Synergistic effect of a LPEMF and SPIONs on BMMSC proliferation, directional migration, and osteoblastogenesis

Abstract

Pulsed electromagnetic fields (PEMFs) represent a new type of physiotherapy that has been shown to be effective for improving bone fracture healing and treating osteoporosis. Targeted therapy with bone marrow mesenchymal stem cells (BMMSCs) has been the focus of several recent studies. The key to such therapy is the effective application of certain nanomaterials in BMMSCs so they achieve an ideal target concentration under the influence of a PEMF. In our present study, the effects of a PEMF on the process of osteoblastogenesis were systematically investigated using superparamagnetic iron oxide nanoparticle (SPION)-labeled BMMSCs. Rat BMMSCs labeled with SPIONs were exposed to a low-frequency pulsed electromagnetic field (LPEMF) of 50 Hz at 1.1 mT. Exposure to the LPEMF resulted in an enhanced proliferation of SPION-labeled BMMSCs when compared with a control group. Furthermore, observations made by transmission electron microscopy (TEM) revealed greater cell concentrations in the central zone with exposure to the LPEMF than in the peripheral zone without LPEMF stimulation, indicating that a LPEMF could induce the migration of SPION-labeled BMMSCs towards a magnetic field. Transwell experiments confirmed that combining SPIONs with a LPEMF could significantly promote the directional migration of BMMSCs. Von Kossa and ALP staining of LPEMF-exposed SPION-labeled cells was more intense, and those cells displayed higher levels of ALP activity than control cells. The SPION-labeled, LPEMF-exposed cells also showed increased levels of osteogenesis-related gene and protein expression (e.g., ALP, OCN, and RUNX2) in PCR and western blot studies. Taken together, our findings suggest that a combination of LPEMF and SPIONs exerts a synergistic effect on promoting the directional migration and osteogenic differentiation of BMMSCs, indicating that application of a LPEMF in conjunction with SPIONs may constitute a method for treating bone defects.

Keywords: Pulsed electromagnetic fields; bone marrow mesenchymal stem cells; osteoblastogenesis; superparamagnetic iron oxide nanoparticles.

Figure 1

The expression levels of CD29, CD90, CD34, and CD45 in rat BMSCs were examined using flow cytometry. An isotype antibody served as a negative control (blue line). The percentages of CD29-positive and CD90-positive cells were 99.44% and 96.40%, respectively. The percentages of CD34-positive and CD45-positive cells were ~2.27% and 1.53%, respectively.

Figure 2

The effect of SPION on BMMSCs. A. TEM image of the synthesized 12 nm SPIONs. The inset shows a strong response of the nanoparticle solution to an external magnetic field. B. Third passage BMMSC morphology as viewed under a bright field microscope (×100 magnification). C. Prussian blue staining of BMMSCs incubated with 50 μg/mL SPIONs (×100 magnification). D. Inhibition of cell growth produced by exposure to different SPION concentrations for 6 consecutive days. Cell proliferation gradually decreased as the SPION concentration increased.

Figure 3

Cell performance on cell growth, cell density and distribution after stimulated by LPEMF. A. Cell growth curves of 50 μg/mL SPION-labeled BMMSCs with or without exposure to the LPEMF. B. Cells were uniformly distributed prior to application of the LPEMF. C. Cells aggregated after 2 h of LPEMF exposure. D. Cells were much sparser in the peripheral zone where the LPEMF wasn’t applied. Scale bar = 100 μm. E. Cell density in the different zones before and after LPEMF stimulation. *P < 0.05 compared with cell density before stimulation. **P < 0.01 compared with cell density before stimulation. Data are presented as the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 compared with the LPEMF(-) group.

Figure 4

Effect of SPIONs and PEMF on Cell migration, von Kossa calcim staning, activity of ALP of BMMSCs. A and B. Transwell experiments showed that the number of migrating BMMSCs in the LPEMF(+)SPION(+) group was significantly greater than those numbers in the other three groups. *P < 0.05, **P < 0.01, #P > 0.05 compared with the control group. C. ALP activity in the four BMSC groups after 21 days of osteogenic induction. *P < 0.01 compared with any other group. D. (Top row) von Kossa calcium staining and (bottom row) ALP staining of the four BMMSC groups after 21 days of osteogenic induction. Visible black crystalline mineral deposits were observed after staining. Combined SPION labeling plus LPEMF exposure.

Figure 5

Expression of Runx2, OCN and ALP after 21 days of osteogenic induction. The qRT-PCR analyses showed that LPEMF exposure significantly increased the expression of genes related to cell differentiation in SPION-labeled cells. Data acquired by the RT-PCR assays are presented as the mean ± SD (n = 3). The relative expression level of each gene was normalized to that of β-actin, and the ratio is given. *P < 0.05, **P < 0.01 compared with the SPION(-)PEMF(-) group. Expression of osteoblastogenesis-related proteins after 21 days of osteogenic induction. Western blot studies showed that LPEMF exposure significantly increased the synthesis of osteoblastogenesis-related proteins in SPIONs-labeled cells. Relative protein content was normalized to that of β-actin.