Electromagnetic fields and nanomagnetic particles increase the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Abstract

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are widely used in a number of cell therapies and have osteogenic differentiation capacity. Exposure to electromagnetic fields (EMFs) increases the osteogenic differentiation of hBM-MSCs. Nanomagnetic particles (MPs) also promote the differentiation potential of stem cells. In the present study, we investigated the effects of EMFs and MPs on the osteogenic differentiation of hBM-MSCs. hBM-MSCs were treated with 50 g/ml of Fe3O4 MPs or exposed to a frequency of 45 Hz and an intensity of 1 mT EMF twice every 8 h per day for 7 days. MP incorporation, EMF exposure and MP incorporation with exposure to EMFs did not induce cytotoxic effects. A strong expression of osteogenic markers (osteocalcin, osteopontin and osteonectin) and von Kossa staining intensity was observed in the cells treated with MPs, the cells exposed to EMFs and in the cells treated with MPs and exposed to EMFs compared with the control group, as shown by immunohistochemical staining. Quantitative RT-PCR revealed that the mRNA expression levels of osteoblast markers [osteocalcin, osteopontin, osteonectin, collagen I, collagen III, bone morphogenetic protein 2 (BMP-2), bone sialoprotein (BSP) and runt-related transcription factor 2 (Runx-2)] were markedly increased in the cells treated with MPs and exposed to EMFs. Furthermore, the mRNA expression of calcium channels (CACNA1C, CACNA1E, CACNA1G and CACNA1I) was activated during osteogenic differentiation. The expression levels of osteogenesis-related proteins (BSP, BMP-2, osteopontin and osteonectin) and phosphorylated extracellular signal-regulated kinase (p-ERK) were increased in the cells treated with MPs, those exposed to EMFs and in the cells treated with MPs and exposed to EMFs compared with the control group, as shown by western blot analysis. Fluorescence-activated cell sorting (FACS) analysis was performed for the hBM-MSC markers, CD73, CD90 and CD105. The expression levels of hBM-MSC surface antigens were decreased in the cells treated with MPs, those exposed to EMFs and in the cells treated with MPs and exposed to EMFs compared with the control group. The cell numbers were determined to be approximately 3.4x10(5) cells in the control group, 3.7x10(5) cells in the MP-treated group, 3.1x10(5) cells in the group exposed to EMFs and 3.9x10(5) cells in the group treated with MPs and exposed to EMFs. The cell mitochondrial activity among the 4 experimental groups was similar. The hBM-MSCs treated with MPs and exposed to EMFs showed an increase in alkaline phosphatase (ALP) activity. Taken together, these results suggest that the treatment of hBM-MSCs with MPs or exposure to EMFs increases osteogenic differentiation, and that treatment with MPs in conjunction with EMF exposure is more effective in increasing osteogenic differentiation.