Mechanisms Of Electromagnetic Fields Regulating Bone Remodeling By Promoting Migration And Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective:After maturation,bone is always in the process of continuous absorption and formation,which we call bone remodeling.The dynamic balance of bone resorption and bone formation is supportive for stress adaptation and injury repair of bone tissue.The maladjustment of bone remodeling is accompanied by various bone diseases.In the process of bone remodeling,cytokines released by osteoclasts after dissolving bone matrix induce mesenchymal stem cells(MSC)to migrate to bone absorption site,then differentiate into osteoblasts,and repair the absorbed bone.Therefore,the migration ability and lineage commitment of MSC are very important for the bone remodeling process.In the orthopedic diseases related to bone mass lost,the migration ability and osteogenic differentiation of MSCs were impaired.As early as 20 years ago,scientists found that electromagnetic field can treat delayed union of fracture.Because of the merit of non-invasive and less side effects,electromagnetic field treatment has great potential in orthopedic diseases.Previous studies have confirmed that electromagnetic field can promote the osteogenic differentiation of MSC,but the mechanisms are still unclear.In addition,whether electromagnetic field has a certain influence on the migration ability of MSC has not been reported.Therefore,we investigate the biological effects of electromagnetic field on h-BMSC migration and osteogenic differentiation.Methods:In this study,human bone marrow mesenchymal stem cells(h-BMSC)were used as a cell model,and the applied electric and magnetic fields were 7.5-75 Hz/1 m T sinusoidal alternating electromagnetic fields.The whole experimental research was divided into two parts.In the first part,we investigated the effect of electromagnetic field on h-BMSC migration and its mechanism.Firstly,we used Transwell migration assay to detect the change of h-BMSC migration ability after electromagnetic stimulation of different frequencies(7.5,15,30,50 and 75 Hz).As 50 Hz/1 m T electromagnetic field has the strongest pro-migration effect on h-BMSC.Then,50 Hz/1 m T electromagnetic field was applied to h-BMSC with the use of 10?M verapamil,an L-type calcium channel blocker,and/or5?M PF573228,an adhesion kinase(FAK)inhibitor,and we detected the intracellular calcium content,the expression of cell adhesion proteins(FAK,Tailin and vinculin),the activity of Rho GTPases(Rho A,Rac1 and Cdc42)and the organization of F-actin cytoskeleton,which in order to clarify the roles of voltage-gated calcium channel(VGCC),FAK and Rho GTPases in the electromagnetic field induced h-BMSC migration and the relationship between them.In the second part,we studied the mechanisms of the promotive effect of electromagnetic field on h-BMSC osteogenic differentiation.Firstly,we measured the expression of P2X7,a calcium channel receptor,in h-BMSC under osteogenic induction and electromagnetic field stimulation of different frequencies(7.5,15,30,50 and 75 Hz).As to the result,15Hz/1 m T was selected for further tests.The h-BMSCs were stimulated with 15 Hz/1 m T electromagnetic field and osteogenic induction medium for 7 days(8 h/d),and the changes of Akt/GSK3?/?-Catenin pathway,which is downstream of P2X7,were detected.Then,with P2X7 specific inhibitor A740003(5?M)and/or PI3K inhibitor LY294002(10?M)employed at the same time with the 15 Hz/1 m T electromagnetic field exposure,we detected the activity of PI3K/Akt/GSK3?/?-catenin signaling pathway and calcium deposition of osteogenic induced h-BMSC.In addition,we also treated the h-BMSC with P2X7 agonists(Bz ATP and ATP)combined with electromagnetic field under osteogenic induction and detected the osteogenic markers,to carry out whether P2X7 agonists and electromagnetic field have synergistic effect on h-BMSC osteogenic differentiation.Results:In the first part,we found that the electromagnetic field with different frequencies(7.5?15?30?50 and 75 Hz)promoted the migration of h-BMSC to some extent,among which the electromagnetic field with 50 Hz/1 m T has the strongest effect.The stimulation of 50 Hz/1m T electromagnetic field could increase the intracellular Ca²⁺content,the expression of cell adhesion proteins(FAK,Tailin,vinculin),the activity of Rho GTPases(Rho A,Rac1,Cdc42)and the formation of F-actin.10?M Verapamil could partially inhibit the electromagnetic field mediated pro-migration effect and the increase of intracellular Ca²⁺content.With the 50 Hz/1 m T electromagnetic field stimulation,Verapamil(10?M)and PF573228(5?M),alone or in combination,could partially inhibit the promotive effects of electromagnetic field on adhesion protein expression,Rho GTPases activity and F-actin formation.And the combination of Verapamil and PF573228 did not show further inhibitory effect than the single use of each ones.In the second part,we found that different frequencies(7.5?15?30?50 and 75 Hz)of electromagnetic fields increased the expression of P2X7 in h-BMSC s under osteogenic induction,but have no significant effect on the expression of P2X7 in h-BMSCs without osteogenic induction.Among them,the effect of 15 Hz/1 m T electromagnetic field on P2X7 expression was the strongest,which was in a time dependent manner,and P2X7expression reached the peak point at 14 days.The expression of RUNX2,ALP and OPN was increased and the formation of calcium nodules was significantly enhanced after stimulation of h-BMSCs with 15 Hz/1 m T electromagnetic field for 7 days.These effects were partially reversed by A740003(5?M)and/or LY294002(10?M).We also detected the expression of proteins in Akt/GSK3?/?-catenin pathway,which is downstream of P2X7,with the use of A740003 and/or LY294002 at the same time as electromagnetic field stimulation.The results showed that 15 Hz/1 m T electromagnetic field stimulation for 7days could significantly increase the levels of phosphorylated Akt,phosphorylated GSK3?and intranuclear?-catenin,but this effect could be inhibited by 5?M A740003 and/or 10?M LY294002,among which 10?M LY294002 showed more fierce inhibitory effect.And there was no significant difference in the inhibitory extent between the two inhibitors when they were used in combination and when they were used alone.Finally,we also found that electromagnetic field and P2X7 agonists(Bz ATP and ATP),alone or in combination,could promote the expression of RUNX2,ALP,OPN in osteogenic induced h-BMSC,and the effect of combined therapy is significantly stronger than that of electromagnetic field or P2X7 agonists alone.Conclusion:Electromagnetic field can promote bone formation and regulate the balance of bone remodeling by enhancing the migration ability and osteogenic differentiation of h-BMSC.In the process of electromagnetic field promoted h-BMSC migration,electromagnetic field activates VGCC,increases intracellular Ca²⁺content.Ca²⁺accumulation activates FAK,which enhances the formation of focal adhesions.At the same time,FAK enhances the formation of cytoskeleton by activating Rho GTPases,which together promotes the migration of h-BMSC.In the process of osteogenic differentiation of h-BMSC,continuous electromagnetic field stimulation induces the expression of P2X7,which can activate the downstream Akt/GSK3?/?-catenin pathway and promote the osteogenic differentiation of h-BMSC.In addition,the combined treatment of P2X7 agonist and electromagnetic field has synergistic effect on promoting h-BMSC osteogenic differentiation.This study is based on previous researches,and complements the patten and mechanisms of electromagnetic field regulation on bone remodeling,and provides theoretical basis for further understanding the biological effect of electromagnetic field and give more chances for clinical application of electromagnetic field on orthopedic diseases.