Effects of 10-week PEMF exposure on tibial Wnt1, LRP5, β-catenin, RANKL, RANK total mRNA expressions in OVX rats by RT-PCR analysis.

<p>(<b>A</b>) representative RT-PCR images for Wnt1, LRP5, β-catenin, RANKL, RANK and β-actin expressions, (<b>B</b>) Wnt1/β-actin ratio (<i>n</i> = 10), (<b>C</b>) LRP5/β-actin ratio (<i>n</i> = 10), (<b>D</b>) β-catenin/β-actin ratio (<i>n</i> = 10), (<b>E</b>) RANKL/β-actin ratio (<i>n</i> = 10) and (<b>F</b>) RANKL/β-actin ratio (<i>n</i> = 10) in rat tibiae of Control, OVX and OVX+PEMF groups. Control, sham-operated control group; OVX, ovariectomy group; OVX+PEMF, ovariectomy with PEMF exposure group; Values are all expressed as mean ± S.D. ^**Significant difference from the OVX group with <i>P</i><0.01.</p

3-D MicroCT images of trabecular bone microarchitecture in the distal femora in Control, OVX and OVX+PEMF rats.

<p>A volume of interest (VOI) with 1.6 mm height was selected for the analysis of trabecular bone microarchitecture, which is represented with yellow color in <b>Fig. 4A</b>. The VOI started at a distance of 0.4 mm (25 slices) from the lowest end of the growth plate and extended to the proximal end of the femur with a distance of 1.6 mm (100 slices). Representative 3-D MicroCT images of femoral trabecular bone microarchitecture determined by the VOI were shown in <b>Fig. 4B∼D</b>. (<b>Fig. 4B</b>): Control group; (<b>Fig. 4C</b>): OVX+PEMF group; (<b>Fig. 4D</b>): OVX group. The femur in the OVX group exhibited significant decrease in the trabecular number, trabecular connectivity and trabecular area as compared with that in the Control group. PEMF exposure partially inhibited OVX-induced trabecular bone loss and significantly improved trabecular bone mass and bone microarchitecture.</p

Electron micrographs of sciatic nerves in Control, DM, and DM+PEMF groups after a 7-week experimental period in all rats (magnification: ×6000).

<p>(<b>A</b>) Control group: Myelinated fiber had normal structure and morphology. Myelin sheath was in integrity and lined up in order. (<b>B</b>) DM group: Demyelination and axon enlargement were observed. Myelin sheath showed infolding, splitting, swelling and deformation, and layers were separated or disappeared. (<b>C</b>) DM+PEMF group: Myelin sheath of sciatic nerve was abnormal, the densities of layer on myelin sheath were uneven and rarefaction, but the damage was slighter than in the DM group.</p

Effects of 10-week PEMF exposure on femoral trabecular MicroCT indices in OVX rats, including (A) bone mineral density (BMD), (B) trabecular number (Tb.N), (C) trabecular thickness (Tb.Th), (D) trabecular separation (Tb.Sp), (E) bone volume per tissue volume (BV/TV) and (F) structure model index (SMI).

<p>Control, sham-operated control group; OVX, ovariectomy group; OVX+PEMF, ovariectomy with PEMF exposure group; Values are all expressed as mean ± S.D. (<i>n</i> = 10). ^bSignificant difference from the Control group with <i>P</i><0.01; ^adSignificant difference from the Control group with <i>P</i><0.05 and OVX group with <i>P</i><0.01.</p

Trends of TWT in Control, DM and DM+PEMF groups in weeks 0, 1, 3, 5 and 7 after PEMF stimulation.

<p>Data are presented as means ± SEM for 8 rats in each group. **P<0.01, statistically significant compared to the Control group, ^#P<0.05, statistically significant compared to the DM group (Bonferroni-adjusted pairwise comparison regarding the main group effect after two-way repeated measures ANOVA).</p

Trends of body weight in Control, DM and DM+PEMF groups in weeks 0, 1, 3, 5 and 7 after PEMF stimulation.

