Erping Luo

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, β‐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.

Circadian rhythm affects the preventive role of pulsed electromagnetic fields on ovariectomy-induced osteoporosis in rats

Pulsed electromagnetic fields (PEMF) have been proved effective in the prevention of osteoporosis both experimentally and clinically. Chronotherapy studies have shown that circadian rhythm (CR) played an important role in the occurrence, development and treatment of several diseases. CR has also been recognized as an essential feature of bone metabolism. Therefore, it is of therapeutic significance to investigate the impact of CR on the efficacy of PEMF in the prevention of osteoporosis. However, this issue has never been discussed previously. The objective of this study was to systematically evaluate the impact of CR on the preventive effect of PEMF on osteoporosis in rats. Thirty-two 3 month old female Sprague-Dawley rats were randomly divided into four different groups: sham-operated control (Sham), ovariectomy (OVX), OVX with PEMF stimulation in daytime (OVX+DPEMF) and OVX with PEMF stimulation in nighttime (OVX+NPEMF) groups. The OVX+DPEMF and OVX+NPEMF groups were subjected to daily PEMF exposure on the 2nd post-operative day, from 9:00 to 15:00, and 0:00 to 6:00, respectively. After 12 weeks, the OVX+DPEMF group presented better efficacy in prevention against OVX-induced bone loss and deterioration of trabecular bone architecture compared with the OVX+NPEMF group. This was evidenced by the increased levels of femoral bone mineral density, trabecular area percentage, trabecular thickness, trabecular number and decreased trabecular separation. Furthermore, the bone turnover biomarkers (serum alkaline phosphatase, serum bone Gla protein and urinary deoxypyridinoline) and the dynamic histomorphometric parameters reflecting the trabecular osteoblast and osteoclast activity (bone formation rate with bone volume as referent, osteoclast number, etc.) in the OVX+DPEMF group decreased to a larger extent compared with the OVX+NPEMF group. In conclusion, the results indicated that CR was an important factor determining the preventive effect of PEMF on osteoporosis and PEMF exposure in the daytime presented better stimulus efficacy in rats. The findings might be helpful for the efficacious use of PEMF mediations, evaluation of PEMF action and experimental design in the future studies of biological effect of electromagnetic fields.

In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading

Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca2+) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca2+ responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid‐induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca2+ spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca2+ oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca2+ oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.—Jing, D., Baik, A. D., Lu, X. L., Zhou, B., Lai, X., Wang, L., Luo, E., Guo, X. E. In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. FASEB J. 28, 28–1582 (1592). www.fasebj.org

The effects of pulsed electromagnetic field on the functions of osteoblasts on implant surfaces with different topographies

The use of pulsed electromagnetic fields (PEMFs) is a promising approach to promote osteogenesis. However, few studies have reported the effects of this technique on the osseointegration of endosseous implants, especially with regard to different implant topographies. We focused on how the initial interaction between cells and the titanium surface is enhanced by a PEMF and the possible regulatory mechanisms in this study. Rat osteoblasts were cultured on three types of titanium surfaces (Flat, Micro and Nano) under PEMF stimulation or control conditions. Protein adsorption was significantly increased by the PEMF. The number of osteoblasts attached to the surfaces in the PEMF group was substantially greater than that in the control group after 1.5h incubation. PEMF stimulation oriented the osteoblasts perpendicular to the electromagnetic field lines and increased the number of microfilaments and pseudopodia formed by the osteoblasts. The cell proliferation on the implant surfaces was significantly promoted by the PEMF. Significantly increased extracellular matrix mineralization nodules were observed under PEMF stimulation. The expression of osteogenesis-related genes, including BMP-2, OCN, Col-1,ALP, Runx2 and OSX, were up-regulated on all the surfaces by PEMF stimulation. Our findings suggest that PEMFs enhance the osteoblast compatibility on titanium surfaces but to different extents with regard to implant surface topographies. The use of PEMFs might be a potential adjuvant treatment for improving the osseointegration process.

Pulsed Electromagnetic Fields Improve Bone Microstructure and Strength in Ovariectomized Rats through a Wnt/Lrp5/β-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 β-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/β-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.

