PEMFs have been used for many years . They reportedly are effective for treating nonunions [1, 7, 10], delayed unions [1, 42, 44], osteotomies , avascular necrosis of the femoral head [5, 34], bone grafts , and spinal fusion . 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 . PEMFs induce cell proliferation in mitogen-stimulated lymphocytes  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 . 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 Ca2+ leading to an increase in cytosolic Ca2+ and an increase in activated cytoskeletal calmodulin . PEMFs induce a dose-dependent increase in bone  and cartilage differentiation [2-4, 33], and upregulation of mRNA expression of extracellular matrix molecules, proteoglycan, and Type II collagen . The acceleration of chondrogenic differentiation is associated with increased expression of TGF-β1 mRNA and protein , suggesting the stimulation of TGF-β1 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 . 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-β transcription during osteogenesis  and inhibition of cyclooxygenase 2 in synovial fibroblasts stimulated with TNFα or lipopolysaccharide . 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.
Materials and Methods
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 μg/mL; Sigma-Aldrich). Cultures were maintained in a 5% CO2 humidified atmosphere at 37°C. For the assay, cells were collected and seeded at a density of 1 × 105 cells/mL in two multiwells (one for the control and one for the treated). Each multiwell was comprised of six wells, 9-cm2, 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 [32-34]. 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 [38-46]. 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 [12-16]. 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 μL 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 1× saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate at 42°C and three times for 5 minutes with 0.1× 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  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)  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 1α2 (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  and in disorders in which osteogenesis needs to be enhanced . 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 . In contrast, Lohmann et al. reported PEMFs enhanced cell differentiation in MG63 cultures and reduced cell proliferation . 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 . HOXA10 controls osteoblastogenesis via RUNX2-promoted osteoprogenitor cell differentiation in immature osteoblasts . This protein also is believed to be involved in activation of alkaline phosphatase, osteocalcin, and sialoprotein genes . We also observed 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 , regulates the transcription of various genes that modulate cell proliferation and differentiation in a cell-specific manner . P2RX7 is a purinergic receptor, which is correlated with calcium channels and interacts with the calmodulin-dependent protein . Activation of P2RX7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells promoting osteogenesis . V-akt murine thymoma viral oncogene homolog 1 (AKT1), is a phosphoinositide-dependent serine-threonine protein kinase, and one of the key players in the signaling of potent bone anabolic factors . The disruption of AKT1 in mice led to low-turnover osteopenia through dysfunction . AKT1 deficiency causes decreased bone mass and formation , impairs RUNX2-dependent differentiation and function of osteoblasts , and impairs bone resorption via dysfunction of osteoblasts and osteoclasts . AKT1 suppresses osteoblasts apoptosis through inhibition of FOXO3a and Bim , and may mediate the osteoblastic bone formation by IGF-1 . The IGF-1/AKT1 pathway might be a common pathway for bone anabolic action of parathyroid, thyroid, and growth hormone .
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 1α2. Collagen Type 1 is the most represented collagen in the human organism and is important for ECM stability . Osteonectin (SPARC), the most abundant noncollagenous protein in bone tissue, modulates cell-matrix interaction and is involved in the tissue-remodeling process . FN1 is important for ECM stability and involved in adhesion and migration cellular processes such as tissue healing . VCL is a cytoskeletal protein associated with the intercellular junctions between the cells and the matrix .
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 . DUSP4 inactivates the superfamily of MAP kinase, which is involved with proliferation and differentiation. DUSP4 downregulation, then, stimulates proliferation . 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 . Stromelysin-1 determines the release and activation ECM-bound latent TGF-β1 and is involved with ECM turnover . Upregulation of CALM1 promotes enhancement of calmodulin1, a protein involved in proliferative cell activation . Calmodulin also is involved in the transduction mechanism of PEMFs .
Our data suggest many effects of PEMFs on human osteoblastlike cells in vitro. PEMFs seem to exert an 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.
1. Aaron, RK., Boyan, BD., Ciombor, DM., Schwartz, Z. and Simon, BJ. Stimulation of growth factor synthesis by electric and electromagnetic fields. Clin Orthop Relat Res.
2004; 419: 30-37. 10.1097/00003086-200402000-00006
2. Aaron, RK. and Ciombor, DM. Acceleration of experimental endochondral ossification by biophysical stimulation of the progenitor cell pool. J Orthop Res.
1996; 14: 582-589. 10.1002/jor.1100140412
3. Aaron, RK., Ciombor, DM. and Jolly, G. Stimulation of experimental endochondral ossification by low-energy pulsing electromagnetic fields. J Bone Miner Res.