<p>Data are presented as means ± SEM for 8 rats in each group. **P<0.01, statistically significant compared to the Control group (Bonferroni-adjusted pairwise comparison regarding the main group effect after two-way repeated measures ANOVA).</p

Trends of blood glucose levels in Control, DM and DM+PEMF groups in weeks 0, 1, 3, 5 and 7 after PEMF stimulation.

<p>Data are presented as means ± SEM for 8 rats in each group. **P<0.01, statistically significant compared to the Control group (Bonferroni-adjusted pairwise comparison regarding the main group effect after two-way repeated measures ANOVA).</p

Schematic drawing of PEMF exposure system and PEMF pulse protocol.

<p>(<b>A</b>) Modified Helmholtz coils consisted of three identical coils with diameters of 800 mm which were in series connection and mounted coaxially at a distance of 304 mm apart. Two cubic plastic rat cages whose length was along O–Y direction (Origin is the center of the middle coil, O–X direction is the axial direction of the coil and the coordinate system meets right-hand rule) were put in the center of every two neighboring coils and cages were supported by stands to let the activities of rats restrict on the XY plane. The modified Helmholtz coils were wired to the GHY-III pulse generator. (<b>B</b>) The pulse stimulator (GHY-III) generated an open-circuit voltage waveform of PEMF with a repetitive burst frequency at 15 Hz (burst width, 5 ms; burst wait, 60 ms; pulse width, 0.2 ms; pulse wait, 0.02 ms; pulse rise and fall time: 0.3 µs, 2.0 µs).</p

Moderate-Intensity Rotating Magnetic Fields Do Not Affect Bone Quality and Bone Remodeling in Hindlimb Suspended Rats

<div><p>Abundant evidence has substantiated the positive effects of pulsed electromagnetic fields (PEMF) and static magnetic fields (SMF) on inhibiting osteopenia and promoting fracture healing. However, the osteogenic potential of rotating magnetic fields (RMF), another common electromagnetic application modality, remains poorly characterized thus far, although numerous commercial RMF treatment devices have been available on the market. Herein the impacts of RMF on osteoporotic bone microarchitecture, bone strength and bone metabolism were systematically investigated in hindlimb-unloaded (HU) rats. Thirty two 3-month-old male Sprague-Dawley rats were randomly assigned to the Control (<i>n</i> = 10), HU (<i>n</i> = 10) and HU with RMF exposure (HU+RMF, <i>n</i> = 12) groups. Rats in the HU+RMF group were subjected to daily 2-hour exposure to moderate-intensity RMF (ranging from 0.60 T to 0.38 T) at 7 Hz for 4 weeks. HU caused significant decreases in body mass and soleus muscle mass of rats, which were not obviously altered by RMF. Three-point bending test showed that the mechanical properties of femurs in HU rats, including maximum load, stiffness, energy absorption and elastic modulus were not markedly affected by RMF. µCT analysis demonstrated that 4-week RMF did not significantly prevent HU-induced deterioration of femoral trabecular and cortical bone microarchitecture. Serum biochemical analysis showed that RMF did not significantly change HU-induced decrease in serum bone formation markers and increase in bone resorption markers. Bone histomorphometric analysis further confirmed that RMF showed no impacts on bone remodeling in HU rats, as evidenced by unchanged mineral apposition rate, bone formation rate, osteoblast numbers and osteoclast numbers in cancellous bone. Together, our findings reveal that RMF do not significantly affect bone microstructure, bone mechanical strength and bone remodeling in HU-induced disuse osteoporotic rats. Our study indicates potentially obvious waveform-dependent effects of electromagnetic fields-stimulated osteogenesis, suggesting that RMF, at least in the present form, might not be an optimal modality for inhibiting disuse osteopenia/osteoporosis.</p></div

Trends of MWT in Control, DM and DM+PEMF groups in weeks 0, 1, 3, 5 and 7 after PEMF stimulation.

<p>Data are presented as means ± SEM for 8 rats in each group. **P<0.01, statistically significant compared to the Control group, ^#P<0.05, statistically significant compared to the DM group (Bonferroni-adjusted pairwise comparison regarding the main group effect after two-way repeated measures ANOVA).</p