Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

Hindawi Stem Cells International Volume 2017, Article ID 2450327, 17 pages https://doi.org/10.1155/2017/2450327 Research Article Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-𝛽 Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation Nagarajan Selvamurugan,1 Zhiming He,2 Daniel Rifkin,3 Branka Dabovic,3 and Nicola C. Partridge2 1 Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, India Department of Basic Science and Craniofacial Biology, New York University College of Dentistry, New York, NY, USA 3 Department of Cell Biology, New York University School of Medicine, New York, NY, USA 2 Correspondence should be addressed to Nicola C. Partridge; Received 23 September 2016; Revised 5 January 2017; Accepted 12 February 2017; Published 23 April 2017 Academic Editor: Elisabetta A. Cavalcanti-Adam Copyright © 2017 Nagarajan Selvamurugan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Pulsed electromagnetic fields (PEMFs) have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs’ cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-𝛽) signaling pathway and microRNA 21 (miR21) were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-𝛽 signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-𝛽 signaling pathway, was found to be miR21-5p’s putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-𝛽 signaling pathway and stimulation of expression of miR21-5p in hBMSCs. 1. Introduction Abundant reports describe the effects of electricity on bone growth and fracture repair, and a variety of pulsed electromagnetic field (PEMF) devices have been developed to produce electromagnetic fields at the fracture site. These widespread PEMF devices utilize noninvasive inductive coupling and can be used along with every method of fracture fixation [1, 2]. The stimulation of bone at the fracture site by the introduction of electromagnetic fields may be similar to the resulting stimulation from mechanical loading [1]. The beneficial therapeutic effects of such selected low energy, time varying PEMF promote fracture healing in nonunions [3], increase lumbar spinal fusion rates [4, 5], and have been found to affect bone metabolism by decreasing bone resorption and increasing bone formation [6–8]. PEMFs have also been reported to stimulate the synthesis of extracellular matrix (ECM) proteins [9] and may also affect several membrane receptors including those for parathyroid hormone, low density lipoprotein, insulin-like growth factor2, insulin, and calcitonin [10]. Several growth factors such as bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4) and transforming growth factor-beta (TGF-𝛽) have been reported to be secreted from osteoblasts upon PEMF treatment [11]. It has been shown that electromagnetic stimulation could raise net Ca2+ flux in human osteoblast-like cells, and the increase in the cytosolic Ca2+ concentration could initiate activation of signaling pathways resulting in regulation of expression 2 of bone matrix genes [12, 13]. Accelerated osteogenesis has been found in bone marrow-derived mesenchymal stem cells by PEMF treatment [14] and this promotion of ECM deposition was more efficient compared with adipose-tissue mesenchymal stem cells [15]. Previously we have reported that both BMP-2 and PEMF (Spinal-Stim by Orthofix, Inc., Lewisville, TX) separately stimulated proliferation of rat primary calvarial osteoblastic cells and stimulated expression of early osteoblast differentiation genes in culture [7]. In this study, we investigated the effects of PEMF (Cervical-Stim by Orthofix, Inc., Lewisville, TX) on human bone marrow stromal cells (hBMSCs) proliferating and differentiated to osteoblastic cells. In addition, the underlying molecular mechanisms by which PEMF stimulates differentiation of hBMSCs into osteoblasts are not well understood. Thus, we also aimed to investigate the PEMF effects on proliferation, differentiation, and mineralization of hBMSCs by Affymetrix microarray analysis. The TGF-𝛽 signaling pathway and microRNA 21 (miR21) were most highly regulated by PEMF. Thus, in this study we systematically investigated the mechanism of action of PEMF effects on osteogenesis via TGF-𝛽 and miR21 using hBMSCs. 2. Materials and Methods 2.1. Cell Culture. Fresh human bone marrows from 21–68year-old women were used. These were either purchased from Lonza (Walkersville, MD) or left over tissue from surgical procedures at New York University Hospital for Joint Diseases. Since these were deidentified, this is not considered Human Subjects Research by the New York University School of Medicine Institutional Review Board. In both cases, the bone marrows were freshly collected, never frozen, and immediately diluted 1 : 1 in Hank’s Balanced Salt Solution (HBSS; GIBCO Laboratories, Grand Island, NY) containing 20 IU/mL of sodium heparin (Sigma Chemical Co., St. Louis, MO). The diluted bone marrow was layered over an equal volume of Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ) and centrifuged at 400𝑔 for 40 min at 18∘ C. The mononuclear cells at the interface layer were collected, washed three times with HBSS, resuspended and seeded into a tissue culture flask, and incubated at 37∘ C in the presence of 5% CO2 overnight. The next day, nonadherent cells were removed from the culture flask. Adherent cells (BMSCs) were grown to confluence then placed in 6-well plates at 6.4 × 104 cells/well for exposure to PEMF or control. All cells were incubated at 37∘ C in the presence of 5% CO2 . The medium used for culturing these cells was 𝛼-MEM (Corning, Tewksbury, MA) containing 15% fetal bovine serum (FBS; GIBCO, Grand Island, NY) and Penicillin-Streptomycin (GIBCO, Grand Island, NY). 2.2. PEMF Exposure. The PEMF was generated as previously described [7] but was set to have similar waveform characteristics to a commercial, c (...truncated)