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

Paul D Benya; Aaron Kavanaugh; Martin Zakarian; Philip Söderlind; Tea Jashashvili; Nianli Zhang; Erik I Waldorff; James T Ryaby; Fabrizio Billi

doi:10.1371/journal.pone.0244223

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

PLoS One. 2021 Feb 4;16(2):e0244223. doi: 10.1371/journal.pone.0244223. eCollection 2021.

Authors

Paul D Benya¹, Aaron Kavanaugh¹, Martin Zakarian¹, Philip Söderlind², Tea Jashashvili³, Nianli Zhang⁴, Erik I Waldorff⁴, James T Ryaby⁴, Fabrizio Billi¹

Affiliations

¹ Department of Orthopaedic Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
² Department of Architecture and Urban Design, University of California Los Angeles, Los Angeles, California, United States of America.
³ Department of Radiology, Molecular Imaging Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
⁴ Orthofix Medical Inc., Lewisville, Texas, United States of America.

Abstract

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Proliferation / radiation effects
Cells, Cultured
Electromagnetic Fields*
Mice
Osteoblasts / radiation effects*
Osteogenesis / radiation effects*

Grants and funding

This research was supported by grants to FB from Orthofix Medical Inc, Lewisville, TX, USA; orthofix.com. PDB, AK, MZ, and FB, received salary support, and PS and TJ received fee-for-service support. NZ, EIW, and JTR are employed by Orthofix. Publication fees were paid by Orthofix. Orthofix participated in study design and preparation of the manuscript through the author contributions of NZ, EIW, and JTR. Support was also provided by grants to FB from the Orthopaedic Institute for Children, Los Angeles, CA, USA, OIC.com. FB received salary and general research support. OIC had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.