The Regulatory Effects Of Pulsed Electromagnetic Fields And Mechanical Vibration On The Behavior And Function Of Osteocytes And Osteoclasts

Osteoporosis is a common systemic metabolic bone disease characterized by decreased bone density,low bone mass,and deteriorated bone microarchitecture,which can lead to increased bone fragility and fracture risk.Due to the issues of the growing aging population in our country,the economic burden resulted from osteoporosis to our society has become increasingly prominent.However,the limitations of the current therapeutic methods for osteoporosis cannot be ignorable,including adverse effects(renal toxicity,esophageal irritation,etc.),poor long-term adherence and high costs.Pulsed electromagnetic fields(PEMF)and mechanical vibration are two non-invasive,inexpensive,safe,and effective methods for the biophysical treatment of osteoporosis.Although their efficacy has been identified in clinics,but their specific mechanisms of action and the optical parameters are still not clear.In this study,two kinds of biophysical factors with different parameters(PEMF and mechanical vibration)were applied to osteocytes and osteoclasts to detect the changes in their behaviors and functions.The underlying regulatory mechanisms of these two biophysical factors were also investigated.The results of the current study suggest that:(1)PEMF exhibit an intensity-dependent effect on the regulation of osteocytes and osteoclasts,and 5 G is the optimal parameter for PEMF to act on both cells to inhibit osteoclastic bone resorption;(2)Mechanical vibration has a frequency dependence on the regulation of two kinds of cells,and 70 Hz is the optimal parameter for inhibiting osteoclast differentiation and bone resorption capability in RAW264.7 cells,while 50 Hz is the optical frenquency for mechanical vibration to effect on osteocytes and then inhibits osteoclastogenesis and bone resorption capability of RAW264.7 cells derived osteoclasts;(3)MLO-Y4 cells regulate the differentiation of RAW264.7 cells into osteoclasts through the secretion of cytokines RANKL and OPG.The two physical factors can affect the secretion of RANKL and OPG and regulate the differentiation process.(4)Primary cilia play an essential role in detecting and sensing external PEMF signals in osteocyte-like MLO-Y4 cells.Part I: Effects of pulsed electromagnetic fields with different intensity on osteoclast formation,apoptosis and bone resorptionBACKGROUND: PEMF can increase bone mass in vivo,and the bone formation rate of osteoporosis animals is significantly increased.Moreover,osteoblast activity and osteoblast mineralization ability are also enhanced according to several in vitro experiments.It has been reported that PEMF stimulation can promote osteoblast formation,but there is still currently a lack of understanding of the regulation of osteoclast activity and function and related mechanisms when PEMF directly acts on osteoclasts.METHODS: RANKL-induced RAW264.7 cells were exposed to different intensities of PEMF(0,5,10,20 and 30 G)to detect the osteoclast formation,apoptosis and bone resorption capabilities.The changes of F-actin cytoskeletal structure of derived osteoclasts were detected by the phalloidin staining.The number of derived osteoclasts was quantified by the TRAP staining.Osteoclastic bone resorption capacity was detected by SEM sccaning.Apoptosis was detected by Annexin v-FITC/PI staining.qRT-PCR was performed to evaluate bone resorption and apoptosis-related gene expression.RESULTS: In the 5 G PEMF group,the number of TRAP positive osteoclasts was fewer than control group.The bone resorption capability and apoptosis of derived osteoclasts was inhibited in 5 G PEMF group.Moreover,most of the derived osteoclasts showed an immature cytoskeletal structure with podosomes.In 20 G and 30 G PEMF group,the cells showed the F-actin cytoskeletal structure of mature osteoclasts,the number of TRAP positive osteoclasts apoptotic osteoclasts increased.In addition,the gene expressions of RANK,NFATc1,TRAP,CTSK,BAX and BAX/BCL-2 were significantly decreased in the 5 G group and significantly increased in the 30 G PEMF group.CONCLUSION: PEMF has an intensity-dependent effect on the regulation of osteoclast formation,apoptosis,and bone resorption capacity.Furthermore,5 G can be considered as the optimal parameter for the osteoclast-associated anti-resorptive function of PEMF as osteoclast formation and bone resorption capability were inhibited.Part II: Osteocytes as mechanosensors in the regulation of osteoclastogenesis under pulsed electromagnetic fields stimulation needing the existence of primary ciliaBACKGROUND: Primary cilia are widely present on various cell surfaces,which can sense the changes of extracellular mechanical and chemical signals and assist their transduction into the interior of cells,and thereby causing a battery of intracellular events.In this experiment,we studied the responses of osteocytes to different intensities of PEMF stimulation.To inhibit the formation of primary cilia,MLO-Y4 cells were treated with si RNA to knockdown the expression of Polaris.This can be used to explore whether primary cilia are necessary for osteocytes to detect and respond to external PEMF signals.METHODS: Different intensities(0,5 and 30 G)of PEMF were used to stimulate MLO-Y4 cells,and their effects on cell proliferation,apoptosis,F-actin cytoskeleton structure,and realted gene expressions were systematically examined.The RAW264.7 cells were cultured in the conditioned media of MLO-Y4 cells in different groups to study osteoclastogenensis and bone resorption capabilities of derived osteoclasts.The proliferation of MLO-Y4 cells was detected by CCK-8.The cell apoptosis was detected by Annexin v-FITC/PI and flow cytometry.The cytoskeletal structure changes were