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Disuse osteoporosis is a metabolic bone disease resulted from skeletal unloading (e.g., during extended bed rest, limb immobilization, and spaceflight), and the slow and insufficient bone recovery during re-ambulation 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 blocking 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 a mechanistic insight into disuse-induced deterioration of bone mechanosensitivity and a promising therapeutic approach to accelerate bone recovery after long-duration disuse.

Radiotherapy increases tumor cure and survival rates; however, radiotherapy-induced bone damage remains a common issue for which effective countermeasures are lacking, especially considering tumor recurrence risks. We report a high-specificity protection technique based on noninvasive electromagnetic field (EMF). A unique pulsed-burst EMF (PEMF) at 15 Hz and 2 mT induces notable Ca 2+ oscillations with robust Ca 2+ spikes in osteoblasts in contrast to other waveforms. This waveform parameter substantially inhibits radiotherapy-induced bone loss by specifically modulating osteoblasts without affecting other bone cell types or tumor cells. Mechanistically, primary cilia are identified as major PEMF sensors in osteoblasts, and the differentiated ciliary expression dominates distinct PEMF sensitivity between osteoblasts and tumor cells. PEMF-induced unique Ca 2+ oscillations depend on interactions between ciliary polycystins-1/2 and endoplasmic reticulum, which activates the Ras/MAPK/AP-1 axis and subsequent DNA repair Ku70 transcription. Our study introduces a previously unidentified method against radiation-induced bone damage in a noninvasive, cost-effective, and highly specific manner.

Memory impairment is one of the neuropsychological effects of hypobaric hypoxia (HH), which can be associated with programmed cell death, such as apoptosis and ferroptosis. Emerging evidence indicates crosstalk between apoptosis and ferroptosis, while the crosstalk between HH-induced apoptosis and ferroptosis in the hippocampus has not been clarified. Here, microarray profiles were extracted to analyze the differentially expressed genes with and without HH exposure, and keratin 18 (Krt18) was found to be a potential gene related to both apoptosis and ferroptosis. Then, we conducted morphological observations that showed that apoptosis and ferroptosis coexisted in the rat hippocampus after HH exposure. Combined with the real-time q-PCR analysis, the mRNA expression of Krt18 decreased significantly after HH exposure for 1 day and 3 days, and Mapk10 (JNK3) was upregulated at the corresponding time points. After exposure for 7 days, Krt18 and JNK3 showed no significant change. In conclusion, Krt18 may regulate apoptosis and ferroptosis simultaneously, possibly via the JNK signaling pathway, which might provide a potential central target for apoptosis and ferroptosis in hippocampal injury after HH exposure.

Type 2 diabetes mellitus (T2DM) results in compromised bone microstructure and quality, and subsequently increased risks of fractures. However, it still lacks safe and effective approaches resisting T2DM bone fragility. Pulsed electromagnetic fields (PEMFs) exposure has proven to be effective in accelerating fracture healing and attenuating osteopenia/osteoporosis induced by estrogen deficiency. Nevertheless, whether and how PEMFs resist T2DM-associated bone deterioration remain not fully identified. The KK-Ay mouse was used as the T2DM model. We found that PEMF stimulation with 2 h/day for 8 wk remarkably improved trabecular bone microarchitecture, decreased cortical bone porosity, and promoted trabecular and cortical bone material properties in KK-Ay mice. PEMF stimulated bone formation in KK-Ay mice, as evidenced by increased serum levels of bone formation (osteocalcin and P1NP), enhanced bone formation rate, and increased osteoblast number. PEMF significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. PEMF exerted beneficial effects on osteoblast- and osteocyte-related gene expression in the skeleton of KK-Ay mice. Nevertheless, PEMF exerted no effect on serum biomarkers of bone resorption (TRAcP5b and CTX-1), osteoclast number, or osteoclast-specific gene expression ( TRAP and cathepsin K). PEMF upregulated gene expression of canonical Wnt ligands (including Wnt1, Wnt3a, and Wnt10b), but not noncanonical Wnt5a. PEMF also upregulated skeletal protein expression of downstream p-GSK-3β and β-catenin in KK-Ay mice. Moreover, PEMF-induced improvement in bone microstructure, mechanical strength, and bone formation in KK-Ay mice was abolished after intragastric administration with the Wnt antagonist ETC-159. Together, our results suggest that PEMF can improve bone microarchitecture and quality by enhancing the biological activities of osteoblasts and osteocytes, which are associated with the activation of the Wnt/β-catenin signaling pathway. PEMF might become an effective countermeasure against T2DM-induced bone deterioration. NEW & NOTEWORTHY PEMF improved trabecular bone microarchitecture and suppressed cortical bone porosity in T2DM KK-Ay mice. It attenuated T2DM-induced detrimental consequence on trabecular and cortical bone material properties. PEMF resisted bone deterioration in KK-Ay mice by enhancing osteoblast-mediated bone formation. PEMF also significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. The therapeutic potential of PEMF on T2DM-induced bone deterioration was associated with the activation of Wnt/ß-catenin signaling.

