Effects of extremely low-frequency-pulsed electromagnetic field on different-derived osteoblast-like cells

The aim of this study is to investigate the effects of extremely low-frequency pulsed electromagnetic field (PEMF) on osteoblast-like cells. PEMF with a magnetic flux density of 1.55 mT at 48 Hz was employed to stimulate the MC3T3-E1 cell and the primary osteoblast cell derived from 2-day-old Sprague Dawley (SD) rat calvaria for different time. MTS method was applied to analyze cell proliferation and flow cytometry to detect cell cycle. The intracellular alkaline phosphatase (ALP) activity was measured by colorimetry. Our results demonstrated that PEMF of 1.55 mT at 48 Hz did not affect cell number of MC3T3-E1 cell, whereas the cell percentage of S and G(2)M phase decreased significantly. Although the cell number of the primary osteoblast cell did not alter by MTS assay after being exposed to PEMF for 24 h continuously, the cell percentage of S and G(2)M phase increased significantly. When culture time extended to 48 h, the cell number increased greatly and the cell percentage of S and G(2)M phase decreased significantly despite of the exposure type. After the primary osteoblast cell was exposed to PEMF for 24 h continuously, the ALP activity decreased significantly, whereas it increased significantly when being exposed to PEMF for 48 h continuously. From the results we concluded that PEMF of 1.55 mT at 48 Hz did not affect proliferation and differentiation of MC3T3-E1 cell, but it promoted proliferation, inhibited differentiation at proliferation stage, and promoted differentiation at differentiation stage of primary osteoblast cells.     

Effect of pulse-burst electromagnetic field stimulation on osteoblast cell activities

Electric stimulation has been used successfully to treat a wide range of bone disorders. However, the mechanism by which the electric fields can influence the bone cells behavior remains poorly understood. The purpose of this research was to assess the possible mechanism of the stimulatory effect of pulsed electromagnetic field (PEMF) on bone cells. A PEMF with a frequency of 15 Hz (1 G [0.1 mT]; electric field strength 2 mV/cm) were applied to neonatal mouse calvarial bone cell cultures for 14 days. The temporal effects of PEMF on the osteoblasts were evaluated by the status of proliferation, differentiation, mineralization, and gene expression on the 3rd, 5th, 7th, and 14th days of culture. Our results demonstrated that PEMF stimulation significantly increased the osteoblasts' proliferation by 34.0, 11.5, and 13.3% over the control group after 3, 5, and 7 days' culture. Although the alkaline phosphatase (ALP) staining and the mineralization nodules formation did not change, the ALP activity of the bone cells decreased significantly after PEMF stimulation. Under the PEMF stimulation, there was no effect on the extracellular matrix synthesis, while the osteoprotegerin (OPG) mRNA expression was up regulated and the receptor activator of NF-kappaB ligand (RANKL) mRNA expression were down regulated, compared to the control. In conclusion, the treatment by PEMF of osteoblasts may accelerate cellular proliferation, but did not affect the cellular differentiation. The effect of PEMF stimulation on the bone tissue formation was most likely associated with the increase in the number of cells, but not with the enhancement of the osteoblasts' differentiation.     

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.     

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.     

Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds

The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair.     

Cytokine release from osteoblasts in response to different intensities of pulsed electromagnetic field stimulation

We use an in-vitro osteoblast cell culture model to investigate the effects of low-frequency (7.5 Hz) pulsed electromagnetic field (PEMF) stimulation on osteoblast population, cytokines (prostaglandin E(2) (PGE(2)), transforming growth factor beta1(TGFbeta1), and alkaline phosphatase (ALP) activity to find the optimal intensity of PEMF for osteoblast growth. The results demonstrate that PEMF can stimulate osteoblast growth, release of TGFbeta1, and, in addition, an increase of ALP activity. The synthesis and release of PGE(2) in the culture medium are reduced with increasing numbers of cells. Higher intensity does not necessarily mean increased osteoblast growth, and the most efficient intensity is about 2 mV/cm in this case. Although the lower intensities of the PEMF are yet to be determined, the results of this study can shed light on the mechanisms of PEMF stimulation on non union fracture therapy and osteoporosis prevention in the future.     

Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF‐A independent mechanism

The clinically beneficial effect of low frequency pulsed electromagnetic fields (ELF‐PEMF) on bone healing has been described, but the exact mechanism of action remains unclear. A recent study suggests that there is a direct autocrine mitogenic effect of ELF‐PEMF on angiogenesis. The hypothesis of this study is that ELF‐PEMF also has an indirect effect on angiogenesis by manipulation of vascular endothelial growth factor (VEGF)‐A‐based paracrine intercellular communication with neighboring osteoblasts. Conditioned media experiments measured fetal rat calvarial cell (FRC) and human umbilical vein endothelial cell (HUVEC) proliferation using tritiated thymidine uptake. We demonstrate that ELF‐PEMF (15 Hz, 1.8 mT, for 8 h) has an indirect effect on the proliferation rate of both endothelial cells and osteoblasts in vitro by altering paracrine mediators. Conditioned media from osteoblast cells stimulated with ELF‐PEMF increased endothelial proliferation 54‐fold, whereas media from endothelial cells stimulated with ELF‐PEMF did not affect osteoblast proliferation. We examined the role of the pro‐angiogenic mediator VEGF‐A in the mitogenic effect of ELF‐PEMF‐stimulated osteoblast media on endothelial cells. The production of VEGF‐A by FRC as measured by ELISA was not changed by exposure to PEMF, and blocking experiments demonstrated that the ELF‐PEMF‐induced osteoblast‐derived endothelial mitogen observed in these studies was not VEGF‐A, but some other soluble angiogenic mediator. Bioelectromagnetics 30:189–197, 2009. © 2008 Wiley‐Liss, Inc.     