Spatiotemporal properties of intracellular calcium signaling in osteocytic and osteoblastic cell networks under fluid flow

Mechanical stimuli can trigger intracellular calcium (Ca(2+)) responses in osteocytes and osteoblasts. Successful construction of bone cell networks necessitates more elaborate and systematic analysis for the spatiotemporal properties of Ca(2+) signaling in the networks. In the present study, an unsupervised algorithm based on independent component analysis (ICA) was employed to extract the Ca(2+) signals of bone cells in the network. We demonstrated that the ICA-based technology could yield higher signal fidelity than the manual region of interest (ROI) method. Second, the spatiotemporal properties of Ca(2+) signaling in osteocyte-like MLO-Y4 and osteoblast-like MC3T3-E1 cell networks under laminar and steady fluid flow stimulation were systematically analyzed and compared. MLO-Y4 cells exhibited much more active Ca(2+) transients than MC3T3-E1 cells, evidenced by more Ca(2+) peaks, less time to the 1st peak and less time between the 1st and 2nd peaks. With respect to temporal properties, MLO-Y4 cells demonstrated higher spike rate and Ca(2+) oscillating frequency. The spatial intercellular synchronous activities of Ca(2+) signaling in MLO-Y4 cell networks were higher than those in MC3T3-E1 cell networks and also negatively correlated with the intercellular distance, revealing faster Ca(2+) wave propagation in MLO-Y4 cell networks. Our findings show that the unsupervised ICA-based technique results in more sensitive and quantitative signal extraction than traditional ROI analysis, with the potential to be widely employed in Ca(2+) signaling extraction in the cell networks. The present study also revealed a dramatic spatiotemporal difference in Ca(2+) signaling for osteocytic and osteoblastic cell networks in processing the mechanical stimulus. The higher intracellular Ca(2+) oscillatory behaviors and intercellular coordination of MLO-Y4 cells provided further evidences that osteocytes may behave as the major mechanical sensor in bone modeling and remodeling processes.

Effects of 180 mT static magnetic fields on diabetic wound healing in rats.

Diabetic wound (DW) problems are becoming a formidable clinical challenge due to the sharp increase in the diabetic population and the high incidence of DW. Static magnetic field (SMF) therapy, an inexpensive and accessible noninvasive method, has been proven to be effective on various tissue repairs. However, the issue of the therapeutic effect of SMF on DW healing has never been investigated. The objective of this study was to systematically evaluate the effect of a 180 mT moderate-intensity gradient SMF on DW healing in streptozotocin-induced diabetic rats. Forty-eight 3-month-old male Sprague-Dawley rats (32 diabetic and 16 non-diabetic rats) were assigned to three equal groups: normal wound, DW, and DW + SMF groups. An open circular wound with 1.5 cm diameter was created in the dorsum. The wound was covered with a dressing and the magnet was fixed on top of the dressing. On days 5, 12, and 19, four rats of each group were euthanized and gross wound area, histology and tensile strength were evaluated. The wound area determination suggested that SMF significantly increased the healing rate and reduced the gross healing time. This result was further confirmed by histological observations. The wound tensile strength, reflecting the amount and quality of collagen deposition, increased to a larger extent in the DW + SMF group on days 12 and 19 compared with the DW group. The results indicated that 180 mT SMF presented a beneficial effect on DW healing, and implied the clinical potential of SMF therapy in accelerating DW repair and releasing the psychological and physical burdens of diabetic patients.

