Pulsed electromagnetic field (PEMF) transiently stimulates the rate of mineralization in a 3-dimensional ring culture model of osteogenesis

Pulsed Electromagnetic Field (PEMF) has shown efficacy in bone repair and yet the optimum characteristics of this modality and its molecular mechanism remain unclear. To determine the effects of timing of PEMF treatment, we present a novel three-dimensional culture model of osteogenesis that demonstrates strong de novo generation of collagen and mineral matrix and exhibits stimulation by PEMF in multiple stages over 62 days of culture. Mouse postnatal day 2 calvarial pre-osteoblasts were cast within and around Teflon rings by polymerization of fibrinogen and cultured suspended without contact with tissue culture plastic. Ring constructs were exposed to PEMF for 4h/day for the entire culture (Daily), or just during Day1-Day10, Day11-Day 27, or Day28-Day63 and cultured without PEMF for the preceding or remaining days, and compared to no-PEMF controls. PEMF was conducted as HF Physio, 40.85 kHz frequency with a 67 ms burst period and an amplitude of 1.19 mT. Osteogenesis was kinetically monitored by repeated fluorescence measurements of continuously present Alizarin Red S (ARS) and periodically confirmed by micro-CT. PEMF treatment induced early-onset and statistically significant transient stimulation (~4-fold) of the mineralization rate when PEMF was applied Daily, or during D1-D10 and D11-D27. Stimulation was apparent but not significant between D28-D63 by ARS but was significant at D63 by micro-CT. PEMF also shifted the micro-CT density profiles to higher densities in each PEMF treatment group. Ring culture generated tissue with a mineral:matrix ratio of 2.0 by thermogravimetric analysis (80% of the calvaria control), and the deposited crystal structure was 50% hydroxyapatite by X-ray diffraction (63% of the calvaria and femur controls), independent of PEMF. These results were consistent with backscatter, secondary electron, and elemental analysis by scanning electron microscopy. Thus, in a defined, strong osteogenic environment, PEMF applied at different times was capable of further stimulation of osteogenesis with the potential to enhance bone repair.

[1]  Jun Liu,et al.  DNA methylation of noncoding RNAs: new insights into osteogenesis and common bone diseases , 2020, Stem Cell Research & Therapy.

[2]  A. Franco-Obregón,et al.  Pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration , 2020, Stem Cell Research & Therapy.

[3]  J. Behravan,et al.  Osteogenesis and bone remodeling: A focus on growth factors and bioactive peptides , 2019, BioFactors.

[4]  A. Nussler,et al.  Translational Insights into Extremely Low Frequency Pulsed Electromagnetic Fields (ELF-PEMFs) for Bone Regeneration after Trauma and Orthopedic Surgery , 2019, Journal of clinical medicine.

[5]  J. Lotz,et al.  Pulsed Electromagnetic Fields Reduce Interleukin-6 Expression in Intervertebral Disc Cells Via Nuclear Factor-κβ and Mitogen-Activated Protein Kinase p38 Pathways. , 2019, Spine.

[6]  N. Sims,et al.  Cellular Processes by Which Osteoblasts and Osteocytes Control Bone Mineral Deposition and Maturation Revealed by Stage-Specific EphrinB2 Knockdown , 2019, Current Osteoporosis Reports.

[7]  W. Jang,et al.  Estradiol-induced RORα expression positively regulates osteoblast differentiation , 2019, Steroids.

[8]  G. Smyth,et al.  Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone , 2019, Nature Communications.

[9]  G. Almeida-Porada,et al.  Targeting Mesenchymal Stromal Cells/Pericytes (MSCs) With Pulsed Electromagnetic Field (PEMF) Has the Potential to Treat Rheumatoid Arthritis , 2019, Front. Immunol..

[10]  D. Perugia,et al.  Biophysical stimulation of bone and cartilage: state of the art and future perspectives , 2019, International Orthopaedics.

[11]  L. Soslowsky,et al.  Effects of pulsed electromagnetic field therapy at different frequencies and durations on rotator cuff tendon-to-bone healing in a rat model. , 2017, Journal of shoulder and elbow surgery.

[12]  N. Selvamurugan,et al.  Pulsed electromagnetic fields inhibit human osteoclast formation and gene expression via osteoblasts. , 2018, Bone.

[13]  B. Pavan,et al.  Carbonate substitution in the mineral component of bone: Discriminating the structural changes, simultaneously imposed by carbonate in A and B sites of apatite. , 2017, Journal of solid state chemistry.

[14]  D. Rifkin,et al.  Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation , 2017, Stem cells international.

[15]  Erik I. Waldorff,et al.  Pulsed electromagnetic field applications: A corporate perspective , 2017, Journal of orthopaedic translation.

[16]  P. Benya,et al.  Keloid‐derived, plasma/fibrin‐based skin equivalents generate de novo dermal and epidermal pathology of keloid fibrosis in a mouse model , 2016, Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[17]  F. Sbrana,et al.  Electro-magnetic field promotes osteogenic differentiation of BM-hMSCs through a selective action on Ca2+-related mechanisms , 2015, Scientific Reports.

