[The effect of concentrated growth factor on the biological performance of human dental pulp stem cells under oxidative stress status].

Abstract

Objective: To investigate the effect of concentrated growth factor (CGF) on the biological performance of human dental pulp stem cells (hDPSCs) under oxidative stress status induced by hydrogen peroxide (H₂O₂). Methods: The hDPSCs were isolated by using tissue block separation method from healthy permanent teeth extracted for orthodontic reason. hDPSCs surface markers CD34, CD45, CD90 and CD105 were detected by flow cytometry. Alkaline phosphatase (ALP), alizarin red S (ARS), oil red O staining and colony formation assay were used to identify hDPSCs. After the cell counting kit-8 (CCK-8) detection, the optimal H₂O₂ concentration was used to construct the hDPSCs oxidative stress model. CGF conditioned medium was prepared by repeated freeze-thaw methods. After CCK-8 detection, the optimum CGF concentration was chosen for the subsequent experiments. The hDPSCs were divided into control group, H₂O₂ (only H₂O₂ processing), H₂O₂+CGF group (H₂O₂ processing in combination with the CGF) and CGF group (only CGF processing). Subsequent experiments were performed according to these groups. The oxidative stress model was verified by reactive oxygen species, β-galactosidase staining and Western blotting. The effects of CGF on the proliferation and migration of hDPSCs under oxidative stress status were detected by CCK-8 and cell scratch assay, respectively. ALP activity and ARS staining were used to detect the effect of CGF on the osteogenic differentiation of hDPSCs under oxidative stress status. The mRNA expression levels of odontogenesis related genes were detected by real-time fluorescence quantitative PCR (RT-qPCR), and the expression levels of odontogenesis and osteogenesis related proteins were detected by Western blotting. Results: Isolated hDPSCs showed positive expression of mesenchymal stem cells surface markers of CD90, CD105, and negative expression of hematopoietic stem cells surface markers CD34, CD45. The hDPSCs were proved to have the capacity of osteogenic, adipogenic differentiation and clone formation. The optimal concentration to construct the oxidative stress model was 200 μmol/L H₂O₂. Twenty percent CGF was the optimal concentration for subsequent experiments. Compared with the control group, the expression of aging protein p53 was significantly up-regulated from (0.82±0.12) to (1.19±0.14) in H₂O₂ group (P<0.05), with deepened β-galactosidase staining and increased fluorescence intensity of reactive oxygen species. The proliferative capacity of cells in H₂O₂+CGF group on day 1, 3, 5 and 7 (0.23±0.01, 0.50±0.02, 1.60±0.07, 1.80±0.21) were all higher than in H₂O₂ group (0.15±0.01, 0.14±0.02, 0.50±0.03, 0.90±0.09) (P<0.01). Cell healing capacity of cells in H₂O₂+CGF group at 12 h and 24 h (0.47±0.07, 0.58±0.44) also increased compared with the H₂O₂ group (0.36±0.02, 0.44±0.02) (P<0.05), and similar results in the activity of ALP and the formation of mineralized nodules. On day 28, the mRNA expressions of dentin sialophosphoprotein (0.52±0.16) and dental matrix protein 1(DMP-1) (0.39±0.13) in H₂O₂ group were all significantly lower than those in H₂O₂+CGF group (0.96±0.24, 0.83±0.30, respectively) and CGF group (1.12±0.18, 1.23±0.19, respectively) (P<0.05). On day 28, the expressions of odontogenesis related protein DMP-1 (0.27±0.04) and osteogenesis related protein Runt-related transcription factor-2 (0.42±0.15) in H₂O₂ group were all significantly lower than those in H₂O₂+CGF group (0.66±0.18, 0.68±0.04) and CGF group (1.15±0.13, 1.06±0.19, respectively) (P<0.05). Conclusions: H₂O₂ can induce oxidative stress in hDPSCs, while CGF can promote proliferation, migration, odontogenic and osteogenic differentiation of hDPSCs under oxidative stress status.