key: cord-0000412-nnhs8k0i
authors: Sollazzo, Vincenzo; Palmieri, Annalisa; Pezzetti, Furio; Massari, Leo; Carinci, Francesco
title: Effects of Pulsed Electromagnetic Fields on Human Osteoblastlike Cells (MG-63): A Pilot Study
date: 2010-04-13
journal: Clin Orthop Relat Res
DOI: 10.1007/s11999-010-1341-5
sha: 834fbf0a08acd3107e6a560519d8ce9e3479f2c4
doc_id: 412
cord_uid: nnhs8k0i

BACKGROUND: Although pulsed electromagnetic fields (PEMFs) are used to treat delayed unions and nonunions, their mechanisms of action are not completely clear. However, PEMFs are known to affect the expression of certain genes. QUESTIONS/PURPOSES: We asked (1) whether PEMFs affect gene expression in human osteoblastlike cells (MG63) in vitro, and (2) whether and to what extent stimulation by PEMFs induce cell proliferation and differentiation in MG-63 cultures. METHODS: We cultured two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control). Gene expression was evaluated throughout cDNA microarray analysis containing 19,000 genes spanning a substantial fraction of the human genome. RESULTS: PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2) and noncollagenous (SPARC) matrix components. However, PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4). CONCLUSIONS AND CLINICAL RELEVANCE: PEMFs appear to induce cell proliferation and differentiation. Furthermore, PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption. Our data suggest specific mechanisms of the observed clinical effect of PEMFs, and thus specific approaches for use in regenerative medicine.

PEMFs have been used for many years [44] . They reportedly are effective for treating nonunions [1, 7, 10] , delayed unions [1, 42, 44] , osteotomies [32] , avascular necrosis of the femoral head [5, 34] , bone grafts [11] , and spinal fusion [36] . Although the therapeutic properties of PEMFs are well known, the sequence of events by which electromagnetic stimulation can bring about its desirable effects on bone healing is not completely understood. PEMFs modify some important physiologic parameters of cells, such as proliferation, transduction, transcription, synthesis, and secretion of growth factors [24] . PEMFs induce cell proliferation in mitogen-stimulated lymphocytes [10] and improve IL-2 receptor expression and IL-2 use in lymphocytes from aged donors, which are characterized by defective production and use of this growth factor [10] . PEMF exposure induces cell proliferation in human osteoblasts and chondrocytes cultured in vitro [18, 20, 38, 44, 45] . PEMFs determine signal transduction by means of intracellular release of Ca 2+ leading to an increase in cytosolic Ca 2+ and an increase in activated cytoskeletal calmodulin [9] . PEMFs induce a dose-dependent increase in bone [2] and cartilage differentiation [2] [3] [4] 33] , and upregulation of mRNA expression of extracellular matrix molecules, proteoglycan, and Type II collagen [3] . The acceleration of chondrogenic differentiation is associated with increased expression of TGF-b1 mRNA and protein [4] , suggesting the stimulation of TGF-b1 may be a mechanism through which PEMFs affect complex tissue behavior such as cell differentiation and through which the effects of PEMFs may be amplified [4] . PEMFs also are postulated to act at a membrane level influencing signal transduction of several hormones or growth factors such as parathyroid hormone, IGF 2, and adenosine A2a, producing the amplification of their transmembrane receptors [1, 19, 21, 23, 31, 46] . Studies of single genes using RT-PCR suggest activation of osteocalcin, osteopontin, and TGF-b transcription during osteogenesis [22] and inhibition of cyclooxygenase 2 in synovial fibroblasts stimulated with TNFa or lipopolysaccharide [21] . A wide analysis of gene expression in cells exposed to PEMFs has not been performed: most studies focus on a few aspects of cell activities or they have been performed using different types of signals in different experimental conditions.

We therefore asked (1) whether PEMFs affected a wide array of genes in human osteoblastlike cells (MG63), and (2) whether and to what extent PEMFs induce proliferation and differentiation of osteoblasts.

