Pulsed electromagnetic fields promote repair of focal articular cartilage defects with engineered osteochondral constructs

Articular cartilage injuries are a common source of joint pain and dysfunction. We hypothesized that pulsed electromagnetic fields (PEMFs) would improve growth and healing of tissue-engineered cartilage grafts in a direction-dependent manner. PEMF stimulation of engineered cartilage constructs was first evaluated in vitro using passaged adult canine chondrocytes embedded in an agarose hydrogel scaffold. PEMF coils oriented parallel to the articular surface induced superior repair stiffness compared to both perpendicular PEMF (p = .026) and control (p = .012). This was correlated with increased glycosaminoglycan deposition in both parallel and perpendicular PEMF orientations compared to control (p = .010 and .028, respectively). Following in vitro optimization, the potential clinical translation of PEMF was evaluated in a preliminary in vivo preclinical adult canine model. Engineered osteochondral constructs (∅ 6 mm × 6 mm thick, devitalized bone base) were cultured to maturity and implanted into focal defects created in the stifle (knee) joint. To assess expedited early repair, animals were assessed after a 3-month recovery period, with microfracture repairs serving as an additional clinical control. In vivo, PEMF led to a greater likelihood of normal chondrocyte (odds ratio [OR]: 2.5, p = .051) and proteoglycan (OR: 5.0, p = .013) histological scores in engineered constructs. Interestingly, engineered constructs outperformed microfracture in clinical scoring, regardless of PEMF treatment (p < .05). Overall, the studies provided evidence that PEMF stimulation enhanced engineered cartilage growth and repair, demonstrating a potential low-cost, low-risk, noninvasive treatment modality for expediting early cartilage repair.     

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells

Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF‐exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm², respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20–60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF‐exposed BMMSC showed multi‐lineage differentiation potential similar to the control group. Bioelectromagnetics 30:251–260, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of localized pulsed electromagnetic fields on tail-suspension osteopenia in growing mice

Pulsed magnetic fields (PEMFs) have been used effectively to treat bone fractures and sciatic-nerve-section-induced osteopenias. Properly applied PEMFs are presumed to stimulate osteogenesis. Mouse-tail suspension has been implemented as a means of inducing an osteopenic response in the long bones of the hind limbs. To evaluate localized PEMF effects, the mouse-suspension model was modified to accommodate the use of miniature wire coils affixed directly to the rear legs. Laterally and axially orientated PEMF effects were compared. Three test groups of mice included (C) control mice, (S) tail-suspended mice with treatment apparatus attached, and (SF) tail-suspended mice with apparatus attached and PEMFs delivered. The SF group was divided into mice receiving axial or lateral PEMFs. Significant bone changes occurred in suspended as compared with control mice after a 2-week test period. The PEMF mice showed significantly fewer osteopenic effects than did untreated, suspended mice. These findings are based on biomechanical measures of stiffness, strength, ductility, and energy as well as whole-bone mass and porosity. The effects of PEMFs on these properties differ for axial and lateral exposures. The results are discussed in terms of mechanisms underlying PEMF effects.     

Effects of Pulsed Electromagnetic Fields on the Proliferation of Lymphocytes from Aids Patients,HIV-Seropositive Subjects,and Seronegative Drug Users

The effect of the exposure of mitogen-stimulated lymphocytes from subjects infected by human immunodeficiency virus to extremely low frequency pulsed electromagnetic fields (PEMFs) was studied, by evaluating the incorporation of tritiated thymidine, the expression of IL-2 receptor, and the amount of activated T lymphocytes. Four groups of subjects were considered patients with acquired immunodeficiency syndrome (AIDS), asymptomatic seropositive subjects, seronegative drug users, and young healthy controls. PEMFs increased cell proliferation only in the group of healthy controls, as measured at the 72nd hour of culture, but an increase in the number of activated T lymphocytes was observed by cytofluorimetric analysis after 18 hrs of PEMF exposure in cultures from AIDS patients.     

