In Vitro And Vivo Studies On The Effects Of Electromagnetic Fields With Different Frequency Spectrum Bands On Ovariectomized Osteoporosis In Mice

Osteoporosis（OP）is a serious health problem that is especially related to aging in postmenopausal women,which is characterized by skeletal fragility and microarchitectural deterioration.As the aging process accelerates,the OP is an increasingly important global public health problem.Bone remodeling is a continuous process between bone resorption（activity of osteoclasts（OCs））and formation（activity of osteoblasts（OBs））.The absence of estrogen induced by the menopause increases the formation and the activity of OCs,which play key roles in bone loss,and OCs ultimately increase the risk of menopausal OP.Therefore,inhibiting the formation and function of OCs and enhance the formation and function of OBs are important therapeutic strategies.Various pharmacological treatments are available for postmenopausal OP,such as estrogen replacement therapy,bisphosphonate and calcitonin.However,long-term use of drugs can cause serious side effects such as breast cancer,endometrial cancer and cardiovascular disease.Thus,the safety of long-term use of medications to treat OP has caused great concern.An alternative therapy which is worthy of consideration in the treatment of OP is electromagnetic fields（EMF）,which has been investigated as a noninvasive alternative method.It has been reported that EMF can increase bone mineral density（BMD）in OP patients,prevent bone loss in OVX induced OP in vivo,and affect bone metabolism in vitro.However,the efficacy of this modality remains uncertain currently.Moreover,the studies also showed us the conflicting results.These different results are usually attributed to the different EMF platforms used by the various research groups,which are explained by different intensity,frequency,waveform and duration of EMF.This led people to question the scientific nature of EMF treatment of OP,so that the clinical application and promotion of EMF therapy in bone is hindered.Therefore,can we discard these numerous and complicated parameters to find other substitutes to accurately and simply describe the EMF,aiming to increase the repeatability and reproducibility of the experiments?There is a report implied that the dominant influence of EMF in OBs is not related to variables of EMF those expressed in the time domain,extension of the analysis to EMF characteristics expressed in the frequency domain should be encouraged.Therefore,focusing on the frequency spectrum of EMF might facilitate to investigate the possible mechanisms of EMF on bone healing.There are good evidences that low frequency EMF can produce resonance interactions that influence ion movements through membrane channels and other biological phenomena when the frequency of EMF matches cyclotron resonance frequencies of critical ions.Investigators have suggested that the physical mechanism underlying these effects is ion cyclotron resonance（ICR）.According to ICR model,the resonant frequencies of many biologically important ions,such as Na⁺,K⁺and Ca²⁺,are intermittent frequency points and fall within 1-100 Hz.Aparting from the fundamental frequency of resonant frequencies,when the frequency of EMF is equal to higher harmonics of the cyclotron frequencies,the biological resonant effectiveness might also be attained.Moreover,these higher harmonics of the cyclotron frequencies of the biologically relevant ions is blow 3,000 Hz.In addition,high frequency EMF is also capable of inducing osteogenic differentiation of osteoprogenitor cells.Therefore,we designed four kinds of EMF with different frequency spectrum bands（1-100 Hz,100-3,000 Hz,3,000-50,000 Hz and 1-50,000 Hz）,among which 1-100 Hz and 100-3,000 Hz are designated as ICR frequency bands.The purpose of this study was to explore the following four issues:（1）To develop a device capable of generating EMF with different frequency bands;（2）Effects of EMF with different frequency spectrum bands on the proliferation and differentiation in MC3T3-E1 osteoblast-like cells;（3）Effects of EMF with different frequency spectrum bands on RANKL-induced osteoclast differentiation and differentiation in RAW 264.7cells;（4）Effects of EMF with different frequency spectrum bands on OP in OVX mice.Part 1:To develop a device capable of generating EMF with different frequency bandsObjective:Based on the theory of ion cyclotron resonance（ICR）,an EMF generating device that can generate ICR frequency band,high frequency band,mixed frequency band including ICR frequency and high-frequency bands needs to be developed.Then,the device was used to investigate whether different frequency bands of EMF affect the proliferation and differentiation of MC3T3-E1 osteoblast-like cells,RANKL-induced osteoclast differentiation and activation in RAW 264.7 cells,and OP of OVX mice.Methods:The EMF signal was programed by the Labview software.Four kinds of EMF signals were utilized in our study,which were generated from uniform white noise by filtering with four kinds of signal filters.USB-6211 converted digital filtered EMF signals to analog signals.XP9900S with two output channels amplifies the EMF signals to drive the Helmholtz coilsResults:The open circuit voltage of EMF were low pass signal filtered at 1-100 Hz（LP）,band pass