We treated osteoblastlike cell cultures (MG-63) with PEMFs for 18 hours, and maintained similar nontreated controls. Gene expression of both groups therefore was evaluated with cDNA microarray analysis, containing 19 ,000 genes spanning a substantial fraction of the human genome. All experiments were performed in triplicate in the same culture conditions for control and treated cells.

Osteoblastlike cells (MG63) were grown in sterile Falcon wells (Becton & Dickinson, Franklin Lakes, NJ) containing Eagle's minimum essential medium supplemented with 10% fetal calf serum (Sigma-Aldrich, St Louis, MO) and antibiotics (penicillin 100 U/mL and streptomycin 100 lg/mL; Sigma-Aldrich). Cultures were maintained in a 5% CO 2 humidified atmosphere at 37°C. For the assay, cells were collected and seeded at a density of 1 9 10 5 cells/mL in two multiwells (one for the control and one for the treated). Each multiwell was comprised of six wells, 9-cm 2 , in which 3-mL of complete medium was added.

After 24 hours, cells were exposed to PEMFs for 18 hours using a PEMF generator system (Igea, Carpi, Italy). The PEMF used in this study is used clinically to treat nonunions or delayed unions and avascular necrosis of the femoral head [32] [33] [34] . The solenoids were powered using a Biostim pulse generator (Igea), a PEMF generator. The electromagnetic bioreactor applied to the cells has the following characteristics: intensity of the magnetic field, 2 ± 0.2 mT; amplitude of the induced electric tension, 5 ± 1 mV; signal frequency, 75 ± 2 Hz; and pulse duration, 1.3 ms. The stimulated multiwell was placed parallel between the two solenoids of the PEMF generator. The solenoids were placed at a distance of 10 cm and the multiwell was located on an acrylic support exactly at the center of the two solenoids. Control cultures were placed in the same incubator; nevertheless, the presence of the electromagnetic field was checked and its value was less than 0.05 mT. This value was ineffective in previous studies [38] [39] [40] [41] [42] [43] [44] [45] [46] . After 18 hours, when cultures were subconfluent, cells were processed for RNA extraction.

For DNA microarray screening and analysis, we used the same protocol as described previously [12] [13] [14] [15] [16] . Briefly, RNA was extracted from cells by using RNAzol. Ten micrograms of total RNA was used for each sample. cDNA was synthesized by using Superscript II (Life Technologies, Invitrogen, Milano, Italy) and amino-allyl dUTP (Sigma-Aldrich). Monoreactive Cy3 and Cy5 esters (Amersham Pharmacia, Little Chalfont, UK) were used for indirect cDNA labeling. RNA extracted from untreated cells was labeled with Cy3 and used as control against the Cy5-labeled treated (PG) cDNA in the first experiment and then switched. For 20 K human DNA microarrays slides (MWG Biotech AG, Ebersberg, Germany), 100 lL of the sample and control cDNAs in DIG Easy hybridization solution (Roche, Basel, Switzerland) were used in a sandwich hybridization of the two slides, constituting the 20 K set at 37°C overnight. Washing was performed three times for 10 minutes with 19 saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate at 42°C and three times for 5 minutes with 0.19 SSC at room temperature. Slides were dried by centrifugation for 2 minutes at 2000 rpm. Hybridized arrays were scanned with a GenePix 4000 scanner (Axon Instruments) at variable photomultiplier tube (PMT) voltage to obtain maximal signal intensities with less than 1% probe saturation.

The Foreground Median intensity for Cy3 and Cy5, Background Median intensity for Cy3 and Cy5, spot size data were imported into BRB-ArrayTools software [43] using the Import wizard function. Global normalization was used to median center the log-ratios on each array r to adjust for differences in labeling intensities of the Cy3 and Cy5 dyes.

The normalized Log ratios also were imported to Significance Analysis of Microarray (SAM) [48] software to identify differentially expressed genes. SAM assigns a score to each gene on the basis of a change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold, SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance-the false discovery rate (FDR). Analysis parameters (Delta) were set to result in zero FDR.