Extremely low frequency pulsed electromagnetic fields increase cell proliferation in lymphocytes from young and aged subjects

The effect of the in vitro exposure to extremely low frequency pulsed electromagnetic fields (PEMFs) on the proliferation of human lymphocytes from 24 young and 24 old subjects was studied. The exposure to PEMFs during a 3-days culture period or during the first 24 hours was able to increase phytohaemagglutinin-induced lymphocyte proliferation in both groups. Such effect was greater in lymphocytes from old people which showed a markedly reduced proliferative capability and, after PEMF exposure, reached values of 3H-TdR incorporation similar to those of young subjects. The relevance of these data for the understanding and the reversibility of the proliferative defects in cells from aged subjects and for the assessment of risk related to the environmental exposure to PEMFs has to be considered.     

The anti-tumor effect of A3 adenosine receptors is potentiated by pulsed electromagnetic fields in cultured neural cancer cells

A(3) adenosine receptors (ARs) play a pivotal role in the development of cancer and their activation is involved in the inhibition of tumor growth. The effects of pulsed electromagnetic fields (PEMFs) on cancer have been controversially discussed and the detailed mechanisms are not yet fully understood. In the past we have demonstrated that PEMFs increased A(2A) and A(3)AR density and functionality in human neutrophils, human and bovine synoviocytes, and bovine chondrocytes. In the same cells, PEMF exposure increased the anti-inflammatory effect mediated by A(2A) and/or A(3)ARs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-tumor effect of A(3)ARs in PC12 rat adrenal pheochromocytoma and U87MG human glioblastoma cell lines in comparison with rat cortical neurons. Saturation binding assays and mRNA analysis revealed that PEMF exposure up-regulated A(2A) and A(3)ARs that are well coupled to adenylate cyclase activity and cAMP production. The activation of A(2A) and A(3)ARs resulted in the decrease of nuclear factor-kappa B (NF-kB) levels in tumor cells, whilst only A(3)ARs are involved in the increase of p53 expression. A(3)AR stimulation mediated an inhibition of tumor cell proliferation evaluated by thymidine incorporation. An increase of cytotoxicity by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation in PC12 and U87MG cells, but not in cortical neurons, was observed following A(3)AR activation. The effect of the A(3)AR agonist in tumor cells was enhanced in the presence of PEMFs and blocked by using a well-known selective antagonist. Together these results demonstrated that PEMF exposure significantly increases the anti-tumor effect modulated by A(3)ARs.     

Effects of 50 Hz pulsed electromagnetic fields on the growth and cell cycle arrest of mesenchymal stem cells: an in vitro study

Mesenchymal stem cells (MSCs) are capable of self-renew and multipotent differatiation which allows them to be sensitive to microenvironment is altered. Pulsed electromagnetic fields (PEMF) can affect cellular physiology of some types of cells. This study was undertaken to investigate the effects of PEMF on the growth and cell cycle arrest of MSCs expanded in vitro. To achieve this, cultured of normal rat MSCs, the treatment groups were respectively irradiated by 50 Hz PEMF at 10 mT of flux densities for 3 or 6 h. The effects of PEMF on cell proliferation, cell cycle arrest, and cell surface antigen phenotype were investigated. Our results showed that exposed MSCs had a significant proliferative capacity (P < 0.05) but the effect of PEMF for 3 and 6 h on cell growth was not different (P>0.05) at an earlier phase after PEMF treatment. Exposure to PEMF had a significant increase the percentage of MSCs in G1 phase compare with the control group, with a higher percentage of cells in G1 phase exposed for 6 h then that for 3 h. At the 16th hour after treatment, PEMF had no significant effect on cell proliferation and cell cycle (P>0.05). These results suggested that PEMF enhanced MSCs proliferation with time-independent and increased the percentage of cells at the G1 phase of the cell cycle in a time-dependent manner, and the effect of PEMF on the cell proliferation and cell cycle arrest of MSCs was temporal after PEMF treatment.     

