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.
PEMF stimulates osteogenesis/mineralization in a variety of circumstances in the ring culture system. The most instructive circumstance was when PEMF was applied on Day 11 (Protocol B) (Fig 5), coincident with the change to Mineralization Medium, which provided for the first time an adequate phosphate source (3 mM beta-glycerol phosphate) to permit the deposition of mineral and ARS to measure it. Importantly, this was preceded by 10 days of culture in Differentiation Medium that drives osteoblast differentiation and the deposition of an extensive collagenous extracellular matrix, required as a template for mineral nucleation and accretion. Following a 3-day lag to Day 14, PEMF demonstrated a rapid increase in the mineral deposition, resulting in an approximately 4-fold higher ARS fluorescence than in the no-PEMF controls on Day 16. After Day 18, PEMF-treated cultures no longer demonstrated an enhanced rate of mineral deposition but accumulated ARS fluorescence at a rate similar to no-PEMF controls (Fig 5). Thus, the effect of PEMF treatment can be characterized in this protocol (B) as a lag, followed by enhanced mineralization, followed by cessation of stimulation of mineralization within 7 days. This character was specific to PEMF treatment since the cessation of stimulation has no parallel in the rate of mineralization in no-PEMF controls.
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