differentiation of jBMSCs was demonstrated in vitro, and 0.1 μmol/L ATRA was identified as the optimal concentration for osteogenic differentiation of jBMSCs in rats, which provided a reference basis for the development of in vivo studies and clinical application of ATRA.

Key words: all-trans retinoic acid (ATRA), jaw, bone marrow mesenchymal stem cell, osteogenic differentiation, dual-directional effect