Static Magnetic Field Promotes Proliferation, Migration, Differentiation, and AKT Activation of Periodontal Ligament Stem Cells

Abstract

Periodontal ligament stem cells (PDLSCs) possess self-renewal and multilineage differentiation potential and exhibit great potential for the treatment of bone tissue defects caused by inflammation. Previous studies have indicated that static magnetic field (SMF) can enhance the proliferation and differentiation of mesenchymal stem cells (MSCs). SMF has been widely used to repair bone defects and for orthodontic and implantation treatment. In this study, we revealed that a 320 mT SMF upregulates the protein expression levels of cytokines such as MCM7 and PCNA in proliferating PDLSCs. Cell counting kit-8 results revealed that the SMF group had higher optical density values than the control group. The ratio of cells in the S phase to those in the G2/M phase was significantly increased after exposure to a 320 mT SMF. In scratch assays, the SMF-treated PDLSCs exhibited a higher migration rate than the sham-exposed group after 24 h of culture, indicating that the SMF promoted the migratory ability of PDLSCs. The activity level of the early differentiation marker alkaline phosphatase and the late marker matrix mineralization, as well as osteoblast-specific gene and protein expression, were enhanced in PDLSCs exposed to the SMF. Furthermore, AKT signaling pathway was activated by SMF. Our data demonstrated that the potential mechanism of action of SMF may enhance PDLSCs proliferation and osteogenic differentiation by activating the phosphorylated AKT pathway. The elucidation of this molecular mechanism may lead to a better understanding of bone repair responses and aid in improved stem cell-mediated regeneration.

Keywords: Migration; Osteogenic differentiation; Proliferation; Protein kinase B; Static magnetic fields.

Fig. 1

SMF promoted the cell proliferation of PDLSCs. a SMF exposure system. b The protein expression levels of PCNA and MCM7 were detected by Western blot after 24 h with or without SMF exposure. c Proliferation of PDLSCs was investigated via CCK-8 on days 1, 3, 5, and 7 with or without SMF. d The cell cycle of PDLSCs was investigated by flow cytometry analysis after 24 h with or without SMF exposure. The expression levels were normalized to the level of β-actin. All experiments were performed in triplicate, and the values are expressed as the mean ± SD. *p < 0.05.

Fig. 2

SMF promoted the migratory ability of PDLSCs. a The sham-exposed cells (1) and SMF-exposed cells (2) were scratched with a 200 μL pipette tip at 0 h. The sham-exposed cells (3) and SMF-treated cells (4) at the edge of the scratch wound started to migrate 12 h after the scratch wound was inflicted. 5, 6 A greater number of leading cells were noted at the scratch edge of the SMF-treated group than of the sham-exposed group after 24 h. b SMF-treated PDLSCs exhibited a higher migration rate than the sham-exposed group after 24 h. Scale bar, 200 μm. *p < 0.05, **p < 0.01.

Fig. 3

SMF promoted the osteogenic differentiation of PDLSCs. a Differentiation was assessed based on ALP staining after cells were cultured for 3 days and ARS after cells were cultured for 14 days (b). All experiments were performed in triplicate, and the values are expressed as the mean ± SD. Scale bars, 100 μm.

Fig. 4

SMF promoted the osteogenic differentiation of PDLSCs. a mRNA expression of ALP, BSP, and RUNX2 was detected by qRT–PCR after cells were cultured for 7 days. b, c The protein expression levels of ALP, BSP, and RUNX2 were detected by Western blot after cells were cultured for 7 and 14 days. All experiments were performed in triplicate, and the values are expressed as the mean ± SD. *p < 0.05, **p < 0.01.

Fig. 5

SMF induced activation of the AKT signaling pathway. a The protein expression level of AKT was determined by Western blot after cells were cultured for 24 h with or without SMF exposure. b The protein expression level of AKT was determined by Western blot after cells were cultured for 14 days with or without SMF exposure. All experiments were performed in triplicate, and the values are expressed as the mean ± SD. *p < 0.05.

Fig. 6

The proliferation of PDLSCs was decreased by an AKT inhibitor. Cells were pretreated for 2 h with LY294002 (20 μM) and then cultured with 320 mT SMF for 24 h. a The suppression efficiency of LY294002 was detected via Western blot. b The protein expression levels of PCNA and MCM7 were detected by Western blot. c Proliferation of PDLSCs with or without SMF exposure on days 1, 3, 5, and 7 was investigated via CCK-8 assay. All experiments were performed in triplicate, and the values are expressed as the mean ± SD. *p < 0.05.

Fig. 7

The proliferation of PDLSCs was decreased by an AKT inhibitor as measured by cell cycle assay. Cells were pretreated for 2 h with LY294002 (20 μM) and then cultured with 320 mT SMF for 24 h. a, b The cell cycle of PDLSCs was investigated by flow cytometry analysis after 24 h with or without SMF exposure. All experiments were performed in triplicate, and the values are expressed as the mean ± SD.