PEMF affected gene expression in MG-63 osteoblastlike cells (Fig. 1) . The genes differentially expressed in cells treated with PEMFs were either upregulated (268 genes) ( Table 1) or downregulated (277 genes) ( Table 2) . PEMF induced osteoblast proliferation and differentiation and regulated genes involved in bone formation in the direction of an enhancement of osteogenesis (Tables 3, 4 ).

In particular, PEMFs induced upregulation of several genes at the transcriptional level like STAT3, homeobox A10 (HOXA10), and V-akt murine thymoma viral oncogene homolog 1 (AKT1). Some genes acting at the transductional level also are upregulated including calmodulin (CALM1), activator protein 1 (AP-1), Nuclear factor kappaB (NF-KB), cAMP response element binding (CREB), and P2RX7 (Table 3) . Several interesting overexpressed genes are components of cytoskeleton and involved in cell adhesion (Table 3) . Examples are fibronectin (FN1) and vinculin (VCL). PEMF also increased the expression of genes encoding for collagenous and noncollagenous extracellular matrix proteins including collagen Type 1a2 (COL1A2), osteonectin (SPARC), and metallopeptidase inhibitor 1 (TIMP1) ( Table 3) . Some genes downregulated by PEMFs are related to degradation of extracellular matrix (ECM) (Table 4) , specifically, matrix metallopeptidase 11 (MMP11), or stromelysin 3 and dual specificity phosphatase 4 (DUSP4).

The improvement of osteogenesis is important because of the wide clinical applications it may have. PEMFs reportedly restart osteogenesis in disorders in which it has stopped [34] and in disorders in which osteogenesis needs to be enhanced [32] . Although considerable basic and clinical research on PEMFs has been reported, their mechanism of action is not completely clear. Moreover, studies in the existing literature have so far focused only on a few aspects of cell activities [9, 10, 46] , or they have been performed by using different types of signals in different experimental conditions [1, 9, 22, 23] . To address these limitations in the literature, we asked (1) whether PEMFs affected a wide array of genes in human osteoblastlike cells (MG63), and (2) whether and to what extent PEMFs induce proliferation and differentiation of osteoblasts.