detected by phalloidin staining.Primary cilia formation was disturbed by si RNA transfection.The number of osteoclasts was observed by TRAP staining.The bone resorption capacity of osteoclasts was detected by SEM and toluidine blue staining.The changes of secretory RANKL and OPG were detected by ELISA.The RANKL/OPG ratio and apoptosis-related genes were detected by qRT-PCR testing.RESULTS: The apoptosis of bone cells and actin cytoskeleton structure were regulated by PEMF stimulation.Osteoclast formation and bone resorption capacity of osteoclasts induced by PEMF-stimulated conditioned medium of MLO-Y4 cells were inhibited when stimulated with 5 G PEMF.After stimulated with PEMF,the expression levels of RANKL,BCL-2 and the ratio of RANKL/OPG were all significantly decreased.After the primary cilia of MLO-Y4 cells were disturbed,the inhibitory effects of 5 G PEMF-stimulated MLO-Y4 conditioned media on osteoclast activity and bone resorption were attenuated.CONCLUSION: Osteocytes can detect and sense external PEMF stimulation and the cell response showed an intensity-dependent manner.5 G is proved to be the optimal parameter for PEMF stimulation to inhibit bone resorption capability.The primary cilia of bone cells play an important role in detecting and processing external PEMF stimulation.Part III: Effects of vibrational stress with different frequencies on osteoclast formation and bone resorption in RAW264.7 cellsBackground: The inhibitive effects of mechanical vibration on bone loss have been systematically identified,whereas the specific regulatory mechanisms remains unclear.Furthermore,critical question regarding whether the osteogenic effects of mechanical vibration is mainly associated with the regulation of osteoblast activities and whether shows relationship with osteoclasts remain unclear.Therefore,investigating the effects of different frequencies of mechanical vibration on osteocytic activity,osteoclast differentiation,and bone resorption ability can be helpful for better understanding the underlying mechanism by which mechanical vibration inhibit the development of osteoporosis.METHODS: This experiment used different frequencies of mechanical vibration(Control,30,50 and 70 Hz)to directly stimulate the RAW264.7 cells.Then,the effects of mechanical vibration on osteoclast differentiation,maturation and bone resorption capabilities were observed.The F-actin cytoskeletal structure changes were detected by the phalloidin staining,and the osteoclast number was quantified by the TRAP staining.The bone resorption capacity of osteoclasts was evaluated by toluidine blue staining,and the changes of bone resorption capacity and apoptosis-related genes were detected by qRT-PCR.RESULTS: After stimulated with mechanical vibration at different frequencies,the numbers of osteoclasts derived by RAW264.7 cells were significantly reduced,the bone resorption capacity was weakened,the cytoskeletal structure was changed,and the cytoskeletal structures at 50 Hz and 70 Hz were highly deformed.At different frequencies,the expression levels of MMP-9,TRAP and CTSK were significantly inhibited.CONCLUSION: Mechanical vibration at different frequencies(0,30,50 and 70 Hz)can directily act on RANKL-induced RAW264.7 cells,and also significantly inhibit osteoclast formation and bone resorption capability,and the extent of inhibition is related to the frequencies of mechanical vibration.As the frequency increases,the effect of mechanical vibration on the number of osteoclasts generated and their bone resorption capacity become more pronounced.Our results highlight that the regulatory effect of mechanical vibration on the treatment of osteoporosis is not only associated with osteoblasts,but also shows close relationship with osteoclasts.Part IV: Effects of mechanical vibration at different frequencies on MLO-Y4 cells and in regulation of osteoclastogenesis and bone resorption capabilityBACKGROUND: This experiment aims to study the cell responses and molecular mechanisms of mechanical vibration in the treatment of osteoporosis by exerting anti-absorption in bones.METHODS: MLO-Y4 cells were exposed to different frequencies(0,30,50 and 70 Hz)of mechanical vibration,and the changes of cell apoptosis,F-actin cytosksleton structure and related gene expressions were systemically examined.The RAW264.7 cells were cultured in the conditioned media of MLO-Y4 cells with different vibiration frequencies to examine the osteoclast formation and bone resorption capabilities.Cell apoptosis was detected by Annexin v-FITC/PI and flow cytometry.The changes in cytoskeletal structure were detected by the phalloidin staining.The number of osteoclasts was observed by the TRAP staining.Osteoclastic bone resorption capability was detected by the toluidine blue staining.The RANKL and OPG secretion were detected by the ELISA assay,and RANKL/OPG pathway and apoptosis-related genes were detected by qRT-PCR.RESULTS: After MLO-Y4 cells were stimulated by mechanical vibrations with different frequencies,the apoptosis,RANKL/OPG ratio,the number of induced osteoclasts,and the bone resorption capacity were all significantly decreased.The inhibition effects were the most obvious in the 50 Hz group.Mechanical vibration with 70 Hz induced the most obvious changes of F-actin cytoskeletal structure of MLO-Y4 cells,as characterized by the cells were shrunkened and the intercellular synaptic junction network was destroyed.Conclusion: Mechanical vibration can inhibit the apoptosis of MLO-Y4 cells and decrease the ratio of RANKL/OPG.Osteocytes can regulate osteoclast differentiation and bone resorption of RAW264.7 cells by secreting soluble factors RANKL and OPG to inhibit osteoclastic activities.Mechanical vibration with 50 Hz can be ultilized as an optimal frequency parameter for osteoporosis treatment.