Effects of pulsed electromagnetic field (PEMF) stimulation on bone tissue like formation are dependent on the maturation stages of the osteoblasts

The effects of pulsed electromagnetic field (PEMF, 15 Hz pulse burst, 7 mT peak) stimulation on bone tissue-like formation on osteoblasts (MC3T3-E1 cell line) in different stages of maturation were assessed to determine whether the PEMF stimulatory effect on bone tissue-like formation was associated with the increase in the number of cells and/or with the enhancement of the cellular differentiation. The cellular proliferation (DNA content), differentiation (alkaline phosphatase activity), and bone tissue-like formation (area of mineralized matrix) were determined at different time points. PEMF treatment of osteoblasts in the active proliferation stage accelerated cellular proliferation, enhanced cellular differentiation, and increased bone tissue-like formation. PEMF treatment of osteoblasts in the differentiation stage enhanced cellular differentiation and increased bone tissue-like formation. PEMF treatment of osteoblasts in the mineralization stage decreased bone tissue-like formation. In conclusion, PEMF had a stimulatory effect on the osteoblasts in the early stages of culture, which increased bone tissue-like formation. This stimulatory effect was most likely associated with enhancement of the cellular differentiation, but not with the increase in the number of cells.     

Effects of different extremely low-frequency electromagnetic fields on osteoblasts

It is well known that the extremely low-frequency electromagnetic field (EMF) can promote the healing of bone fractures, but its mechanism remains poorly understood. The purpose of this study was to examine the response of neonatal rat calvarial bone cells to the rectangular electromagnetic field (REMF), triangular electromagnetic field (TEMF), sinusoidal electromagnetic field (SEMF), and pulsed electromagnetic field (PEMF). The stimulatory effects of EMF were evaluated by the proliferation (methyltetrazolium colorimetric assay), differentiation (alkaline phosphatase (ALP) activity), and mineralization (area of mineralized nodules of the cells). REMF treatment of osteoblasts increased cellular proliferation and decreased ALP activity (p < 0.05). TEMF had an accelerative effect on the cellular mineralized nodules (p < 0.05). SEMF treatment of osteoblasts decreased the cellular proliferation, increased ALP activity, and suppressed mineralized nodules formation (p < 0.05). PEMF promoted the proliferation of osteoblasts, inhibited their differentiation, and increased the mineralized nodules formation (p < 0.05). Moreover, the effects of PEMF on osteoblasts were concerned with the extracellular calcium, P2 receptor on the membrane, and PLC pathway, but the response of osteoblasts on SEMF was only related to PLC pathway. The results suggested that the waveforms of EMF were the crucial parameters to induce the response of osteoblasts.     

Effects of PEMF and glucocorticoids on proliferation and differentiation of osteoblasts

Pulsed electromagnetic fields (PEMF) can promote bone healing, while use of dexamethasone induces bone loss and osteoporosis. There is no report available on the combined effects of PEMF and dexamethasone on the activity of osteoblasts. Here, we investigated the effects of PEMF and dexamethasone on the proliferation and differentiation of MC3T3-E1 osteoblasts. Our results showed that PEMF and dexamethasone respectively increased and decreased the proliferation of MC3T3-E1 osteoblasts, meanwhile PEMF eliminated the effect of dexamethasone on MC3T3-E1 osteoblasts. Moreover, we also found that dexamethasone combined with PEMF upregulated the mRNA expression of IGF-1 at the early stage after the stimulation of PEMF and improved the decrease of COX-2 mRNA expression induced by dexamethasone at the late stage after the stimulation of PEMF. PEMF may be beneficial to improve dexamethasone-induced bone loss and osteoporosis.     

In vitro effects of low frequency electromagnetic fields on osteoblast proliferation and maturation in an inflammatory environment

An in vitro model was set up to investigate the effects of low frequency pulsed electromagnetic fields (PEMF) and its induced electric fields on osteoblast cells under inflammatory conditions. Osteoblasts (7F2) were seeded on top of chitosan scaffolds and co-cultured with macrophage cells (RAW 264.7) growing on the bottom of culture wells, stimulated by lipopolysaccharide to release reactive oxygen species including nitric oxide (NO). The co-culture was exposed to PEMF (magnitude of the magnetic field = 1.5 mT; induced electric voltage = 2.5 mV; frequency = 75 Hz; pulse duration = 1.3 ms) for 9 h. The osteoblasts were examined for their proliferation, viability, alkaline phosphatase (ALP) activity, and genetic expressions of type I collagen (COL I) and osteocalcin (OC), immediately and 7 days after PEMF exposure (days 0 and 7). Macrophage cell viability and NO concentration in the medium were monitored before and after PEMF exposure. The PEMF-exposed co-culture released a significantly higher amount of NO (65 μM) compared to control (17 μM) on day 7. Despite the high level of NO in the medium that was reported to be cytotoxic, PEMF-exposed osteoblasts had enhanced cell proliferation (23%), viability (36%), and COL I mRNA expression (3.4-fold) compared to the controls. The osteoblasts subjected to the PEMF had 41% less ALP activity than the control, which was associated with the active cell proliferation and COL I expression. The expression of OC mRNA was not seen in either the PEMF or control group, indicating cells had not entered the mineralization stage by day 7.     