Generally, for healthy adults, the entropy of electroencephalogram (EEG) signals gradually decreases from wake to sleep stages N1, N2, to N3, and increases during REM. However, some researchers found that multiscale entropy curves of sleep and wakefulness intercept, a cross-over phenomenon whose origin remains unexplored. The objective of the present work is to trace the origin of the cross-over phenomenon and to propose a workaround strategy. We simulated EEG by generating 1/f broadband signal and chirp signals with continuously varying frequencies. We then retrieved the rhythmic component from simulated EEG and real-world EEG and conducted MSE analysis of the instantaneous frequency variation (IFV) of the rhythmic component. The simulation revealed that this interception was ubiquitous in the MSE analysis of simulated EEG with rhythmic components of different frequencies. The cross-over point moved toward larger scale factors with the increasing sampling rate. We found that the MSE curve of IFV from real-world EEG for the wakefulness group was higher than that for sleep, showing no interception. These results suggest that (1) for a rhythmic signal like EEG, MSE analysis of the raw signal is highly affected by the rhythmic component, presenting artificial cross-over curves in sleep EEG study, (2) frequency variation of rhythmic components are complex signal which differs between wakefulness and sleep, in accordance with the complexity loss theory.

Quantifying the complexity of physiologic time series has long attracted interest from researchers. The multiscale entropy (MSE) algorithm is a prevailing method to quantify the complexity of signals in a variety of research fields. However, the MSE method assigns increased complexity to the mixed signal of a physiologic time series added with white noise, although the mixed signal should become less complex due to the broken correlation. In addition, the MSE method needs users to visually examine its scale dependence (shape) to better characterize the complexity of a physiologic process, which is sometimes not feasible. In this paper, we proposed a new method, namely the power-law exponent modulated multiscale entropy (pMSE), as a complexity measure for physiologic time series. We tested the pMSE method on simulated data and real-world physiologic interbeat interval time series and demonstrated that it could solve the above two difficulties of the MSE method. We expect that the proposed pMSE method or its future variants could serve as a useful complement to the MSE method for the complexity analysis of physiologic time series.

Intracerebral hemorrhage causes high mortality and morbidity, but its therapy methods are limited. In the present study, pulsed electromagnetic field (PEMF) was demonstrated to have beneficial effects on an intracerebral hemorrhage (ICH) model. This study explored the effects and underlying mechanisms of PEMF in a mouse model of ICH and cultured BV2 cells. PEMF was applied 4 hours after collagenase-induced ICH at day 0 and 4 hours per day for seven consecutive days. The expression levels of proinflammatory factors were assessed by ELISA kits and western blotting. Hematoma volume was measured by histological analysis. The effects of PEMF on phagocytosis of the erythrocytes were observed in cultured BV2 cells and ICH mouse models. Seven days after ICH, the hematoma volume was significantly reduced in PEMF-treated animals compared to nontreated mice. We found that PEMF decreased the hematoma volume and the expression levels of proinflammatory factors after ICH. Moreover, PEMF enhanced the erythrophagocytosis of microglia via CD36. Furthermore, we found that downregulation CD36 with Genistein blocked the effects of PEMF-induced hematoma clearance and anti-inflammations effects. Thus, the PEMF-mediated promotion of neurological functions may at least partly involve anti-inflammatory processes and hematoma clearance. These results suggest that PEMF treatment promoted the hematoma clearance and alleviated the inflammation after ICH.

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-3β and β-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.