signal filtered at 100-3,000 Hz（BP）,high pass signal filtered at 3,000-50,000Hz（HP）and all pass signal filtered at 1-50,000 Hz（AP）.Each frequency component of these EMF had the same amplitude（-40 dB）.Conclusion:The device makes full use of the hardware and software resources of the computer,and has the advantages of strong functions,high cost performance,good scalability,and easy operation.Researchers can operate the virtual instrument panel through the graphical user interface,which greatly facilitates the development of experimental research.Part 2:Effects of EMF with different frequency spectrum bands on the proliferation and differentiation in MC3T3-E1 osteoblast-like cellsObjective:This study aimed to investigate the effects of four kinds of EMF with different frequency bands on the osteogenesis of MC3T3-E1 osteoblast-like cells.Methods:Five groups including Control,LP,BP,HP and AP were included in the experiments.LP,BP,HP and AP groups were stimulated with four kinds of EMF respectively three hours every day for six days.The study examined the cell proliferation,differentiation and osteogenesis-related genes and proteins.Results:Our results showed that LP,BP and AP increased the osteogenesis of the cells,evidenced by increased proliferation,alkaline phosphatase（ALP）activity and osteogenesis-related mRNA and protein expressions.No difference in proliferation and differentiation of MC3T3-E1 cells was present between LP and BP.Moreover,AP had more enhancement effects than LP and BP on the proliferation and differentiation of MC3T3-E1 cells.Besides,LP,BP and AP also enhanced the osteoclastogenesis of the cells,evidenced by increased RANKL/OPG mRNA and protein ratio.HP inhibited both osteogenesis and osteoclastogenesis of the cells.Conclusion:The results of the study showed that the LP and BP containing ionic ICR frequencies not only promoted the differentiation of MC3T3-E1 cells into OBs,but also indirectly promoted the formation of osteoclasts.HP,which does not contain the ICR frequencies,inhibits the differentiation of MC3T3-E1 cells into OBs.At the same time,the AP has accumulative effect of LP,BP and HP,which significantly promotes the differentiation of MC3T3-E1 cells into OBs,and indirectly promotes the osteoclasts caused by the enhanced ability of OBs formation.Part 3:Effects of EMF with different frequency spectrum bands on RANKL-induced osteoclast differentiation and differentiation in RAW 264.7 cells Objective:This study aimed to investigate the effects of four kinds of EMF with different frequency bands on RANKL-induced osteoclast differentiation and activation in RAW264.7 cells.Methods:Six groups including Control,RL,RL+LP,RL+BP,RAL+HP and RL+AP were used in the experiments.After the cells were plated into culture plates and allowed to confluence,receptor activator of NF-κB ligand（RANKL,50 ng/ml）were added to RANKL,RANKL+LP,RANKL+BP,RANKL+HP and RANKL+AP groups except Control group.Then,RANKL+LP,RANKL+BP,RANKL+HP and RANKL+AP were stimulated for three hours every day for six days with four kinds of different EMF respectively,except Control and RANKL group.The study examined the cell proliferation,apoptosis,tartrate-resistant acid phosphatase（TRAP）staining,actin rings and hoechst33342 staining and osteoclastogenesis-related protein expression.Results:After 2 days of exposure,there was no difference in cell proliferation between the groups.After 6 days of exposure,there was no significant difference in cell proliferation between Control,RL,RL+LP and RL+BP groups.LP and BP had no effect on RANKL-induced OCs proliferation.HP and AP significantly inhibited RANKL-induced OCs proliferation（P<0.01）.EMF with four kinds of frequency bands had no effect on RANKL-induced apoptosis of OCs for 3 days.Six days after exposure,LP and BP had no effect on RANKL-induced apoptosis of OCs.HP and AP significantly promoted early apoptosis of OCs induced by RANKL and had no effect on late apoptosis.RANKL could induce mononuclear RAW 264.7 osteoclast precursor cells to differentiate into mature multinuclear OCs.Six days of LP and BP exposure had no effect on the formation ability of OCs.HP and AP inhibited the formation of OCs.The RANKL-induced OCs have a clear and regular skeleton distribution,and many filopodia are formed around the cells,and intracellular F-actin fluorescence is strong.LP and BP have no effect on the OCs skeleton.HP,AP shrink the cytoskeleton,F-actin fluorescence is more or less,and some appear nuclear lysis.Western blot results showed that the RANK protein was not expressed in the Control group.RANKL induced the formation of multinuclear OCs,and RANKL induced high expression of RANK protein.Compared with RANKL-induced group,LP and BP did not change RANK expression（P>0.05）,and there was no difference between LP and BP.In addition,compared with RANKL-induced group,HP and AP significantly decreased RANK expression（P<0.01）,while there was no difference between HP and AP.Conclusion:RANKL can induce mononuclear RAW 264.7 osteoclast precursor cells to differentiate into mature multinuclear OCs.LP and BP have no effect on RANKL-induced proliferation of RAW 264.7 osteoclast precursor cells and have no effect on OCs formation ability.HP and AP inhibition RANKL-induced RAW 264.7 osteoclast precursor cell proliferation and inhibition of OCs