The effects of pulsed electromagnetic fields on the cellular activity of SaOS-2 cells

Although pulsed electromagnetic fields (PEMFs) have been used for treatments of nonunion bone fracture healing for more than three decades, the underlying cellular mechanism of bone formation promoted by PEMFs is still unclear. It has been observed that a series of parameters such as pulse shape and frequency should be carefully controlled to achieve effective treatments. In this article, the effects of PEMFs with repetitive pulse burst waveform on the cellular activity of SaOS-2 osteoblast-like cells were investigated. In particular, cell proliferation and mineralization due to the imposed PEMFs were assessed through direct cell counts, the MTT assay, tissue nonspecific alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. PEMF stimulation with repetitive pulse burst waveform did not affect metabolic activity and cell number. However, the ALP activity of SaOS-2 cells and mineral nodule formation increased significantly after PEMF stimulation. These observations suggest that repetitive pulse burst PEMF does not affect cellular metabolism; however, it may play a role in the enhancement of SaOS-2 cell mineralization. We are currently investigating cellular responses under different PEMF waveforms and Western blots for protein expression of bone mineralization specific proteins.     

Effect of calcitonin gene-related peptide on osteoblast differentiation in an osteoblast and endothelial cell co-culture system

We have investigated the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on the differentiation of OB (osteoblasts) in co-culture with HUVEC (human umbilical vein endothelial cells). Primary human MOB (mandibular OB) and OB-like cells (MG-63) were either cultured directly or indirectly co-cultured with HUVEC at a 1:1 ratio. Expression of OC (osteocalcin) was measured by ELISA, and expression of ALP (alkaline phosphatase) and collagen mRNA was measured by quantitative fluorescent PCR. For mineralization nodus, OB were stained with Alizarin Red-S. When co-cultured with HUVEC, expression of OC and ALP mRNA were increased in MG-63 (P<0.01), and the expression of OC, ALP and collagen mRNA were increased in MOB (P<0.01 or 0.05). When treated with CGRP, OC and ALP mRNA and mineralization nodus numbers were increased in the MG-63 co-culture system (P<0.01 or 0.05); OC, ALP and collagen mRNA, and mineralization nodus numbers were increased in the MOB co-culture system (P<0.01 or 0.05). The effect of CGRP regulation on the differentiation of OB is not only direct but also indirect, via its effect on HUVEC and stimulation of OB.     

Role of diabetes- and obesity-related protein in the regulation of osteoblast differentiation

Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men.     

Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites.     

Nitric oxide mediates the effects of pulsed electromagnetic field stimulation on the osteoblast proliferation and differentiation.

The purpose of this research was to investigate whether the effects of pulsed electromagnetic field (PEMF) stimulation on the osteoblast proliferation and differentiation are mediated by the increase in the nitric oxide (NO, nitrogen monoxide) synthesis. The osteoblasts (MC3T3-E1 cell line) were cultured in the absence (-NMMA group) or in the presence (+NMMA group) of the NO synthase inhibitor L-NMMA. First, osteoblasts were subjected to PEMF stimulation (15 Hz and 0.6 mT) up to 15 days. The DNA content and the NO concentration in the conditioned medium were determined on the 3rd, 7th, and 15th days of culture. Following, osteoblasts were stimulated in the proliferation (P-NMMA and P+NMMA groups) or in the differentiation (D-NMMA and D+NMMA groups) stages of maturation, and the alkaline phosphatase (AlPase) activity was determined on the 15th day of culture for all groups. PEMF stimulation increased significantly the nitrite concentration in the -NMMA group on the 3rd, 7th, and 15th days of culture. However, this effect was partially blocked in the +NMMA group. The DNA content in the -NMMA group, but not in the +NMMA group, increased significantly on the 3rd and 7th days of culture. The AlPase activity in the P-NMMA and D-NMMA groups, but not in the P+NMMA and D+NMMA groups, also increased significantly. In conclusion, the PEMF stimulatory effects on the osteoblasts proliferation and differentiation were mediated by the increase in the NO synthesis.     

620nm低能量红光对骨髓间充质干细胞成骨分化的影响

为研究发光二极管(LED)发射的红光对大鼠骨髓间充质干细胞的成骨分化的影响,体外培养SD大鼠骨髓间充质干细胞,620 nm波长的LED置于细胞上方2 cm处,照射剂量分别为0,1,2和4J/cm2.采用CCK-8法检测照射后第2、4天的增殖活性,碱性磷酸酶(ALP)活性试剂盒与von kossa矿化结节染色法检测骨髓间...     

Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.     

Pulsed electromagnetic fields affect osteoblast proliferation and differentiation in bone tissue engineering

Bone tissue engineering is an interdisciplinary field involving both engineers and cell biologists, whose main purpose is to repair bone anatomical defects and maintain its functions. A novel system that integrates pulsed electromagnetic fields (PEMFs) and bioreactors was applied to bone tissue engineering for regulating osteoblast proliferation and differentiation in'vitro. Osteoblasts were acquired from the calvaria of newborn Wistar rats and isolated after sequential digestion. Poly(DL-lactic-co-glycolic acid) (PLGA) scaffolds were made by the solvent merging/particulate leaching method. Osteoblasts were seeded into porous PLGA scaffolds with 85% porosity and cultured in bioreactors for the 18-day culture period. Cells were exposed to PEMF pulsed stimulation with average (rms) amplitudes of either 0.13, 0.24, or 0.32 mT amplitude. The resulting induced electric field waveform consisted of single, narrow 300 micros quasi-rectangular pulses with a repetition rate of 7.5'Hz. The results showed that PEMF stimulation for 2 and 8 h at .13 mT increased the cell number on days 6 and 12, followed by a decrease on day 18 using 8 h stimulation. However, ALP activity was decreased and then increased on days 12 and 18, respectively. On the other hand, PEMF-treated groups (irrespective of the stimulation time) at 0.32 mT inhibited cell proliferation but enhanced ALP activity during the culture period. These findings suggested that PEMF stimulation with specific parameters had an effect on regulating the osteoblast proliferation and differentiation. This novel integrated system may have potential in bone tissue engineering.     

Nobiletin promotes osteogenic differentiation of human osteoblastic cell line (MG-63) through activating the BMP-2/RUNX-2 signaling pathway

Nobiletin (NOB) is polymethoxy flavonoids, which plentifully there in Citrus depressa and they demonstrate numerous pharmacological effects. NOB has an anti-proliferative effect, attenuates ovalbumin-treated eosinophilic airway inflammation and Type II collagen treated arthritis. NOB noticeably inhibits bone resorption and renovates bone loss in mice model, but role of NOB in bone metabolism is unclear. Human bone is a important organ that sustains its homeostasis among bone resorpting osteoclasts and bone developing osteoblasts. The balances of among these two kind of cell outcomes are implicated in bone remodeling. The current study designed to explore possessions of NOB on differentiation and proliferation of MG-63 cells and contribution of morphogenetic protein signaling. Cell proliferation was analyzed by MTT, mineralization analysis by alizarin red staining and morphogenetic signaling protein by RT-PCR. No stimulus outcome of NOB on cell proliferation was found at days of 1, 3 and 7. Accumulation of calcium was augmented after that treatment of NOB. The mRNA expression of BMP-2, COL-I, ALP, OCN, RUNX2 and COL1A1 augmented markedly with NOB supplement. Hence, NOB can stimulate osteogenic differentiation of MG-63, almost certainly by promoting RUNX2 and BMP-2 signaling and this result might provide to its action on stimulation of osteoblast development, differentiation and augments of bone mass.     

Intermittent pulsed electromagnetic field stimulation activates the mTOR pathway and stimulates the proliferation of osteoblast‐like cells

Pulsed electromagnetic fields (PEMFs) have been shown to be a noninvasive physical stimulant for bone fracture healing. However, PEMF stimulation requires a relatively long period of time and its mechanism of action has not yet been fully clarified. Recently, the mammalian target of rapamycin (mTOR) pathway has been shown to be involved in bone formation. This study aimed to investigate the effects of PEMFs on osteoblastic MC3T3‐E1 cells by examining various cellular responses including changes in the mTOR pathway. Continuous PEMF stimulation induced a transient phosphorylation of the mTOR pathway, whereas intermittent PEMF stimulation (1 cycle of 10 min stimulation followed by 20 min of stimulation pause) revitalized the reduced phosphorylation. Moreover, PEMF stimulation stimulated cell proliferation (bromodeoxyuridine incorporation) rather than differentiation (alkaline phosphatase activity), with a more notable effect in the intermittently stimulated cells. These results suggest that intermittent PEMF stimulation may be effective in promoting bone fracture healing by accelerating cell proliferation, and in shortening stimulation time. Bioelectromagnetics. 2019;40:412–421. © 2019 Bioelectromagnetics Society.     

Pulsed electromagnetic fields regulate osteocyte apoptosis,RANKL/OPG expression,and its control of osteoclastogenesis depending on the presence of primary cilia

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).