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, and is related to disturbed lipid metabolism and redox homeostasis. However, a definitive drug treatment has not been approved for this disease. Studies have found that electromagnetic fields (EMFs) can ameliorate hepatic steatosis and oxidative stress. Nevertheless, the mechanism remains unclear.Methods: NAFLD models were established by feeding mice a high-fat diet. Simultaneously, EMF exposure is performed. The effects of the EMF on hepatic lipid deposition and oxidative stress were investigated. Additionally, the AMPK and Nrf2 pathways were analysed to confirm whether they were activated by the EMF.Results: Administration of the EMF decreased the body weight, liver weight and serum triglyceride (TG) levels and restrained the excessive hepatic lipid accumulation caused by feeding the HFD. This EMF function is achieved by boosting CaMKKβ protein expression, activating AMPK phosphorylation and suppressing mature SREBP-1c protein expression. Meanwhile, the activity of GSH-Px was enhanced following an increase in nuclear Nrf2 protein expression by EMF. However, no change was observed in the activities of SOD and CAT. Consequently, EMF reduced hepatic reactive oxygen species (ROS) and MDA levels, which means that EMF relieved liver damage by oxidative stress in HFD-fed mice.Conclusions: EMF can activate the CaMKKβ/AMPK/SREBP-1c and Nrf2 pathways to control hepatic lipid deposition and oxidative stress. This investigation indicates that EMF may be a novel therapeutic method for NAFLD.

Following radiotherapy, patients have decreased bone mass and increased risk of fragility fractures. Diabetes mellitus (DM) is also reported to have detrimental effects on bone architecture and quality. However, no clinical or experimental study has systematically characterized the bone phenotype of the diabetic patients following radiotherapy. After one month of streptozotocin injection, three-month-old male rats were subjected to focal radiotherapy (8 Gy, twice, at days 1 and 3), and then bone mass, microarchitecture, and turnover as well as bone cell activities were evaluated at 2 months post-irradiation. Micro-computed tomography results demonstrated that DM rats exhibited greater deterioration in trabecular bone mass and microarchitecture following irradiation compared with the damage to bone structure induced by DM or radiotherapy. The serum biochemical, bone histomorphometric, and gene expression assays revealed that DM combined with radiotherapy showed lower bone formation rate, osteoblast number on bone surface, and expression of osteoblast-related markers (ALP, Runx2, Osx, and Col-1) compared with DM or irradiation alone. DM plus irradiation also caused higher bone resorption rate, osteoclast number on bone surface, and expression of osteoclast-specific markers (TRAP, cathepsin K, and calcitonin receptor) than DM or irradiation treatment alone. Moreover, lower osteocyte survival and higher expression of Sost and DKK1 genes (two negative modulators of Wnt signaling) were observed in rats with combined DM and radiotherapy. Together, these findings revealed a higher deterioration of the diabetic skeleton following radiotherapy, and emphasized the clinical importance of health maintenance.

Cai, Jing, Junyong Ruan, Xi Shao, Yuanjun Ding, Kangning Xie, Chi Tang, Zedong Yan, Erping Luo, and Da Jing. Oxygen enrichment mitigates high-altitude hypoxia-induced hippocampal neurodegeneration and memory dysfunction associated with attenuated tau phosphorylation.

Microdamage accumulation contributes to impaired skeletal mechanical integrity. The bone can remove microdamage by initiating targeted bone remodeling. However, the spatiotemporal characteristics of microdamage initiation and propagation and their relationship with bone remodeling in response to fatigue loading, especially for more physiologically relevant daily bouts of compressive loading, remain poorly understood. The right forelimbs of 24 rats were cyclically loaded with a ramp waveform for 1,500 cycles/day, and contralateral ulnae were not loaded as the controls. The rats were divided into four equal groups and loaded for 1, 4, 7, and 10 days, respectively. We demonstrated that linear microcracking accumulation exhibited a non-linear time-varying process within 10 days of loading with peaked microcrack density at Day 7. Disrupted canaliculi surrounding linear microcracks showed high similarity with the temporal changes of linear microcracking accumulation. Observable intracortical resorption regions were found on Day 10. We found more linear microcracks accumulated in the tensile cortex, but longer cracks were observed in the compressive sides. Increased accumulation of diffuse microdamage was observed from Day 4, but no obvious peak was observed within the 10-day loading period. Diffuse damage first initiated in the compressive cortices but extended to tension from Day 7. The diffuse damage exhibited no impacts on the surrounding osteocyte integrity. Together, our findings revealed a time-dependent, bone remodeling-mediated varying process of linear microcracking accumulation following daily bouts of fatigue loading (with observable peak at Day 7 under our loading regime). Our study also identified distinct spatial accumulation of linear and diffuse microdamage in rat ulnae with tensile and compressive strains. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2112-2121, 2019.