Pulsed electromagnetic fields promote osteogenesis and osseointegration of porous titanium implants in bone defect repair through a Wnt/β-catenin signaling-associated mechanism

Treatment of osseous defects remains a formidable clinical challenge. Porous titanium alloys (pTi) have been emerging as ideal endosseous implants due to the excellent biocompatibility and structural properties, whereas inadequate osseointegration poses risks for unreliable long-term implant stability. Substantial evidence indicates that pulsed electromagnetic fields (PEMF), as a safe noninvasive method, inhibit osteopenia/osteoporosis experimentally and clinically. We herein investigated the efficiency and potential mechanisms of PEMF on osteogenesis and osseointegration of pTi in vitro and in vivo. We demonstrate that PEMF enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton for in vitro osteoblasts seeded in pTi. PEMF promoted gene expressions in Runx2, OSX, COL-1 and Wnt/β-catenin signaling. PEMF-stimulated group exhibited higher Runx2, Wnt1, Lrp6 and β-catenin protein expressions. In vivo results via μCT and histomorphometry show that 6-week and 12-week PEMF promoted osteogenesis, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defect. PEMF promoted femoral gene expressions of Runx2, BMP2, OCN and Wnt/β-catenin signaling. Together, we demonstrate that PEMF improve osteogenesis and osseointegration of pTi by promoting skeletal anabolic activities through a Wnt/β-catenin signaling-associated mechanism. PEMF might become a promising biophysical modality for enhancing the repair efficiency and quality of pTi in bone defect.

Pulsed electromagnetic fields promote in vitro osteoblastogenesis through a Wnt/β-catenin signaling-associated mechanism

Substantial evidence indicates that pulsed electromagnetic fields (PEMF) could accelerate fracture healing and enhance bone mass, whereas the unclear mechanism by which PEMF stimulation promotes osteogenesis limits its extensive clinical application. In the present study, effects and potential molecular signaling mechanisms of PEMF on in vitro osteoblasts were systematically investigated. Osteoblast-like MC3T3-E1 cells were exposed to PEMF burst (0.5, 1, 2, or 6 h/day) with 15.38 Hz at various intensities (5 Gs (0.5 mT), 10 Gs (1 mT), or 20 Gs (2 mT)) for 3 consecutive days. PEMF stimulation at 20 Gs (2 mT) for 2 h/day exhibited most prominent promotive effects on osteoblastic proliferation via Cell Counting kit-8 analyses. PEMF exposure induced well-organized cytoskeleton, and promoted formation of extracellular matrix mineralization nodules. Significantly increased proliferation-related gene expressions at the proliferation phase were observed after PEMF stimulation, including Ccnd 1 and Ccne 1. PEMF resulted in significantly increased gene and protein expressions of alkaline phosphatase and osteocalcin at the differentiation phase of osteoblasts rather than the proliferation phase via quantitative reverse transcription polymerase chain reaction and Western blotting analyses. Moreover, PEMF upregulated gene and protein expressions of collagen type 1, Runt-related transcription factor 2 and Wnt/β-catenin signaling (Wnt1, Lrp6, and β-catenin) at proliferation and differentiation phases. Together, our present findings highlight that PEMF stimulated osteoblastic functions through a Wnt/β-catenin signaling-associated mechanism and, hence, regulates downstream osteogenesis-associated gene/protein expressions. Bioelectromagnetics. 37:152-162, 2016.

Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects.

Emerging evidence substantiates the potential of porous titanium alloy (pTi) as an ideal bone-graft substitute because of its excellent biocompatibility and structural properties. However, it remains a major clinical concern for promoting high-efficiency and high-quality osseointegration of pTi, which is beneficial for securing long-term implant stability. Accumulating evidence demonstrates the capacity of low-amplitude whole-body vibration (WBV) in preventing osteopenia, whereas the effects and mechanisms of WBV on osteogenesis and osseointegration of pTi remain unclear. Our present study shows that WBV enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton of primary osteoblasts in pTi. WBV upregulated osteogenesis-associated gene and protein expression in primary osteoblasts, including OCN, Runx2, Wnt3a, Lrp6 and β-catenin. In vivo findings demonstrate that 6-week and 12-week WBV stimulated osseointegration, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defects via μCT, histological and histomorphometric analyses. WBV induced higher ALP, OCN, Runx2, BMP2, Wnt3a, Lrp6 and β-catenin, and lower Sost and RANKL/OPG gene expression in rabbit femora. Our findings demonstrate that WBV promotes osteogenesis and osseointegration of pTi via its anabolic effect and potential anti-catabolic activity, and imply the promising potential of WBV for enhancing the repair efficiency and quality of pTi in osseous defects.