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.     

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.     

不同强度50 Hz脉冲电磁场促进大鼠颅骨成骨细胞矿化成熟最佳参数的筛选

为研究不同强度脉冲电磁场(pulse electromagnetic fields,PEMFs)对大鼠颅骨成骨细胞（rat skull osteoblasts,OB）增殖及成熟矿化的影响,将大鼠颅骨成骨细胞随机分为 7 组. 检测大鼠颅骨成骨细胞的增殖,细胞内碱性磷酸酶（ALP）活性变化,细胞沉积钙盐的情况,组织化学染色以及成骨细胞内标志性分子表达量的改变.结果显示,0.6 mT组促细胞增殖作用最强（P <0.01）;0.6 mT、1.8 mT、3.0 mT和3.6 mT均能提高ALP活性,其中0.6 mT ALP活性最高（P＜0.01）;在磁场处理4 ～12 d时细胞沉积钙盐逐渐增加,6种强度的脉冲电磁场均能促进钙盐沉积,尤以0.6 mT水平最高; ALP 染色、茜素红染色0.6 mT 组均显著高于对照组（P<0.01）;0.6 mT组 Bmp-2和Collagen-1 mRNA 的表达明显（P<0.01）高于对照组,磁场处理组Rankl mRNA 的表达均比对照组低. 0.6 mT 50 Hz 脉冲电磁场是促进成骨细胞增殖和矿化成熟的最佳参数,这为采用脉冲电磁场治疗骨质疏松症提供了治疗参数的基础支持.     

IN VITRO EFFECTS OF PEMFs ON BONE CELL CULTURES OF NORMAL AND OSTEOPENIC RAT

The aim of this study was to examine the effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on cell proliferation and differentiation in rat osteoblast primary cultures. Cells were obtained from normal and osteopenic rat bone and were named NB and OB, respectively. The osteoblastic phenotype was assessed by stimulation with 1,25(OH₂) vitamin D₃. NB and OB cells were seeded in multiwell plates and exposed to PEMFs for two different periods. Control cultures of both groups were incubated under the same conditions, with the pulse generator off. Assessment of PEMF effects was performed for the following parameters for each culture: alkaline phosphatase (ALP) activity, osteocalcin level, and MTT test. Results showed that OB and NB cell proliferation was significantly improved (p < 0.03, p = 0.04 respectively) after 48 h of PEMF exposure. Osteocalcin production of OB after 5 days of PEMF exposure was significantly higher than normal (p = 0.007) and osteopenic (p = 0.033) bone-derived controls. These results show that PEMFs act on osteopenic bone-derived osteoblasts, stimulating proliferation of cells and then, after a longer exposure, activating them.     

The biocompatibility of calcium phosphate cements containing alendronate-loaded PLGA microparticles in?vitro

The composite of poly-lactic-co-glycolic acid (PLGA) and calcium phosphate cements (CPC) are currently widely used in bone tissue engineering. However, the properties and biocompatibility of the alendronate-loaded PLGA/CPC (APC) porous scaffolds have not been characterized. APC scaffolds were prepared by a solid/oil/water emulsion solvent evaporation method. The morphology, porosity, and mechanical strength of the scaffolds were characterized. Bone marrow mesenchymal stem cells (BMSCs) from rabbit were cultured, expanded and seeded on the scaffolds, and the cell morphology, adhesion, proliferation, cell cycle and osteogenic differentiation of BMSCs were determined. The results showed that the APC scaffolds had a porosity of 67.43 ± 4.2% and pore size of 213 ± 95 µm. The compressive strength for APC was 5.79 ± 1.21 MPa, which was close to human cancellous bone. The scanning electron microscopy, cell counting kit-8 assay, flow cytometry and ALP activity revealed that the APC scaffolds had osteogenic potential on the BMSCs in vitro and exhibited excellent biocompatibility with engineered bone tissue. APC scaffolds exhibited excellent biocompatibility and osteogenesis potential and can potentially be used for bone tissue engineering.     