This study was designed to investigate the neuroprotective effect of hyperoxygenate hydrogen-rich saline (HOHS) against brain injury induced by carbon monoxide (CO) poisoning in rats. A rat model of CO poisoning was established by administering CO via intraperitoneal injection to male Sprague-Dawley rats. Forty-eight adult male rats were randomly divided into the following groups: normal control group (NG), CO poisoning group (CO), HOS treatment group (hyperoxygenated solution, HOS) and HOHS treatment group (HOHS). After CO poisoning, the carboxyhemoglobin (COHb) contents in the blood of rats in all the CO poisoning groups were increased significantly. However, HOS and HOHS significantly decreased COHb contents, furthermore, the HOHS group had lower COHb contents than the HOS group. Arterial oxygen partial pressure (PaO2) and arterial oxygen saturation (SaO2) results showed that HOS and HOHS could improve the oxygenation of the rats with CO poisoning. Compared with the CO group, the HOS group and the HOHS group had persistently neuroprotective effect on CO-induced brain injury, as assessed by modified neurological severity score (mNSS), furthermore, the HOHS group had better neurological functional recovery than the HOS group. The neuronal apoptosis induced by CO was also evaluated. Except the NG group, all the CO-poisoning groups had varying degrees of neuronal apoptosis. There was lesser degree of neuronal apoptosis in both the HOS group and the HOHS group than that in the CO group. Moreover, the HOHS group had more minor degree of neuronal apoptosis than the HOS group. Compared with the CO group, the free radicals production in the HOS group and the HOHS group were significantly inhibited. In addition, there were significantly difference in the free radicals production between the HOS group and the HOHS group. We could conclude that HOHS exerted a stronger neuroprotective effect against CO-induced brain injury than HOS, and the neuroprotective mechanism of HOHS may be related with inhibition of both neuronal apoptosis and free radicals.

Chronic high-altitude hypoxia (HAH) results in compensatory pathological adaptations, especially in the cardiorespiratory system. The oxygen enrichment technology can provide long-lasting oxygen supply and minimize oxygen toxicity, which has proven to be effective to increase oxygen saturation, decrease heart rate, and improve human exercise performance after ascending to high altitudes. Nevertheless, it remains unknown whether oxygen enrichment can resist chronic HAH-induced cardiorespiratory alterations. Thirty-six male rats were equally assigned to the normal control (NC), HAH, and HAH with oxygen enrichment (HAHO) groups. The HAH and HAHO rats were housed in a hypobaric hypoxia chamber equivalent to 5,000 m for 4 weeks. The HAHO rats were exposed to oxygen-enriched air for 8 h/day. We found that oxygen enrichment mitigated the augmented skin blood flow and improved the locomotor activity of HAH-exposed rats. Oxygen enrichment inhibited HAH-induced increase in the production of red blood cells (RBCs). The hemodynamic results showed that oxygen enrichment decreased right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) in HAH-exposed rats. HAH-associated right ventricular hypertrophy and cardiomyocyte enlargement were ameliorated by oxygen enrichment. Oxygen enrichment inhibited HAH-induced excessive expression of cytokines associated with cardiac hypertrophy and myocardial fibrosis [angiotensin-converting enzyme (ACE)/angiotensin-converting enzyme 2 (ACE2), angiotensin II (Ang II), collagen type I alpha 1 (Col1α1), collagen type III alpha 1 (Col3α1), and hydroxyproline] in the right ventricle (RV). Oxygen enrichment inhibited medial thickening, stenosis and fibrosis of pulmonary arterioles, and cytokine expression related with fibrosis (Col1α1, Col3α1, and hydroxyproline) and pulmonary vasoconstriction [endothelin-1(ET-1)] in HAH-exposed rats. This study represents the first effort testing the efficacy of the oxygen enrichment technique on cardiopulmonary structure and function in chronic HAH animals, and we found oxygen enrichment has the capability of ameliorating chronic HAH-induced cardiopulmonary alterations.