[18]  X. Lou.  Induced Pluripotent Stem Cells as a new Strategy for Osteogenesis and Bone Regeneration , 2015, Stem Cell Reviews and Reports.

[19]  A. Boyde,et al.  An Investigation of the Mineral in Ductile and Brittle Cortical Mouse Bone , 2015, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[20]  J. J. van den Beucken,et al.  Signaling pathways involved in osteogenesis and their application for bone regenerative medicine. , 2015, Tissue engineering. Part B, Reviews.

[21]  M D McKee,et al.  Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone. , 2015, Bone.

[22]  B. Cronstein,et al.  Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein‐2 , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  M. Morris,et al.  Contributions of Raman spectroscopy to the understanding of bone strength. , 2015, BoneKEy reports.

[24]  J. Jansen,et al.  Signaling pathways involved in osteogenesis and their application for bone regenerative medicine. , 2015, Tissue engineering. Part B, Reviews.

[25]  T. Anada,et al.  Bone matrix calcification during embryonic and postembryonic rat calvarial development assessed by SEM–EDX spectroscopy, XRD, and FTIR spectroscopy , 2014, Journal of Bone and Mineral Metabolism.

[26]  L. Fassina,et al.  A Comparative Analysis of the In Vitro Effects of Pulsed Electromagnetic Field Treatment on Osteogenic Differentiation of Two Different Mesenchymal Cell Lineages , 2013, BioResearch open access.

[27]  K. Varani,et al.  Pulsed Electromagnetic Fields Increased the Anti-Inflammatory Effect of A2A and A3 Adenosine Receptors in Human T/C-28a2 Chondrocytes and hFOB 1.19 Osteoblasts , 2013, PloS one.

[28]  F. Otsuka,et al.  Estrogen facilitates osteoblast differentiation by upregulating bone morphogenetic protein-4 signaling , 2013, Steroids.

[29]  Sarah L Dallas,et al.  The osteocyte: an endocrine cell ... and more. , 2013, Endocrine reviews.

[30]  Roland Baron,et al.  WNT signaling in bone homeostasis and disease: from human mutations to treatments , 2013, Nature Medicine.

[31]  H. Mckellop,et al.  The John Charnley Award: An Accurate and Extremely Sensitive Method to Separate, Display, and Characterize Wear Debris Part 2: Metal and Ceramic Particles , 2012, Clinical orthopaedics and related research.

[32]  Sarah L Dallas,et al.  Live imaging of bone cell and organ cultures. , 2012, Methods in molecular biology.

[33]  L. Bonewald,et al.  Cell line IDG‐SW3 replicates osteoblast‐to‐late‐osteocyte differentiation in vitro and accelerates bone formation in vivo , 2011, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[34]  F. Luyten,et al.  Does tranexamic acid stabilised fibrin support the osteogenic differentiation of human periosteum derived cells? , 2011, European cells & materials.

[35]  Y. Mikami,et al.  Dexamethasone modulates osteogenesis and adipogenesis with regulation of osterix expression in rat calvaria‐derived cells , 2011, Journal of cellular physiology.

[36]  L. Bonewald,et al.  Dynamics of the transition from osteoblast to osteocyte , 2010, Annals of the New York Academy of Sciences.

[37]  K. Foley,et al.  Randomized, prospective, and controlled clinical trial of pulsed electromagnetic field stimulation for cervical fusion. , 2004, The spine journal.

[38]  N. Selvamurugan,et al.  Effects of BMP‐2 and pulsed electromagnetic field (PEMF) on rat primary osteoblastic cell proliferation and gene expression , 2007, Journal of Orthopaedic Research.

[39]  A. Rodríguez-Navarro.  XRD2DScan: new software for polycrystalline materials characterization using two‐dimensional X‐ray diffraction , 2006 .

[40]  Wei-zhong Chang,et al.  Runx2 Integrates Estrogen Activity in Osteoblasts* , 2003, Journal of Biological Chemistry.

[41]  A. Boyde,et al.  Volumes From Which Calcium and Phosphorus X-Rays Arise in Electron Probe Emission Microanalysis of Bone: Monte Carlo Simulation , 2003, Calcified Tissue International.

[42]  Michael D Morris,et al.  Mineralization of Developing Mouse Calvaria as Revealed by Raman Microspectroscopy , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[43]  A. Chamson,et al.  Monitoring of ascorbate at a constant rate in cell culture: Effect on cell growth , 2001, In Vitro Cellular & Developmental Biology - Animal.

[44]  J. Clement,et al.  Age and temperature related changes to the ultrastructure and composition of human bone mineral , 1995, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[45]  V. Mooney.  Pulsed electromagnetic fields: an adjunct to interbody spinal fusion surgery in the high risk patient. , 1993, Surgical technology international.

[46]  D. Garland,et al.  Long-term follow-up of fracture nonunions treated with PEMFs. , 1991, Contemporary orthopaedics.

[47]  V. Mooney,et al.  A Randomized Double-Blind Prospective Study of the Efficacy of Pulsed Electromagnetic Fields for Interbody Lumbar Fusions , 1990, Spine.