Electrical stimulation modulates osteoblast proliferation and bone protein production through heparin‐bioactivated conductive scaffolds

Electrical fields are known to interact with human cells. This principle has been explored to regulate cellular activities for bone tissue regeneration. In this work, Saos‐2 cells were cultured on conductive scaffolds made of biodegradable poly(L ‐lactide) and the heparin‐containing, electrically conducting polypyrrole (PPy/HE) to study their reaction to electrical stimulation (ES) mediated through such scaffolds. Both the duration and intensity of ES enhanced cell proliferation, generating a unique electrical intensity and temporal “window” within which osteoblast proliferation was upmodulated in contrast to the downmodulation or ineffectiveness in other ES regions. The favourable ES intensity (200 mV/mm) was further investigated in terms of the gene activation and protein production of two important osteoblast markers characterised by extracellular matrix maturation and mineralisation, that is alkaline phosphatase (ALP) and osteocalcin (OC). Both genes were found activated and the relevant protein production increased significantly following ES. In contrast, ES in the down‐modulation region (400 mV/mm) suppressed the production of both ALP and OC. This work demonstrated that important osteoblast markers can be modulated with specific ES parameters mediated through conductive polymer substrates, providing a unique strategy for bone tissue engineering. Bioelectromagnetics 34:189–199, 2013. © 2012 Wiley Periodicals, Inc.     

The frequency window effect of sinusoidal electromagnetic fields in promoting osteogenic differentiation and bone formation involves extension of osteoblastic primary cilia and activation of protein kinase A

Electromagnetic fields (EMFs) have emerged as a versatile means for osteoporosis treatment and prevention. However, its optimal application parameters are still elusive. Here, we optimized the frequency parameter first by cell culture screening and then by animal experiment validation. Osteoblasts isolated from newborn rats (ROBs) were exposed 90 min/day to 1.8 mT SEMFs at different frequencies (ranging from 10 to 100 Hz, interval of 10 Hz). SEMFs of 1.8 mT inhibited ROB proliferation at 30, 40, 50, 60 Hz, but increased proliferation at 10, 70, 80 Hz. SEMFs of 10, 50, and 70 Hz promoted ROB osteogenic differentiation and mineralization as shown by alkaline phosphatase (ALP) activity, calcium content, and osteogenesis-related molecule expression analyses, with 50 Hz showing greater effects than 10 and 70 Hz. Treatment of young rats with 1.8 mT SEMFs at 10, 50, or 100 Hz for 2 months significantly increased whole-body bone mineral density (BMD) and femur microarchitecture, with the 50 Hz group showing the greatest effect. Furthermore, 1.8 mT SEMFs extended primary cilia lengths of ROBs and increased protein kinase A (PKA) activation also in a frequency-dependent manner, again with 50 Hz SEMFs showing the greatest effect. Pretreatment of ROBs with the PKA inhibitor KT5720 abolished the effects of SEMFs to increase primary cilia length and promote osteogenic differentiation/mineralization. These results indicate that 1.8 mT SEMFs have a frequency window effect in promoting osteogenic differentiation/mineralization in ROBs and bone formation in growing rats, which involve osteoblast primary cilia length extension and PKA activation.     

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

Correlation between pulsed electromagnetic fields exposure time and cell proliferation increase in human osteosarcoma cell lines and human normal osteoblast cells in vitro

We have exposed cultured bone cells to a pulsed electromagnetic field (PEMF) for different times to find the minimal exposure time necessary to stimulate an increase of DNA synthesis. We used two different human osteosarcoma cell lines, TE-85 and MG-63, and human normal osteoblast cell (NHOC) obtained from surgical bone specimens. The cells were placed in multiwell plates and set in a tissue culture incubator between a pair of Helmoltz coils powered by a pulse generator (1.3-ms pulse, repeated at 75 Hz) for different periods of time. [3H]Thymidine incorporation was used to evaluate cell proliferation. The two osteosarcoma cell lines increase their thymidine incorporation when exposed to a PEMF for at least 30 min, both in a medium containing 10% fetal calf serum and in a serum-free medium. NHOC are known to increase their cell proliferation when exposed to PEMF but only if cultured in the presence of 10% fetal calf serum. In this experimental condition, three of the four cell lineages studied required at least 9 h of PEMF exposure to increase their DNA synthesis, whereas one cell lineage increased its cell proliferation after 6 h of PEMF exposure. Our observations confirm the hypothesis that the proliferative responses of NHOC and human osteosarcoma cell lines to PEMF exposure are quite different. Moreover, NHOC required minimal exposure times to PEMF to increase their cell proliferation, similar to that needed to stimulate bone formation in vivo.     

Growth and differentiation of PC6 cells: the effects of pulsed electromagnetic fields (PEMF)

Previous studies in our laboratory showed that neurite outgrowth in vitro and nerve regeneration in vivo were stimulated by 2 Hz, 0.3 mT (3 G) pulsed electromagnetic fields (PEMF). To learn more about the effects of PEMF on nerve cells, we exposed PC6 cells, a standard neuronal-like cell model, to the same pulsed electromagnetic fields for 2 h/day for 2 days and asked whether two different cell processes, proliferation and differentiation, were affected. The cells were also treated with a differentiating agent, nerve growth factor (NGF), to further define any interactive effects. We found that proliferation was unaffected by either PEMF or NGF alone or in combination. Differentiation, expressed as neurite outgrowth, was strongly upregulated with NGF, but this NGF response was significantly depressed in cells treated with PEMF.     

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.