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 (

Background: Conventional manual sleep stage classification is time-consuming and relies on the knowledge and experience of the specialists. The emergence of automatic sleep stage classification greatly improves the classification efficiency. The feature extraction in automatic sleep stage classification is particularly important, which usually uses the linear methods based on techniques in the time domain, frequency domain, or time-frequency domain. Electroencephalograms (EEGs) contain a wealth of physiological information, have been widely used for the classification of sleep stage. Due to the nonlinear, non-stationary, and multifractal characteristics of EEGs, some nonlinear methods have been used to extract features of sleep stages in recent years, such as complexity, multifractal theory, and chaos theory. The Wavelet Leader Multifractal Formalism (WLMF) of the multifractal theory is widely applied to different physiological signals. The current researches focus on discussing the mean H¨older exponent (h0) and the width of the multifractal singularity spectrum (WD(h)) estimated by the WLMF method. However, in the field of sleep staging, a number of researches focused on h0, but few studies on WD(h). Results: This paper aims to assess the multifractal characteristic for sleep EEG time series from the Sleep-EDF Expanded Database by the WLMF method. In the young group, the mean h0 increased from the Wake stage to the S3 stage (pConclusions: The result shows that WD(h) was influenced by aging. The gender and location of brain regions did not show significant influence on the multifractal characteristics of wakefulness and sleeping. This finding extends the application of the multifractal singularity spectrum on sleep staging, and raises a fundamental question on what might be the underlying mechanisms of the WD(h) reversion.

Background Bone marrow mesenchymal stem cells (BMSCs) have been used as important cell-based tools for clinical applications. Oxidative stress-induced apoptosis causes a low survival rate after transplantation, and the underlying mechanisms remain unknown. The endoplasmic reticulum (ER) and mitochondria are vital organelles regulated by adenosine monophosphate (AMP)-activated protein kinase (AMPK), especially during oxidative stress injury. Melatonin exerts an antioxidant effect by scavenging free radicals. Here, we aimed to explore whether cytoprotective melatonin relieves ER stress-mediated mitochondrial dysfunction through AMPK in BMSCs after oxidative stress injury. Methods Mouse BMSCs were isolated and exposed to H2O2 in the absence or presence of melatonin. Thereafter, cell damage, oxidative stress levels, mitochondrial function, AMPK activity, ER stress-related proteins, and apoptotic markers were measured. Additionally, the involvement of AMPK and ER stress in the melatonin-mediated protection of BMSCs against H2O2-induced injury was investigated using pharmacologic agonists and inhibitors. Results Melatonin improved cell survival and restored mitochondrial function. Moreover, melatonin intimately regulated the phosphorylation of AMPK and molecules associated with ER stress pathways. AMPK activation and ER stress inhibition following melatonin administration improved the mitochondrial membrane potential (MMP), reduced mitochondria-initiated oxidative damage, and ultimately suppressed apoptotic signaling pathways in BMSCs. Cotreatment with N-acetyl-l-cysteine (NAC) significantly enhanced the antioxidant effect of melatonin. Importantly, pharmacological AMPK activation/ER stress inhibition promoted melatonin-induced cytoprotection, while pharmacological AMPK inactivation/ER stress induction conferred resistance to the effect of melatonin against H2O2 insult. Conclusions Our data also reveal a new, potentially therapeutic mechanism by which melatonin protects BMSCs from oxidative stress-mediated mitochondrial apoptosis, possibly by regulating the AMPK-ER stress pathway.

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).

The effects of load-induced interstitial fluid shear stress (FSS) on instantaneous signaling response of osteocytes (e.g., calcium signaling) have been well documented. FSS can also initiate the release of many important messenger molecules of osteocytes (e.g., ATP and PGE2 ). However, the effects of FSS on cellular function and bone metabolism-modulating cytokine expression of osteocytes have not been fully identified (some inconsistent/conflicting results have been documented). Herein, osteocyte-like MLO-Y4 cells were stimulated with 1 Pa, 2-h FSS, and the effects of FSS on cellular morphology, cytoskeletal microstructure, biological activity, and gene and protein expression of important cytokines were investigated. SEM and cytoskeleton staining revealed that FSS induced well-organized cytoskeleton and increased filopodia processes. The osteocytic viability was sustained and apoptosis was inhibited via flow cytometry. FSS promoted Wnt3a and β-catenin gene and protein expression in 0-, 3-, and 6-h (sample collection time post FSS) groups. The FSS-stimulated cells in the 3-h group exhibited more significant effects on the promotion of OCN and Cx43 and inhibition of DKK1 and SOST expression than the 0- and 6-h groups. The 3-h group with FSS stimulation also showed the most prominent effects on suppressing RANKL and RANKL/OPG gene and protein expression. This study revealed a direct regulatory effect of FSS on osteocytic morphology and apoptotic characteristics, and showed that osteocyte-secreted bone metabolism-modulating molecule expression was regulated by FSS in a time-dependent manner. This study not only enriches our basic knowledge for understanding osteocytic mechanotransduction, but also provides important evidence for more scientific experimental design.

