Fluid shear stress improves morphology, cytoskeleton architecture, viability, and regulates cytokine expression in a time-dependent manner in MLO-Y4 cells.

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 ß-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.

Effects of extremely low frequency pulsed magnetic fields on diabetic nephropathy in streptozotocin-treated rats.

BACKGROUND: Extremely low frequency pulsed magnetic fields (ELFPMF) have been shown to induce Faraday currents and measurable effects on biological systems. A kind of very high frequency electromagnetic field was reported that it improved the symptoms of diabetic nephropathy (DN) which is a major complication of diabetes. However, few studies have examined the effects of ELFPMF DN at the present. The present study was designed to investigate the effects of ELFPMF on DN in streptozotocin (STZ)-induced type 1 diabetic rats. METHODS: Adult male SD rats were randomly divided into three weight-matched groups: Control (non-diabetic rats without DN), DN + ELFPMF (diabetic rats with DN exposed to ELFPMF, 8 h/days, 6 weeks) and DN (diabetic rats with DN exposed to sham ELFPMF). Renal morphology was examined by light and electron microscopy, vascular endothelial growth factor (VEGF)-A and connective tissue growth factor (CTGF) were measured by enzyme linked immune sorbent assay. RESULTS: After 6 weeks' ELFPMF exposure, alterations of hyperglycemia and weight loss in STZ-treated rats with DN were not found, while both positive and negative effects of ELFPMF on the development of DN in diabetic rats were observed. The positive one was that ELFPMF exposure attenuated the pathological alterations in renal structure observed in STZ-treated rats with DN, which were demonstrated by slighter glomerular and tubule-interstitial lesions examined by light microscopy and slighter damage to glomerular basement membrane and podocyte foot processes examined by electron microscopy. And then, the negative one was that ELFPMF stimulation statistically significantly decreased renal expression of VEGF-A and statistically significantly increased renal expression of CTGF in diabetic rats with DN, which might partially aggravate the symptoms of DN. CONCLUSION: Both positive and negative effects of ELFPMF on the development of DN in diabetic rats were observed. The positive effect induced by ELFPMF might play a dominant role in the procession of DN in diabetic rats, and it is suggested that the positive effect should be derived from the correction of pathogenic diabetes-induced mediators.

Hierarchical Yolk-Shell Porous Ionic Liquids with Lower Viscosity for Efficient C3H6/C3H8 Adsorption and Separation.

Yolk-shell metal-organic framework (YS-MOF) liquids are candidate materials in large-size species with high-efficiency separation, owing to their hierarchical porosity, faster mass transfer, better compatibility, and higher solution processability than MOF liquids with micropores. Nevertheless, facile synthesis strategies of yolk-shell porous ionic liquids (YSPILs) with regulations of size and morphology are an ongoing challenge. Herein, we propose a general strategy to construct YSPILs based on Z67@PDA with tunable core sizes and morphologies. Benefiting from the unique hierarchical yolk-shell structure, as-prepared YSPILs exhibit promise in C3H6/C3H8 capture and separation with the increased sizes of core in yolk-shell ZIF-67@PDA. Advanced YS-MOF liquids have improved the adsorption properties and increased our ability to tailor chemical composition and pore architecture. Impressively, the adsorption capacity of C3H6 and C3H8 of YSPILs exhibits an approximately 3-fold enhancement compared with that of the neat ILs, confirming that the accessible porosities are retained. Effective C3H6/C3H8 separation performance of YSPILs over PILs based on ZIF-67, revealing the hierarchical porosity of YS-Z67@PDA liquids, benefits larger-size gas separation. Therefore, we believe that this work can not only help us to rationally design novel hierarchically porous ionic liquids but also promote candidate applications in large-size species separation, catalysis, and nanoreactors.

High-specificity protection against radiation-induced bone loss by a pulsed electromagnetic field.

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 Ca2+ oscillations with robust Ca2+ 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 Ca2+ 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.

Radiotherapy-induced bone deterioration is exacerbated in diabetic rats treated with streptozotocin.

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.

Pulsed electromagnetic fields (PEMF) attenuate changes in vertebral bone mass, architecture and strength in ovariectomized mice.

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 ß-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/ß-catenin and OPG/RANKL/RANK signaling pathways.

Radiotherapy-induced bone deterioration is exacerbated in diabetic rats treated with streptozotocin

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.

Moderate-intensity rotating magnetic fields do not affect bone quality and bone remodeling in hindlimb suspended rats.

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 (n = 10), HU (n = 10) and HU with RMF exposure (HU+RMF, n = 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.

Pulsed electromagnetic fields partially preserve bone mass, microarchitecture, and strength by promoting bone formation in hindlimb-suspended rats.

