Glucose- and glutamine-dependent bioenergetics sensitize bone mechanoresponse after unloading by modulating osteocyte calcium dynamics.

Disuse osteoporosis is a metabolic bone disease resulting from skeletal unloading (e.g., during extended bed rest, limb immobilization, and spaceflight), and the slow and insufficient bone recovery during reambulation remains an unresolved medical challenge. Here, we demonstrated that loading-induced increase in bone architecture/strength was suppressed in skeletons previously exposed to unloading. This reduction in bone mechanosensitivity was directly associated with attenuated osteocytic Ca2+ oscillatory dynamics. The unloading-induced compromised osteocytic Ca2+ response to reloading resulted from the HIF-1α/PDK1 axis-mediated increase in glycolysis, and a subsequent reduction in ATP synthesis. HIF-1α also transcriptionally induced substantial glutaminase 2 expression and thereby glutamine addiction in osteocytes. Inhibition of glycolysis by blockade of PDK1 or glutamine supplementation restored the mechanosensitivity in those skeletons with previous unloading by fueling the tricarboxylic acid cycle and rescuing subsequent Ca2+ oscillations in osteocytes. Thus, we provide mechanistic insight into disuse-induced deterioration of bone mechanosensitivity and a promising therapeutic approach to accelerate bone recovery after long-duration disuse.

Pulsed Electromagnetic Fields Ameliorate Skeletal Deterioration in Bone Mass, Microarchitecture, and Strength by Enhancing Canonical Wnt Signaling-Mediated Bone Formation in Rats with Spinal Cord Injury.

Spinal cord injury (SCI) leads to extensive bone loss and high incidence of low-energy fractures. Pulsed electromagnetic fields (PEMF) treatment, as a non-invasive biophysical technique, has proven to be efficient in promoting osteogenesis. The potential osteoprotective effect and mechanism of PEMF on SCI-related bone deterioration, however, remain unknown. The spinal cord of rats was transected at vertebral level T12 to induce SCI. Thirty rats were assigned to the control, SCI, and SCI+PEMF groups (n = 10). One week after surgery, the SCI+PEMF rats were subjected to PEMF (2.0 mT, 15 Hz, 2 h/day) for eight weeks. Micro-computed tomography results showed that PEMF significantly ameliorated trabecular and cortical bone microarchitecture deterioration induced by SCI. Three-point bending and nanoindentation assays revealed that PEMF significantly improved bone mechanical properties in SCI rats. Serum biomarker and bone histomorphometric analyses demonstrated that PEMF enhanced bone formation, as evidenced by significant increase in serum osteocalcin and P1NP, mineral apposition rate, and osteoblast number on bone surface. The PEMF had no impact, however, on serum bone-resorbing cytokines (TRACP 5b and CTX-1) or osteoclast number on bone surface. The PEMF also attenuated SCI-induced negative changes in osteocyte morphology and osteocyte survival. Moreover, PEMF significantly increased skeletal expression of canonical Wnt ligands (Wnt1 and Wnt10b) and stimulated their downstream p-GSK3ß and ß-catenin expression in SCI rats. This study demonstrates that PEMF can mitigate the detrimental consequence of SCI on bone quantity/quality, which might be associated with canonical Wnt signaling-mediated bone formation, and reveals that PEMF may be a promising biophysical approach for resisting osteopenia/osteoporosis after SCI in clinics.

Differential skeletal response in adult and aged rats to independent and combinatorial stimulation with pulsed electromagnetic fields and mechanical vibration.

Pulsed electromagnetic fields (PEMFs) and whole-body vibration (WBV) are proved to partially preserve bone mass/strength in hindlimb-unloaded and ovariectomized animals. However, the potential age-dependent skeletal response to either PEMF or WBV has not been fully investigated. Moreover, whether the coupled "mechano-electro-magnetic" signals can induce greater osteogenic potential than single stimulation remains unknown. Herein, 5-month-old or 20-month-old rats were assigned to the Control, PEMF, WBV, and PEMF + WBV groups. After 8-week treatment, single PEMF/WBV enhanced bone mass, strength, and anabolism in 5-month-old rats, but not in 20-month-old rats. PEMF + WBV induced greater increase of bone quantity, quality, and anabolism than single PEMF/WBV in young adult rats. PEMF + WBV also inhibited bone loss in elderly rats by primarily improving osteoblast and osteocyte activity, but had no effects on bone resorption. PEMF + WBV upregulated the expression of various canonical Wnt ligands and downstream molecules (p-GSK-3ß and ß-catenin), but had no impacts on noncanonical Wnt5a expression in aged skeleton, revealing the potential involvement of canonical Wnt signaling in bone anabolism of PEMF + WBV. This study not only reveals much weaker responsiveness of aged skeleton to single PEMF/WBV relative to young adult skeleton, but also presents a novel noninvasive approach based on combinatorial treatment with PEMF + WBV for improving bone health and preserving bone quantity/quality (especially for age-related osteoporosis) with stronger anabolic effects.

Differential intensity-dependent effects of pulsed electromagnetic fields on RANKL-induced osteoclast formation, apoptosis, and bone resorbing ability in RAW264.7 cells.

Pulsed electromagnetic fields (PEMF) have been proven to be effective for promoting bone mass and regulating bone turnover both experimentally and clinically. However, the exact mechanisms for the regulation of PEMF on osteoclastogenesis as well as optical exposure parameters of PEMF on inhibiting osteoclastic activities and functions remain unclear, representing significant limitations for extensive scientific application of PEMF in clinics. In this study, RAW264.7 cells incubated with RANKL were exposed to 15 Hz PEMF (2 h/day) at various intensities (0.5, 1, 2, and 3 mT) for 7 days. We demonstrate that bone resorbing capacity was significantly decreased by 0.5 mT PEMF mainly by inhibiting osteoclast formation and maturation, but enhanced at 3 mT by promoting osteoclast apoptosis. Moreover, gene expression of RANK, NFATc1, TRAP, CTSK, BAX, and BAX/BCL-2 was significantly decreased by 0.5 mT PEMF, but increased by 3 mT. Our findings reveal a significant intensity window for low-intensity PEMF in regulating bone resorption with diverse nature for modulating osteoclastogenesis and apoptosis. This study not only enriches our basic knowledge for the regulation of PEMF in osteoclastogenesis, but also may lead to more efficient and scientific clinical application of PEMF in regulating bone turnover and inhibiting osteopenia/osteoporosis. Bioelectromagnetics. 38:602-612, 2017. © 2017 Wiley Periodicals, Inc.