The effects of exogenous pulsed electromagnetic field (PEMF) stimulation on T1DM-associated osteopathy were investigated in alloxan-treated rabbits. We found that PEMF improved bone architecture, mechanical properties, and porous titanium (pTi) osseointegration by promoting bone anabolism through a canonical Wnt/β-catenin signaling-associated mechanism, and revealed the clinical potential of PEMF stimulation for the treatment of T1DM-associated bone complications. Type 1 diabetes mellitus (T1DM) is associated with deteriorated bone architecture and impaired osseous healing potential; nonetheless, effective methods for resisting T1DM-associated osteopenia/osteoporosis and promoting bone defect/fracture healing are still lacking. PEMF, as a safe and noninvasive method, have proven to be effective for promoting osteogenesis, whereas the potential effects of PEMF on T1DM osteopathy remain poorly understood. We herein investigated the effects of PEMF stimulation on bone architecture, mechanical properties, bone turnover, and its potential molecular mechanisms in alloxan-treated diabetic rabbits. We also developed novel nontoxic Ti2448 pTi implants with closer elastic modulus with natural bone and investigated the impacts of PEMF on pTi osseointegration for T1DM bone-defect repair. The deteriorations of cancellous and cortical bone architecture and tissue-level mechanical strength were attenuated by 8-week PEMF stimulation. PEMF also promoted osseointegration and stimulated more adequate bone ingrowths into the pore spaces of pTi in T1DM long-bone defects. Moreover, T1DM-associated reduction of bone formation was significantly attenuated by PEMF, whereas PEMF exerted no impacts on bone resorption. We also found PEMF-induced activation of osteoblastogenesis-related Wnt/β-catenin signaling in T1DM skeletons, but PEMF did not alter osteoclastogenesis-associated RANKL/RANK signaling gene expression. We reveal that PEMF improved bone architecture, mechanical properties, and pTi osseointegration by promoting bone anabolism through a canonical Wnt/β-catenin signaling-associated mechanism. This study enriches our basic knowledge for understanding skeletal sensitivity in response to external electromagnetic signals, and also opens new treatment alternatives for T1DM-associated osteopenia/osteoporosis and osseous defects in an easy and highly efficient manner.

Pulsed electromagnetic fields (PEMF) has been investigated as a noninvasive alternative method to prevent bone loss for postmenopausal osteoporosis (OP), and the bone tissue involved in these studies are usually long bones such as femur and tibia in OP patients or rat models. However, few studies have investigated the effects of PEMF on the vertebral bone in mice with OP. This study aimed to investigate whether PEMF preserve lumbar vertebral bone mass, microarchitecture and strength in ovariectomized (OVX) mouse model of OP and its associated mechanisms. Thirty 3-month-old female BALB/c mice were randomly divided into three groups (n=10): sham-operated control (Sham), ovariectomy (OVX), and ovariectomy with PEMF treatment (OVX+PEMF). The OVX+PEMF group was exposed to 15Hz, 1.6 mT PEMF for 8h/day, 7days/week. After 8weeks, the mice were sacrificed. The OVX+PEMF group showed lower body weight gain of mice induced by estrogen deficiency compared with OVX group. Biochemical analysis of serum demonstrated that serum bone formation markers including bone specific alkaline phosphatase (BALP), serum osteocalcin (OCN), osteoprotegerin (OPG) and N-terminal propeptide of type I procollagen (P1NP) were markedly higher in OVX+PEMF group compared with OVX group. Besides, serum bone resorption markers including tartrate-resistant acid phosphatase 5b (TRAP-5b) and C-terminal crosslinked telopeptides of type I collagen (CTX-I) were markedly lower in OVX+PEMF group compared with OVX group. Biomechanical test observed that OVX+PEMF group showed higher compressive maximum load and stiffness of the lumbar vertebrae compared with OVX group. Micro-computed tomography (μCT) and histological analysis of lumbar vertebrae revealed that PEMF partially prevented OVX-induced decrease of trabecular bone mass and deterioration of trabecular bone microarchitecture in lumbar vertebrae. Real-time PCR showed that the canonical Wnt signaling pathway of the lumbar vertebrae, including Wnt3a, LRP5 and β-catenin were markedly up-regulated in OVX+PEMF group compared with OVX group. Moreover, the mRNA expressions of RANKL and OPG were markedly up-regulated in OVX+PEMF group compared with OVX group, whereas no statistical difference in RANKL/OPG mRNA ratio was found between OVX+PEMF group and OVX group. Besides, our study also found that the RANK mRNA expression was down-regulated in OVX+PEMF group compared with OVX group. Taken together, we reported that long-term stimulation with PEMF treatment was able to alleviate lumbar vertebral OP in postmenopausal mice through a combination of increased bone formation and suppressed bone resorption related to regulating the skeletal gene expressions of Wnt3a/LRP5/β-catenin and OPG/RANKL/RANK signaling pathways.

