Biological Effects And Mechanism Of Sinusoidal Electromagnetic Fields On Osteoarthritis Chondrocytes

Part? Effects of SEMF on the Characteristics and Morphology of Rat Chondrocytes and Determination of the Optimal Parameters of SEMFObjective: To investigate the effects of sinusoidal electromagnetic fields on the characteristics and morphology of chondrocytes in rat,and to determine the optimal parameters of sinusoidal electromagnetic fields.Methods: 1.Cell culture of the rat chondrocytes Chondrocytes were isolated from articular cartilage of rats(male Sprague Dawley,4–5 weeks)as approved by the Animal Care and Use Committee for Teaching and Research of Huazhong University of Science and Technology.2.Generate SEMF and exposure SEMF was generated by a pair of Helmhotz coil pair,amplifier,and waveform generator.An oscilloscope was used to verify the frequency as consistent and sinusoidal.3.Cell viability The effects of SEMF with different parameters on cell viability were determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazdium bromide(MTT)assay at specific time intervals of 1,3,5,7,14 and 21 days.4.Morphological observation of chondrocytes:To detect the effects of SEMF with different parameters on the morphology of chondrocytes,optical microscopy and phalloidin staining methods were used to detect chondrocytes morphology and cytoskeleton changes under SEMF exposure.5.Chondrocytes cartilage characteristicsTe invest the effects of SEMF with different parameters on the cartilage characteristics of chondrocytes,the DNA of chondrocytes and glycosaminoglycan content were detected and by using RT-q PCR technique and Western-blot to detect the cartilage cell characteristic differentiation gene(type II collagen,aggrecan and SOX-9)and dedifferentiation genes(type I collagen)expression changes.6.To determine the best parameters of SEMF on chondrocytes.Results The experimental results showed that different parameters of SEMF could maintain cartilage cell proliferation,which played a positive role in promoting the total content of DNA,cartilage cells,cartilage glycosaminoglycan content and related characteristics of cell differentiation gene,while the expression of dedifferentiated chondrocytes gene was inhibited.It was helpful to maintain the chondrocytes morphology and cytoskeleton.Among them,75 Hz 2.3m T was the most effective electromagnetic field parameter.Conclusions: The sinusoidal electromagnetic field has a positive effect on the characteristics and morphology of chondrocytes in articular cartilage,and the effect of 75 Hz,2.3m T is the most obvious.Part? Effects and Mechanism of SEMF on the Metabolism of Lubricin of Rat ChondrocytesObjective: To investigate the effects and mechanism of exposure for different durations with 75 Hz,2.3m T sinusoidal EMFs(SEMFs)on the metabolism of lubricin of rat chondrocytes cultured in vitro.Methods: 1.Cell culture of the rat chondrocytes Chondrocytes were isolated from articular cartilage of rats(male Sprague Dawley,4–5 weeks)as approved by the Animal Care and Use Committee for Teaching and Research of Huazhong University of Science and Technology.2.Generate SEMF and exposure SEMF was generated by a pair of Helmhotz coil pair,amplifier,and waveform generator.An oscilloscope was used to verify the frequency as consistent and sinusoidal.3.Cell viability The effects of SEMF with different duration on cell viability were determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazdium bromide(MTT)assay at specific time intervals of 1,3,7,14 and 21 days.4.The effect of SEMF on the lubricin metabolism of chondrocytes: The expression of lubricin in rat chondrocytes under different durations with SEMF stimulation was detected by RT-PCR,Western-blot and ELISA techniques.5.To study the relevant signaling pathway: RT-q PCR was used to detect the expression of TGF-?1 under different durations with SEMF stimulation;TGF-?1 kinase inhibitor SB431542 was used to block TGF signaling pathway,to detect the expression of lubricin in chondrocytes under the stimulation of SEMF;application of Western-blot to detect the expression of Smads protein to study the function of TGF-?/Smads signaling pathway in the process of the stimulation of SEMF.Results The experiment results showed that SEMF could maintain cartilage cell proliferation and promote the secretion of lubricin in chondrocytes,and these effects were proportional to the exposure time.In addition,after SEMF stimulation,the synthesis of TGF-?1 increased.The use of TGF-?1 kinase inhibitor SB431542 partially eliminated the stimulatory effect of SEMF on lubricin secretion.Signal transduction studies suggested that SEMF could promote the synthesis of Smad2,but the inhibitory effect of Smads(Smad6 and Smad7)expression was not observed.Conclusions We have shown that SEMF exposure can promote lubricin synthesis of rat chondrocytes in a time-dependent manner and TGF-?/Smads signaling pathway plays a partial role in this effect.Furthermore,to our knowledge,this is the first time the changes in lubricin properties caused by SEMF exposure were measured,and our results represent the first evidence that SEMF exposure has the ability to promote the synthesis of lubricin.