A large body of evidence indicates that pulsed electromagnetic fields (PEMF), as a safe and noninvasive method, could promote in vivo and in vitro osteogenesis. Thus far, the effects and underlying mechanisms of PEMF on disuse osteopenia and/or osteoporosis remain poorly understood. Herein, the efficiency of PEMF on osteoporotic bone microarchitecture, bone strength, and bone metabolism, together with its associated signaling pathway mechanism, was systematically investigated in hindlimb-unloaded (HU) rats. Thirty young mature (3-month-old), male Sprague-Dawley rats were equally assigned to control, HU, and HU + PEMF groups. The HU + PEMF group was subjected to daily 2-hour PEMF exposure at 15 Hz, 2.4 mT. After 4 weeks, micro-computed tomography (µCT) results showed that PEMF ameliorated the deterioration of trabecular and cortical bone microarchitecture. Three-point bending test showed that PEMF mitigated HU-induced reduction in femoral mechanical properties, including maximum load, stiffness, and elastic modulus. Moreover, PEMF increased serum bone formation markers, including osteocalcin (OC) and N-terminal propeptide of type 1 procollagen (P1NP); nevertheless, PEMF exerted minor inhibitory effects on bone resorption markers, including C-terminal crosslinked telopeptides of type I collagen (CTX-I) and tartrate-resistant acid phosphatase 5b (TRAcP5b). Bone histomorphometric analysis demonstrated that PEMF increased mineral apposition rate, bone formation rate, and osteoblast numbers in cancellous bone, but PEMF caused no obvious changes on osteoclast numbers. Real-time PCR showed that PEMF promoted tibial gene expressions of Wnt1, LRP5, ß-catenin, OPG, and OC, but did not alter RANKL, RANK, or Sost mRNA levels. Moreover, the inhibitory effects of PEMF on disuse-induced osteopenia were further confirmed in 8-month-old mature adult HU rats. Together, these results demonstrate that PEMF alleviated disuse-induced bone loss by promoting skeletal anabolic activities, and imply that PEMF might become a potential biophysical treatment modality for disuse osteoporosis.

Therapeutic effects of 15 Hz pulsed electromagnetic field on diabetic peripheral neuropathy in streptozotocin-treated rats.

Although numerous clinical studies have reported that pulsed electromagnetic fields (PEMF) have a neuroprotective role in patients with diabetic peripheral neuropathy (DPN), the application of PEMF for clinic is still controversial. The present study was designed to investigate whether PEMF has therapeutic potential in relieving peripheral neuropathic symptoms in streptozotocin (STZ)-induced diabetic rats. Adult male Sprague-Dawley rats were randomly divided into three weight-matched groups (eight in each group): the non-diabetic control group (Control), diabetes mellitus with 15 Hz PEMF exposure group (DM+PEMF) which were subjected to daily 8-h PEMF exposure for 7 weeks and diabetes mellitus with sham PEMF exposure group (DM). Signs and symptoms of DPN in STZ-treated rats were investigated by using behavioral assays. Meanwhile, ultrastructural examination and immunohistochemical study for vascular endothelial growth factor (VEGF) of sciatic nerve were also performed. During a 7-week experimental observation, we found that PEMF stimulation did not alter hyperglycemia and weight loss in STZ-treated rats with DPN. However, PEMF stimulation attenuated the development of the abnormalities observed in STZ-treated rats with DPN, which were demonstrated by increased hind paw withdrawal threshold to mechanical and thermal stimuli, slighter demyelination and axon enlargement and less VEGF immunostaining of sciatic nerve compared to those of the DM group. The current study demonstrates that treatment with PEMF might prevent the development of abnormalities observed in animal models for DPN. It is suggested that PEMF might have direct corrective effects on injured nerves and would be a potentially promising non-invasive therapeutic tool for the treatment of DPN.

Pulsed electromagnetic fields improve bone microstructure and strength in ovariectomized rats through a Wnt/Lrp5/ß-catenin signaling-associated mechanism.

Growing evidence has demonstrated that pulsed electromagnetic field (PEMF), as an alternative noninvasive method, could promote remarkable in vivo and in vitro osteogenesis. However, the exact mechanism of PEMF on osteopenia/osteoporosis is still poorly understood, which further limits the extensive clinical application of PEMF. In the present study, the efficiency of PEMF on osteoporotic bone microarchitecture and bone quality together with its associated signaling pathway mechanisms was systematically investigated in ovariectomized (OVX) rats. Thirty rats were equally assigned to the Control, OVX and OVX+PEMF groups. The OVX+PEMF group was subjected to daily 8-hour PEMF exposure with 15 Hz, 2.4 mT (peak value). After 10 weeks, the OVX+PEMF group exhibited significantly improved bone mass and bone architecture, evidenced by increased BMD, Tb.N, Tb.Th and BV/TV, and suppressed Tb.Sp and SMI levels in the MicroCT analysis. Three-point bending test suggests that PEMF attenuated the biomechanical strength deterioration of the OVX rat femora, evidenced by increased maximum load and elastic modulus. RT-PCR analysis demonstrated that PEMF exposure significantly promoted the overall gene expressions of Wnt1, LRP5 and ß-catenin in the canonical Wnt signaling, but did not exhibit obvious impact on either RANKL or RANK gene expressions. Together, our present findings highlight that PEMF attenuated OVX-induced deterioration of bone microarchitecture and strength in rats by promoting the activation of Wnt/LRP5/ß-catenin signaling rather than by inhibiting RANKL-RANK signaling. This study enriches our basic knowledge to the osteogenetic activity of PEMF, and may lead to more efficient and scientific clinical application of PEMF in inhibiting osteopenia/osteoporosis.