Type 1 diabetes mellitus (T1DM) is associated with reduced bone mass, increased fracture risk, and impaired bone defect regeneration potential. These skeletal complications are becoming important clinical challenges due to the rapidly increasing T1DM population, which necessitates developing effective treatment for T1DM-associated osteopenia/osteoporosis and bone trauma. This study aims to investigate the effects of whole-body vibration (WBV), an easy and non-invasive biophysical method, on bone microstructure, tissue-level mechanical properties and porous titanium (pTi) osseointegration in alloxan-diabetic rabbits. Six non-diabetic and twelve alloxan-treated diabetic rabbits were equally assigned to the Control, DM, and DM with WBV stimulation (WBV) groups. A cylindrical drill-hole defect was established on the left femoral lateral condyle of all rabbits and filled with a novel non-toxic Ti2448 pTi. Rabbits in the WBV group were exposed to 1h/day WBV (0.3g, 30Hz) for 8weeks. After sacrifice, the left femoral condyles were harvested for histological, histomorphometric and nanoindentation analyses. The femoral sample with 2-cm height above the defect was used for qRT-PCR analysis. The right distal femora were scanned with μCT. We found that all alloxan-treated rabbits exhibited hyperglycemia throughout the experimental period. WBV inhibited the deterioration of cancellous and cortical bone architecture and tissue-level mechanical properties via μCT, histological and nanoindentation examinations. T1DM-induced reduction of bone formation was inhibited by WBV, as evidenced by elevated serum OCN and increased mineral apposition rate (MAR), whereas no alteration was observed in bone resorption marker TRACP5b. WBV also stimulated more adequate ingrowths of mineralized bone tissue into pTi pore spaces, and improved peri-implant bone tissue-level mechanical properties and MAR in T1DM bone defects. WBV mitigated the reductions in femoral BMP2, OCN, Wnt3a, Lrp6, and β-catenin and inhibited Sost mRNA expression but did not alter RANKL or RANK gene expression in T1DM rabbits. Our findings demonstrated that WBV improved bone architecture, tissue-level mechanical properties, and pTi osseointegration by promoting canonical Wnt signaling-mediated skeletal anabolic response. This study not only advances our understanding of T1DM skeletal sensitivity in response to external mechanical cues but also offers new treatment alternatives for T1DM-associated osteopenia/osteoporosis and osseous defects in an economic and highly efficient manner.

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.

Osteoporosis is a skeletal metabolic disease characterized by reduced bone mass and a high susceptibility to fractures, in which osteoblasts and osteoclasts are highly involved in the abnormal bone remodeling processes. Recently, low‑magnitude, high‑frequency whole‑body vibration has been demonstrated to significantly reduce osteopenia experimentally and clinically. However, the underlying mechanism regarding how osteoblastic activity is altered when bone tissues adapt to mechanical vibration remains elusive. The current study systematically investigated the effect and potential molecular signaling mechanisms in mediating the effects of mechanical vibration (0.5 gn, 45 Hz) on primary osteoblasts in vitro. The results of the present study demonstrated that low‑level mechanical stimulation promoted osteoblastic proliferation and extracellular matrix mineralization. In addition, it was also revealed that mechanical vibration induced improved cytoskeleton arrangement in primary osteoblasts. Furthermore, mechanical vibration resulted in significantly increased gene expression of alkaline phosphatase, bone morphogenetic protein 2 and osteoprotegerin, and suppressed sclerostin gene expression, as determined by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analyses. Mechanical vibration was observed to upregulate gene and protein expression levels of osteogenesis‑associated biomarkers, including osteocalcin and Runt‑related transcription factor 2. In addition, RT‑qPCR and western blotting analysis demonstrated that mechanical vibration promoted gene and protein expression of canonical Wnt signaling genes, including Wnt3a, low‑density lipoprotein receptor‑related protein 6 and β‑catenin. In conclusion, the present study demonstrated that mechanical vibration stimulates osteoblastic activities and may function through a potential canonical Wnt signaling‑associated mechanism. These findings provided novel information that improves the understanding of the molecular mechanisms involved in osteoblastic activities in response to mechanical vibration, which may facilitate the scientific application of mechanical vibration for the treatment of osteoporosis in the clinic.