We acknowledge several limitations. First, the experiment was performed using a human osteosarcoma cell line (MG63), whereas the use of a primary human osteoblast cell culture might better replicate what happens in humans in vivo. We chose the MG63 cell line because these cells show a phenotype similar to that of normal human osteoblasts, while also providing a reproducible experimental model suitable for the microarray analysis. Second, as it is still difficult to explain the roles of all genes, whose expression was modified, we focused on the role of genes with well-known functions related to osteogenesis. Third, although microarray technology is widely accepted as a valid approach to describe changes induced by a factor on cell environment, additional research using, for example RT-PCR, might be useful to provide supplementary support for the results obtained. Fourth, we studied responses at only one time. We chose 18 hours exposure time on the basis of a previous time experiment, in which a peak in DNA synthesis was seen after 18 hours of stimulation in MG63 cultures maintained in the presence of 10% FCS [45] . In contrast, Lohmann et al. reported PEMFs enhanced cell differentiation in MG63 cultures and reduced cell proliferation [30] . The differences existing between the two sets of data regarding cell proliferation could be related to the different experimental conditions used. Lohmann et al. exposed MG63 cultures when they reached confluence. When cultures are confluent they stop to proliferate. We exposed cells to PEMF when cultures were subconfluent, therefore, they responded with an enhancement of proliferation. We cannot extrapolate our findings to shorter or longer exposures to PEMFs. PEMFs appear to act on bone formation by inducing upregulation of several genes related to osteoblast proliferation and differentiation. Among those genes, HOXA10, a transcriptional factor that acts positively on RUNX2, is the main transcriptional regulator of osteoblast differentiation [25] . HOXA10 controls osteoblastogenesis via RUNX2-promoted osteoprogenitor cell differentiation in immature osteoblasts [25] . This protein also is believed to be involved in activation of alkaline phosphatase, osteocalcin, and sialoprotein genes [25] . We also observed * SAM assigns a score to each gene on the basis of a change in gene expression relative to the standard deviation of repeated measurements. STAT3, P2RX7, and AKT1 upregulation. It has been suggested that osteoblast-specific disruption of STAT3 results in an osteopenic phenotype [27, 41] . STAT3, involved in bone turnover [27] , regulates the transcription of various genes that modulate cell proliferation and differentiation in a cell-specific manner [27] . P2RX7 is a purinergic receptor, which is correlated with calcium channels and interacts with the calmodulin-dependent protein [37] . Activation of P2RX7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells promoting osteogenesis [37] . V-akt murine thymoma viral oncogene homolog 1 (AKT1), is a phosphoinositidedependent serine-threonine protein kinase, and one of the key players in the signaling of potent bone anabolic factors [29] . The disruption of AKT1 in mice led to low-turnover osteopenia through dysfunction [29] . AKT1 deficiency causes decreased bone mass and formation [29] , impairs RUNX2-dependent differentiation and function of osteoblasts [29] , and impairs bone resorption via dysfunction of osteoblasts and osteoclasts [29] . AKT1 suppresses osteoblasts apoptosis through inhibition of FOXO3a and Bim [29] , and may mediate the osteoblastic bone formation by IGF-1 [29] . The IGF-1/AKT1 pathway might be a common pathway for bone anabolic action of parathyroid, thyroid, and growth hormone [29] . We also observed upregulation of genes involved in connective and bone tissue formation (COL1A2) and noncollagenous extracellular matrix (ECM) synthesis (SPARC, FN1, VCL). COL1A2 encodes for collagen Type 1a2. Collagen Type 1 is the most represented collagen in the human organism and is important for ECM stability [6] . Osteonectin (SPARC), the most abundant noncollagenous protein in bone tissue, modulates cell-matrix interaction and is involved in the tissue-remodeling process [47] . FN1 is important for ECM stability and involved in adhesion and migration cellular processes such as tissue healing [39] . VCL is a cytoskeletal protein associated with the intercellular junctions between the cells and the matrix [49] .

The effect of TIMP1 upregulation and of MMP-11 and DUSP4 downregulation can be interpreted as a decrease in the degradation process. TIMP1 promotes apposition of ECM by inhibiting collagen and other components of ECM degradation operated by the metalloproteinase [26] . DUSP4 inactivates the superfamily of MAP kinase, which is involved with proliferation and differentiation. DUSP4 downregulation, then, stimulates proliferation [17] . MMPs potentially can degrade almost all components of the periprosthetic ECM and contribute to prosthetic loosening and osteolysis through pathologic ECM degradation and bone remodeling around prostheses [28, 35] . The stromelysins especially have broad substrate specificity, including proteoglycans, laminin, and fibronectin [35] . Stromelysin-1 determines the release and activation ECMbound latent TGF-ß1 and is involved with ECM turnover [8] . Upregulation of CALM1 promotes enhancement of calmodulin1, a protein involved in proliferative cell activation [40] . Calmodulin also is involved in the transduction mechanism of PEMFs [9] .

Our data suggest many effects of PEMFs on human osteoblastlike cells in vitro. PEMFs seem to exert an 

Inactivates the superfamily of MAP kinase Inhibition of proliferation and differentiation Caunt et al. [17] anabolic effect on cells. In particular, they are consistent with abundant preclinical and clinical findings showing a positive effect of PEMFs on osteogenesis. Stimulation by PEMFs induces bone healing in patients, shortens the time of healing processes, and stimulates healing of nonunions. Exposure to PEMFs acts on cell behavior in different ways. More specifically, PEMFs stimulate cell proliferation and induce osteoblastogenesis and differentiation of osteoblasts. Moreover, PEMFs promote ECM apposition and mineralization, and decrease degradation and absorption processes of ECM. These data suggest a more comprehensive explanation of the observed clinical effect of PEMFs on the induction of osteogenesis. Given their broad effects, PEMFs might be useful in other fields such as regenerative medicine.

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