Narrow-band ultraviolet B (NB UV-B) exposures improve mood in healthy individuals differently depending on chronotype

ABSTRACT

The evening chronotype is associated with psychological symptoms such as depressed mood, while skin exposure to ultraviolet radiation (UVR) may affect mood and behavior through neural and humoral routes. This pilot study aimed to investigate the impact of whole-body narrow-band (NB) UV-B exposure on current mood state and circulating 25-hydroxyvitamin D3 (25(OH)D₃), interleukin-6 (IL-6), cortisol and β-endorphin (β-END) levels in healthy participants. Here, eleven healthy women received full-body NB UV-B exposures on four afternoons, and the chronotype was assessed with a shortened version of Horne and Östberg’s Morningness-Eveningness Questionnaire (MEQ). Perceived mood was evaluated using the Visual Analogue Scale (VAS), and serum 25(OH)D₃, IL-6, cortisol and β-END concentrations were monitored daily. Decreasing VAS values showed mood to improve significantly over the five days after the four suberythematous NB UV-B exposures (p = .038), and the more the circadian preference was inclined toward eveningness, the greater the improvement in the mood dimension of wellbeing (p = .021). Baseline mood state was correlated with baseline 25(OH)D₃ (r = ?0.54, 95% CI: ?0.86 to ?0.09) and with baseline cortisol (r = ?0.57, 95% CI: ?0.87 to ?0.04). During the NB UV-B exposures, 25(OH)D₃ increased significantly, as expected, and IL-6 declined significantly by ?0.35 (95% CI: ?0.69 to ?0.07) pg/mL from the initial values of 1.12 ± 0.66 pg/mL (p = .025). In conclusion, in our pilot study, NB UV-B exposure improved mood, especially among those with evening preference for their daily activities, as well as circulating 25(OH)D₃ levels, whereas circulating IL-6 levels decreased.

Abbreviations: UVR: Ultraviolet radiation; NB UV-B: narrow-band UV-B; VAS: Visual Analogue Scales; β-END: β-endorphin; IL-6: Interleukin-6     

脉冲电磁场对家猪淋巴细胞的细胞遗传学效应

以家猪外周血淋巴细胞为材料,研究了脉冲电磁场（pulsing electromagnetic fields,简称PEMFS)树细胞的遗传学效应,实验发现,100和200kHz的PEMFs对家猪的淋巴细胞照射培养12,24,48h后,染色体畸变（包括非整倍体,染色体断裂等）频率明显高于对照组（P＜0．05）,其中,56％的染色体或染色单体断裂和42％的间隙发生在家猪常见染色体脆性位点部位,同时, 经100kHz和200kHz的PEMFs照射48h后,淋巴细胞姐妹染色单体交换（SCE）频率也明显高于对照组（P＜0．05）,实验结果表明,PEMFS能诱导DNA损伤和染色体畸变。     

In Vitro Evaluation of the Effects of Electromagnetic Fields used for Bone Healing

The use of therapeutic electromagnetic fields (EMF) for bone healing has positive clinical effects but may have adverse biologic effects. For this reason, EMF exposure has been repeatedly investigated to exclude the possibility of genotoxic effects and tumour risk. This paper describes the effects of EMFs on cell cultures. We analyzed the effects of EMF (28 gauss, 75 Hz) on growth and metabolic activities in four different cell types: L929 fibro-blasts, osteoblast-like HOS/TE85 cells, human lymphocytes, and rabbit chondrocytes. We found no cytotoxic or mutagenic effects on cultures exposed to EMF compared with unexposed controls. Results of cell proliferation showed a statistically significant increase for all cultures exposed to EMF with respect to controls (L929 +45%, p = 0.002; HOS/TE85 +32%, p = 0.001; chondrocytes +40%, p = 0.0003; lymphocytes +39%, p = 0.0002). Biochemical and enzymatic tests gave different results, depending on cell types: all tested values were increased after EMF exposure, even if only some of them reached statistical significance (total proteins: HOS/TE85 p = 0.004, chondrocytes p = 0.003; alkaline phosphatase: L929p = 0.0003, HOS/RE85 p = 0.0001, chondrocytes p = 0.009, lymphocytes p = 0.006; lactate dehydrogenase: chondrocytes p = 0.0002, lymphocytes p = 0.0005). Biochemical and enzymatic tests and cell proliferation results suggest a more active metabolism in cartilage and bone cells after EMF exposure. These effects could be relevant for bone healing in clinical practice.     