formation.Part 4:Effects of EMF with different frequency spectrum bands on ovariectomizedosteoporosis in miceObjective:This study aimed to investigate the effects of EMF with different frequency spectrum bands on ovariectomized（OVX）osteoporosis（OP）in mice.Methods:Forty-eight 3-month-old female BALB/c mice were subjected either a sham surgery or bilateral ovariectomy,and all mice were randomly divided into the following six groups（eight mice in each group）:sham-operated control group（Sham）,ovariectomy group（OVX）,OVX+LP exposure group,OVX+BP exposure group,OVX+HP exposure group and OVX+AP exposure group.OVX+LP,OVX+BP,OVX+HP and OVX+AP groups were exposed to four kinds of EMF respectively for 3 h/day,7 days/week,for 8weeks.The body weights were recorded weekly,at the end of 8-week exposure period,all the mice were sacrificed by cervical dislocation after anesthesia.Biochemical analysis of serum,biomechanical examination of right femur,μCT and histological analysis of left femur and real-time PCR of right humerus were employed in our study.Results:Bone formation was slightly enhanced in OVX mice.Microscopically slightly elevated serum bone formation marker levels（BALP,OCN,OPG,and P1NP）and osteogenesis-related gene expression levels（ALP,BMP-2,COL-1,OCN,OSX,Runx2,Wnt1,β-catenin,LPR5,OPG and RANKL）compared to the sham group were confirmed.In addition,bone resorption was significantly enhanced in OVX mice,with greater bone resorption than bone formation,and microscopically significantly elevated serum bone resorption marker levels（TRAP-5b and CTX-I）and osteoclastogenesis-related gene expression levels（RANK and RANKL/OPG）compared to the sham group were confirmed.Ultimately,the accelerated bone remodeling induced by OVX results in decreased bone mass,bone microarchitecture,and mechanical strength degradation,which can be confirmed macroscopically by biomechanics,μCT,and histomorphological features of the bone.Compared with OVX,LP and BP significantly increased serum bone formation marker and osteogenesis-related gene expression.In addition,compared with OVX,LP and BP exposure can increase the ratio of RANKL/OPG mRNA expression in the bone of OVX mice.This study hypothesized that the enhanced bone formation capacity induced by LP and AP can indirectly lead to enhanced osteoclastogenesis.Thus,LP,BP has limited effects on reduced bone mass and degraded bone microarchitecture and mechanical strength in OVX-induced OP mice.Compared with OVX,HP significantly reduced serum bone formation and resorption markers and gene expression associated with osteogenesis and osteoclastogenesis at the same time.HP reduced bone mass and degraded bone microstructure in OVX-induced OP mice.The effect of mechanical strength is still limited.AP has a cumulative effect of LP,BP,and HP,which significantly increases bone formation and decreases bone resorption compared to OVX,ultimately reducing OVX-induced bone loss in OP mice.Conclusion:OVX resulted in the decrease of bone mass and deterioration of bone microarchitecture and mechanical strength in mice,and this condition were not altered by LP,BP and HP exposure.However,AP stimulation attenuated OVX-induced decrease of bone mass and deterioration of bone microarchitecture and mechanical strength in mice by promoting bone formation and inhibiting bone resorption.Bone mass reflects the balanced state of bone formation and bone resorption,which involves coordinated growth of OBs and OCs at the cellular level.ICR-related EMF has positive effects on OBs,and high-frequency EMF has side effects on OBs and OCs.Reasonably combining the effects of different frequency bands on osteogenesis and osteotomy is the key to the treatment of OP.SummaryBy using the"engineering-medicine,micro-macro,in vitro-in vivo"experimental scheme,the effects of EMF with different frequency bands on the OP of OVX mice were studied.The study found that（1）the frequency components of ICR significantly promote bone formation activity,and directly and indirectly significantly promote bone resorption activity.However,OVX-induced bone resorption is still dominant in OP mice,so LP,BP have limited effects on reduced bone mass and degraded bone microarchitecture and mechanical strength in OVX-induced OP mice.（2）High-frequency components not only drastically reduce bone formation activity,but also significantly reduce bone resorption activity.Thus,the effect of HP on the reduced bone mass and degraded bone microstructure and mechanical strength in OVX-induced OP mice was poor.（3）AP containing both ICR-associated frequency and high-frequency components not only significantly promoted bone formation activity but also significantly decreased bone resorption activity,and ultimately reduced OVX-induced bone loss in OP mice.The main innovations of this study are as follows:（1）An EMF method based on white-noise frequency domain features that can be used for bone research was designed,and EMF experimental parameters were screened.（2）The the effects of EMF with different frequency bands on osteogenesis and osteoclastogenesis were verified systemically,providing new ideas and experimental evidence for revealing the mechanism of non-thermal biological effects of EMF on bone.（3）Animal experiments show that the EMF method proposed in this paper can better improve the OP in OVX mice.