1989; 4: 227-233. 10.1002/jbmr.5650040215
4. Aaron, RK., Ciombor, DM., Keeping, H., Wang, S., Capuano, A. and Polk, C. Power frequency fields promote cell differentiation coincident with an increase in transforming growth factor-beta(1) expression. Bioelectromagnetics.
1999; 20: 453-458. 10.1002/(SICI)1521-186X(199910)20:7<453::AID-BEM7>3.0.CO;2-H
5. Aaron, RK., Lennox, D., Bunce, GE. and Ebert, T. The conservative treatment of osteonecrosis of the femoral head: a comparison of core decompression and pulsing electromagnetic fields. Clin Orthop Relat Res.
1989; 249: 209-218.
6. Antoniv, TT., Tanaka, S., Sudan, B., Val, S., Liu, K., Wang, L., Wells, DJ., Bou-Gharios, G. and Ramirez, F. Identification of a repressor in the first intron of the human alpha2(I) collagen gene (COL1A2). J Biol Chem.
2005; 280: 35417-35423. 10.1074/jbc.M502681200
7. Bassett, CA., Mitchell, SN. and Gaston, SR. Treatment of ununited tibial diaphyseal fractures with pulsing electromagnetic fields. J Bone Joint Surg Am.
1981; 63: 511-523.
8. Boyan, BD. and Schwartz, Z.1,25-Dihydroxy vitamin D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60-activated matrix vesicle matrix metalloproteinases. Cells Tissues Organs.
2009; 189: 70-74. 10.1159/000152916
9. Brighton, CT., Wang, W., Seldes, R., Zhang, G. and Pollack, SR. Signal transduction in electrically stimulated bone cells. J Bone Joint Surg Am.
2001; 83: 1514-1523.
10. Cadossi, R., Bersani, F., Cossarizza, A., Zucchini, P., Emilia, G., Torelli, G. and Franceschi, C. Lymphocytes and low-frequency electromagnetic fields. FASEB J.
1992; 6: 2667-2674.
11. Capanna, R., Donati, D., Masetti, C., Manfrini, M., Panozzo, A., Cadossi, R. and Campanacci, M. Effect of electromagnetic fields on patients undergoing massive bone graft following bone tumor resection: a double blind study. Clin Orthop Relat Res.
1994; 306: 213-221.
12. Carinci, F., Pezzetti, F., Volinia, S., Francioso, F., Arcelli, D., Farina, E. and Piattelli, A. Zirconium oxide: analysis of MG63 osteoblast-like cell response by means of a microarray technology. Biomaterials.
2004; 25: 215-228. 10.1016/S0142-9612(03)00486-1
13. Carinci, F., Pezzetti, F., Volinia, S., Francioso, F., Arcelli, D., Marchesini, J., Caramelli, E. and Piattelli, A. Analysis of MG63 osteoblastic-cell response to a new nanoporous implant surface by means of a microarray technology. Clin Oral Implants Res.
2004; 15: 180-186. 10.1111/j.1600-0501.2004.00997.x
14. Carinci, F., Pezzetti, F., Volinia, S., Laino, G., Arcelli, D., Caramelli, E., Degidi, M. and Piattelli, A. P-15 cell-binding domain derived from collagen: analysis of MG63 osteoblastic-cell response by means of a microarray technology. J Periodontol.
2004; 75: 66-83. 10.1902/jop.2004.75.1.66
15. Carinci, F., Piattelli, A., Stabellini, G., Palmieri, A., Scapoli, L., Laino, G., Caputi, S. and Pezzetti, F. Calcium sulfate: analysis of MG63 osteoblast-like cell response by means of a microarray technology. J Biomed Mater Res B Appl Biomater.
2004; 71: 260-267. 10.1002/jbm.b.30133
16. Carinci, F., Volinia, S., Pezzetti, F., Francioso, F., Tosi, L. and Piattelli, A. Titanium-cell interaction: analysis of gene expression profiling. J Biomed Mater Res B Appl Biomater.
2003; 66: 341-346. 10.1002/jbm.b.10021
17. Caunt, CJ., Rivers, CA., Conway-Campbell, BL., Norman, MR. and McArdle, CA. Epidermal growth factor receptor and protein kinase C signaling to ERK2: spatiotemporal regulation of ERK2 by dual specificity phosphatases. J Biol Chem.
2008; 283: 6241-6252. 10.1074/jbc.M706624200
18. Ciombor, DM., Aaron, RK., Wang, S. and Simon, B. Modification of osteoarthritis by pulsed electromagnetic field: a morphological study. Osteoarthritis Cartilage.