The skeleton of type 1 diabetes mellitus (T1DM) has deteriorated mechanical integrity and increased fragility, whereas the mechanisms are not fully understood. Load-induced microdamage naturally occurs in bone matrix and can be removed by initiating endogenous targeted bone remodeling. However, the microdamage accumulation in diabetic skeleton and the corresponding bone remodeling mechanisms remain poorly understood. Herein, streptozotocin-induced T1DM rats and age-matched non-diabetic rats were subjected to daily uniaxial ulnar loading for 1, 4, 7, and 10 days, respectively. The SPECT/CT and basic fuchsin staining revealed significant higher-density spatial accumulation of linear and diffuse microdamage in diabetic ulnae than non-diabetic ulnae. Linear microcracks increased within 10-day loading in diabetic bone, whereas peaked at Day 7 in non-diabetic bone. Moreover, diabetic fatigued ulnae had more severe disruptions of osteocyte canaliculi around linear microcracks. Immunostaining results revealed that diabetes impaired targeted remodeling in fatigued bone at every key stage, including increased apoptosis of bystander osteocytes, decreased RANKL secretion, reduced osteoclast recruitment and bone resorption, and impaired osteoblast-mediated bone formation. This study characterizes microdamage accumulation and abnormal remodeling mechanisms in the diabetic skeleton, which advances our etiologic understanding of diabetic bone deterioration and increased fragility from the aspect of microdamage accumulation and bone remodeling.

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well-recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO-Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO-Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG-induced apoptosis in MLO-Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG-incubated MLO-Y4 cells were observed after mechanical vibration via the F-actin fluorescence staining. The real-time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, β-catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor-κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme-linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E2 and OPG, and inhibited the secretion of tumor necrosis factor-α and RANKL in the supernatant of HG-treated MLO-Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.

Traumatic brain injury (TBI) is common and often fatal in current times. The role of poly(adenosine diphosphate-ribose) polymerase (PARP)-induced cell death (parthanatos) in TBI has not been well studied. Our past study showed that oxidative stress-induced cell death includes parthanatos by confirming the occurrence of PARP activation and nuclear translocation of apoptosis-inducing factor (AIF). As oxidative stress plays a key role in pathological progression after TBI, we believe TBI may also be alleviated by the expression of Iduna, which is the only known endogenous regulator of parthanatos. Thus, a transection model in HT-22 cells was established for present study. Downregulation of Iduna aggravated the cell damage caused by mechanical cell injury, whereas upregulation of Iduna reduced mitochondrial dysfunction induced by mechanical cell injury but exerted no effect on apoptosis associated with mitochondrial dysfunction. By contrast, Iduna prevented parthanatos by reducing PARP activation and nuclear translocation of AIF. We also investigated 2 novel p53-MDM2 pathway inhibitors, AMG 232 and Nutlin-3, which substantially reduced the protective effects of Iduna. These findings indicate that Iduna might prevent TBI by specifically inhibiting parthanatos and promoting mitochondrial function, with the p53-MDM2 pathway playing a critical role.

Periodontal ligament stem cells (PDLSCs) have been confirmed to have self-renewal capacity and multidifferentiation potential and are good candidates for periodontal tissue regeneration. Pulsed electromagnetic field (PEMF) has been demonstrated to promote osteogenesis in non-union fractures, partly by regulating mesenchymal stem cells or osteoblast activity. However, there is no report about the osteo-inductive effect of PEMF stimulation on human PDLSCs (hPDLSCs). Thus, we tested the hypothesis that PEMF biophysical stimulation alone has an influence on the proliferation and osteogenic differentiation of hPDLSCs. To detect the osteo-inductive potential of bone morphogenetic protein (BMP9), we transfected the STRO-1(+) /CD146(+) hPDLCSs with BMP9-expressing recombinant adenoviruses. We examined the proliferation and osteogenic differentiation of hPDLSCs treated with either PEMF (15 Hz, 1 h daily, different intensities), or BMP9, or both stimuli. Cell counting kit-8 (CCK-8) assay showed that PEMF of different intensities had no effect on the proliferation of hPDLSCs and did not enhance the proliferative capability of BMP9-transfected cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting showed that the combination of both PEMFs (1.8 or 2.4 mT) and BMP9 stimulation had a synergistic effect on early and intermediate osteogenic genes and protein expressions of runt-related transcription factor 2, alkaline phosphatase, osteopontin, and late mineralized extracellular matrix formation in hPDLSCs. Bioelectromagnetics. 38:63-77, 2017. © 2016 Wiley Periodicals, Inc.