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.     

Optimum Parameters of Specific 7.5 Hz Single Pulsed Electromagnetic Field Stimulation on Osteoblast Growth

This study examines the response of different time constant 7.5 Hz pulsed electromagnetic field (PEMF) stimulation on rat osteoblasts and tries to determine the shortest exposure time to the selected time constant PEMF that is necessary to increase cell viability in vitro. We use an in vitro rat osteoblast model to investigate, for different periods of time (1, 2, or 3 days), rat osteoblasts to 7.5 Hz PEMF of different time constants (694, 432, and 268 µsec) or exposure time (20 min, 1, 3, 9, and 24 hr) and have evaluated the field's effects on the cell viability by colorimetric tetrazolium (MTT) assay and PGE₂ concentrations by enzyme‐linked immunosorbent assay (ELISA). It was shown that time constant was not the dominant parameter affecting osteoblast growth, and a short time exposure of PEMF 20 min/day could increase cell viability and PGE₂ secretion significantly.     

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.     

Pulsed electromagnetic fields promote bone formation by activating the sAC–cAMP–PKA–CREB signaling pathway

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP–PKA–CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC–cAMP–PKA–CREB signaling pathway of osteoblasts directly or indirectly.     

Changes in polyamines, c-myc and c-fos gene expression in osteoblast-like cells exposed to pulsed electromagnetic fields

Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.     

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.     

Changes in apoptotic rate and cell viability in three fish epidermis cultures after exposure to nonylphenol and to a wastewater sample containing low concentrations of nonylphenol

Three types of epidermal cultures of fish were used for toxicological investigations, a primary cell culture and a tissue culture prepared from the rainbow trout Oncorhynchus mykiss Walbaum and the cell line EPC, derived from a skin tumour of the carp Cyprinus carpio L. Two studies were carried out to compare the different culture systems. In the first cultures were incubated with nonylphenol and in the second set of experiments the cell cultures were exposed to a wastewater sample containing low concentrations of nonylphenol (NP). Both cell cultures were similarly sensitive to nonylphenol with respect to the endpoints cell viability (LC₅₀ (24 h) 47.1 μM NP (primary cell culture) and 44.2 μM NP (EPC)) values and apoptotic rate (significantly increased apoptotic rate after exposure to 50 μM NP for 24 h, p < 0.001 (primary cell culture), p = 0.008 (EPC)). The explant culture was slightly less sensitive (increased apoptotic rate after exposure to 50 μM NP for 24 h, but not significant: p = 0.385), which could be due to the capabilities of a differentiated tissue, providing more protective repair mechanisms, compared with single cells. All cultures revealed a concentration–response relationship for the endpoint apoptotic rate after the application of nonylphenol for 24 h. After wastewater exposure, a significant decrease in the apoptotic rate was measured in the primary cell culture (dilution wastewater : medium 1:1:p = 0.018; dilution wastewater : medium 1:2:p = 0.003), whereas the cell line EPC did not reveal any effects. Our results show that the endpoint apoptotic rate is more sensitive than the parameter cell viability for detecting adverse effects of a wastewater sample.     

INFLUENCE OF LOW-FREQUENCY ELECTRIC FIELDS ON PROLIFERATION AND IL-8 RELEASE OF HL-60 CELLS

The influence of capacitively coupled extremely low-frequency (ELF) electric fields on proliferation and on interleukin (IL)-8 release of exponentially growing HL-60 cells was examined. The cell suspensions were treated with the field component of interferential current (IFC) using different exposure protocols. Modulation frequencies of 10 and 100 Hz were applied with field strengths between 0.075 and 11.54 V_pp/cm for 48 hr using a 5-min exposure time at every 3 hr. At a field strength of 1 V_pp/cm, the influence of the time between two exposure sessions was examined for different modulation frequencies. All exposure protocols applied have no effect on cell proliferation (p>0.05), but statistical significant reduction (p<0.05) of the IL-8 release at selected modulation frequencies and interval times could be observed.