2003; 11: 455-462. 10.1016/S1063-4584(03)00083-9
19. Clark, AN., Youkey, R., Liu, X., Jia, L., Blatt, R., Day, YJ., Sullivan, GW., Linden, J. and Tucker, AL. A1 adenosine receptor activation promotes angiogenesis and release of VEGF from monocytes. Circ Res.
2007; 101: 1130-1138. 10.1161/CIRCRESAHA.107.150110
20. Mattei, M., Caruso, A., Traina, GC., Pezzetti, F., Baroni, T. and Sollazzo, V. Correlation between pulsed electromagnetic fields exposure time and cell proliferation increase in human osteosarcoma cell lines and human normal osteoblast cells in vitro. Bioelectromagnetics.
1999; 20: 177-182. 10.1002/(SICI)1521-186X(1999)20:3<177::AID-BEM4>3.0.CO;2-#
21. Mattei, M., Varani, K., Masieri, FF., Pellati, A., Ongaro, A., Fini, M., Cadossi, R., Vincenzi, F., Borea, PA. and Caruso, A. Adenosine analogs and electromagnetic fields inhibit prostaglandin E2 release in bovine synovial fibroblasts. Osteoarthritis Cartilage.
2009; 17: 252-262. 10.1016/j.joca.2008.06.002
22. Fassina, A., Vasai, L., Benazzo, F., Benedetti, L., Calligaro, A., Angelis, MG., Farina, A., Maliardi, V. and Magenes, G. Effects of electromagnetic stimulation on calcified matrix production by SAOS-2 cells over a polyurethane porous scaffold. Tissue Eng.
2006; 12: 1985-1999. 10.1089/ten.2006.12.1985
23. Fitzsimmons, RG., Ryaby, JT., Magee, FP. and Baylink, DJ. IGF-II receptor number is increased in TE-85 osteosarcoma cells by combined magnetic fields. J Bone Miner Res.
1995; 10: 812-819. 10.1002/jbmr.5650100519
24. Goodman, EM., Greenebaum, B. and Marron, MT. Effects of electromagnetic fields on molecules and cells. Int Rev Cytol.
1995; 158: 279-338. 10.1016/S0074-7696(08)62489-4
25. Hassan, MQ., Tare, R., Lee, SH., Mandeville, M., Weiner, B., Montecino, M., Wijnen, AJ., Stein, JL., Stein, GS. and Lian, JB. HOXA10 controls osteoblastogenesis by directly activating bone regulatory and phenotypic genes. Mol Cell Biol.
2007; 27: 3337-3352. 10.1128/MCB.01544-06
26. Hatori, K., Sasano, Y., Takahashi, I., Kamakura, S., Kagayama, M. and Sasaki, K. Osteoblasts and osteocytes express MMP2 and -8 and TIMP1, -2, and -3 along with extracellular matrix molecules during appositional bone formation. Anat Rec A Discov Mol Cell Evol Biol.
2004; 277: 262-271. 10.1002/ar.a.20007
27. Itoh, S., Udagawa, N., Takahashi, N., Yoshitake, F., Narita, H., Ebisu, S. and Ishihara, K. A critical role for interleukin-6 family-mediated Stat3 activation in osteoblast differentiation and bone formation. Bone.
2006; 39: 505-512. 10.1016/j.bone.2006.02.074
28. Jones, GC. and Riley, GP. ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis. Arthritis Res Ther.
2005; 7: 160-169. 10.1186/ar1783
29. Kawamura, N., Kugimiya, F., Oshima, Y., Ohba, S., Ikeda, T., Saito, T., Shinoda, Y., Kawasaki, Y., Ogata, N., Hoshi, K., Akiyama, T., Chen, WS., Hay, N., Tobe, K., Kadowaki, T., Azuma, Y., Tanaka, S., Nakamura, K., Chung, UI. and Kawaguchi, H. Akt1 in osteoblasts and osteoclasts controls bone remodelling. PloS ONE.
2007; 2: e1058. 10.1371/journal.pone.0001058
30. Lohmann, CH., Schwartz, Z., Liu, Y., Guerkov, H., Dean, DD., Simon, B. and Boyan, BD. Pulsed electromagnetic field stimulation of MG63 osteoblast-like cells affects differentiation and local factor production. J Orthop Res.
2000; 18: 637-646. 10.1002/jor.1100180417
31. Luben, RA., Cain, CD., Chen, MC., Rosen, DM. and Adey, WR. Effects of electromagnetic stimuli on bone and bone cells in vitro: inhibition of responses to parathyroid hormone by low-energy, low-frequency fields. Proc Natl Acad Sci USA.
1982; 79: 4180-4184. 10.1073/pnas.79.13.4180
32. Mammi, GI., Rocchi, R., Cadossi, R., Massari, L. and Traina, GC. The electrical stimulation of tibial osteotomies: double-blind study. Clin Orthop Relat Res.
1993; 288: 246-253.
33. Massari, L., Benazzo, F., Mattei, M., Setti, S., Fini, M. CRES Study GroupEffects of electrical physical stimuli on articular cartilage. J Bone Joint Surg Am.
2007; 899: ((suppl 3)):152-161. 10.2106/JBJS.G.00581
34. Massari, L., Fini, M., Cadossi, R., Setti, S. and Traina, GC. Biophysical stimulation with pulsed electromagnetic fields in osteonecrosis of the femoral head. J Bone Joint Surg Am
2006; 88: (suppl 3):56-60. 10.2106/JBJS.F.00536
35. Matziari, M., Dive, V. and Yiotakis, A. Matrix metalloproteinase 11 (MMP-11; stromelysin-3) and synthetic inhibitors. Med Res Rev.
2007; 27: 528-552. 10.1002/med.20066
36. Mooney, V. A randomized double-blind prospective study of the efficacy of pulsed electromagnetic fields for interbody lumbar fusions. Spine (Phila PA 1976)
1990; 15: 708-712.
37. Ohlendorff, SD., Tofteng, CL., Jensen, JE., Petersen, S., Civitelli, R., Fenger, M., Abrahamsen, B., Hermann, AP., Eiken, P. and Jørgensen, NR. Single nucleotide polymorphisms in the P2X7 gene are associated to fracture risk and to effect of estrogen treatment. Pharmacogenet Genomics.
2007; 17: 555-567. 10.1097/FPC.0b013e3280951625
38. Pezzetti, F., Mattei, M., Caruso, A., Cadossi, R., Zucchini, P., Carinci, F., Traina, GC. and Sollazzo, V. Effects of pulsed electromagnetic fields on human chondrocytes: an in vitro study. Calcif Tissue Int.
1999; 65: 396-401. 10.1007/s002239900720
39. Potts, JR. and Campbell, ID. Structure and function of fibronectin modules. Matrix Biol
1996; 15: 313-320. 10.1016/S0945-053X(96)90133-X
40. Rhymer, JA., Ottiger, M., Wicki, R., Greenwood, TM. and Strehler, EE. Structure of the human CALM1 calmodulin gene and identification of two CALM1-related pseudogenes CALM1P1 and CALM1P2. Eur J Biochem.
1994; 225: 71-82. 10.1111/j.1432-1033.1994.00071.x
41. Scott, MJ., Godshall, CJ. and Cheadle, WG. Jaks, STATs, cytokines, and sepsis. Clin Diagn Lab Immunol.
2002; 9: 1153-1159.
42. Sharrard, WJ. A double-blind trial of pulsed electromagnetic fields for delayed union of tibial fractures. J Bone Joint Surg Br.
1990; 72: 347-355.
43. Simon, R., Lam, A., Li, MC., Ngan, M., Menenzes, S. and Zhao, Y. Analysis of gene expression data using BRB-array tools. Cancer Inform.
2007; 3: 11-17.
44. Sollazzo, V., Massari, L., Caruso, C., Mattei, M. and Pezzetti, P. Effects of low-frequency pulsed electromagnetic fields on human osteoblast-like cells in vitro. Electro- and Magnetobiology.
1996; 15: 75-83.
45. Sollazzo, V., Traina, GC., DeMattei, M., Pellati, A., Pezzetti, F. and Caruso, A. Responses of human MG-63 osteosarcoma cell line and human osteoblast-like cells to pulsed electromagnetic fields. Bioelectromagnetics.
1997; 18: 541-547. 10.1002/(SICI)1521-186X(1997)18:8<541::AID-BEM2>3.0.CO;2-2
46. Varani, K., Gessi, S., Merighi, S., Iannotta, V., Cattabriga, E., Spisani, S., Cadossi, R. and Borea, PA. Effect of low frequency electromagnetic fields on A2A adenosine receptors in human neutrophils. Br J Pharmacol.
2002; 136: 57-66. 10.1038/sj.bjp.0704695
47. Yan, Q. and Sage, EH. SPARC, a matricellular glycoprotein with important biological functions. J Histochem Cytochem.
1999; 47: 1495-1506.
48. Tusher, VG., Tibshirani, R. and Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA.
2001; 98: 5116-5121. 10.1073/pnas.091062498
49. Ziegler, WH., Liddington, RC. and Critchley, DR. The structure and regulation of vinculin. Trends Cell Biol.
2006; 16: 453-460. 10.1016/j.tcb.2006.07.004