Role And Mechanism Of Mechanically Sensitive Ion Channel TRPM7 In LIPUS Promoting Osteogenic Differentiation Of MSCs

Background: Fracture healing is a complex biological process.Despite the continuous development of modern orthopedic treatment techniques,about 10% of fractures will eventually have delayed healing or nonunion,often requiring bone graft surgery.There are many risk factors for delayed bone healing,so it is necessary to find a safer and more effective way to accelerate fracture healing.Studies have found that some biophysical therapies are being used for adjuvant therapy in the bone healing process.Low intensity pulsed ultrasound(LIPUS)can produce non-invasive mechanical energy for transcutaneous delivery,which can accelerate and strengthen the healing of fresh fractures,and has a good effect on delayed healing and nonunion.Bone marrow mesenchymal stem cells(BMSCs)are a class of pluripotent stem cells with strong proliferative capacity and multi-directional differentiation potential,which play an important role in the process of fracture healing.Studies suggest that LIPUS promotes osteogenic differentiation ofBMSCs.However,the specific mechanism of action is still unclear.As a potential stem cell,dental pulp mesenchymal stem cells(DPSCs)have high proliferation ability and multi-directional differentiation;their sources are abundant and there is no ethical dispute.As a kind of excellent seed cells,DPSCs may open a new way for bone tissue engineering.The TRPM(Transient Receptor Potential Melastatin,TRPM)protein family is a widely-existing ion channel expressed in a variety of mammalian cells.In recent years,they have been found to play a key role in maintaining some specific physiological functions,and are closely related to the occurrence and development of certain diseases in humans..TRPM7 is the seventh member of the TRPM family.It is a membrane protein containing a dual structure of cation channels and protein kinases.It is widely distributed in various tissues and organs and can sense various stimuli from the surrounding environment(e.g.osmotic pressure,temperature,machinery,etc.)and has mechanical sensitivity.Studies have shown that TRPM7 defects can lead to bone formation disorders,and TRPM7 acts as a mechanically sensitive plasma membrane calcium permeability channel,which plays an active role in mechanical stimulation of BMSCs osteogenesis and can mediate cytoplasm increased intracellular calcium up-regulates RUNX2 expression in BMSCs.The cytoskeleton can sense mechanical signals and conduct into the cells.Certain mechanical stimuli can cause depolymerization and rearrangement of the cytoskeleton F-actin,while cytoskeletal depolymerization and rearrangement affect the osteogenic differentiation of MSCs.This study will investigate whether LIPUS effectively promotes the osteogenic differentiation of BMSCs and DPSCs and the role of TRPM7 in osteogenic differentiation,and initially explores the changes in intracellular calcium signaling affected by TRPM7 and the depolymerization and rearrangement of microfilaments under the action of LIPUS,in order to further understand the molecular mechanism of action of LIPUS in regulating osteogenic differentiation of MSCs.Part I LIPUS promotes osteogenic differentiation of BMSCs andDPSCsObjective: To explore the effect of LIPUS on promoting osteogenic differentiation of BMSCs and DPSCs.Methods: Mouse BMSCs and human DPSCs were cultured respectively.Flow cytometry was used to detect the expression of two cell-specific surface markers.ALP staining and alizarin red staining were used to detect osteogenic differentiation.Alixin blue and oil red O staining to detect chondrogenic and adipogenic differentiation.Under theaction of osteogenic differentiation induction medium,each cell was then divided into two groups: control group(LIPUS untreated group)and LIPUS treated group.ALP staining,alizarin red staining and ALP activity reading were used to compare the difference of osteogenic differentiation ability between control group and LIPUS treatment.The expression of osteogenic differentiation genes RUNX2,OPN and OCN was detected by qPCR after LIPUS treatment.Results: Successfully cultured BMSCs and DPSCs in vitro and identified them,BMSCs have osteogenic and adipogenic differentiation ability,while DPSCs have adipogenic,osteogenic and cartilage-forming ability.Compared with the control group,ALP staining and alizarin red staining were significantly enhanced after LIPUS treatment of BMSCs and DPSCs;ALP activity readings were significantly increased(*P<0.05,**P<0.01);the expression of OPN,OCN and RUNX2 genes related to osteogenic differentiation increased significantly(P < 0.05).Conclusion: LIPUS promotes osteogenic differentiation of BMSCs and DPSCs.Part II The role of TRPM7 in promoting osteogenic differentiation of BMSCs and DPSCs by LIPUSObjective: To investigate the role of mechanically sensitive ion channel TRPM7 in the process of osteogenic differentiation of BMSCs and DPSCs by LIPUS.Methods: Mouse BMSCs and human DPSCs were cultured separately.Each cell was divided into control group and LIPUS treatment group.The difference of TRPM7 mR NA expression level of the 7th member of TRPM subfamily after 1 day,2 days and 3 days of LIPUS treatment was detected.The expression sites and levels of intracellular TRPM7 were detected by immunofluorescence after LIPUS treament after LIPUS treatment of BMSCs1 d,2d and 3d.It was then divided into four groups: control group,LIPUS group,LIPUS+DMSO group and LIPUS+2-APB group.ALP activity readings,ALP staining and alizarin red staining were used to detect the effect of TRPM7 channel inhibitor2-APB on LIPUS-induced osteogenic differentiation of BMSCs and DPSCs,and WB to detect differences in protein expression of osteogenic differentiation-related genes.Construction of mouse TRPM7 overexpression and siRNA interference with adenoviral vectors,WB to verify the overexpression and interference efficiency of the two viruses.Divided into six groups:(Ad-RFP group and Ad-RFP+LIPUS group),(Ad-TRPM7 group and Ad-Trpm7+LIPUS group),(Ad-siTRPM7 group and Ad-siTrpm7+LIPUS group),two pairs corresponding.ALP activity readings,ALP staining and alizarin red staining were used to detect the bone differentiation ability of each group of cells.WB was used to detect the difference in TRPM7 and osteogenic differentiation-related gene protein expression between the Ad-RFP+LIPUS group and the Ad-siTRPM7+LIPUS group.Results: BMSCs and DPSCs were obtained in vitro.Compared with the control group,qPCR showed that the expression of TRPM7 was significantly increased in both cells after LIPUS treatment(P < 0.05).Immunofluorescence staining showed that compared with the control group,the expression of TRPM7 was increased after LIPUS treatment of BMSCs,mainly expressed in the cytoplasm.Subsequently,it was divided into four groups.The ALP readings were significantly increased in the LIPUS group and the LIPUS+ DMSO group compared with the control group.The positive blue-violet staining and red calcium nodules were significantly increased by ALP staining and alizarin red staining;compared with the LIPUS+ DMSO group,the ALP readings of LIPUS+2-APB group were significantly decreased,ALP staining and alizarin red positive staining were significantly reduced;WB assay showed that the protein expression of genes involved in osteogenic differentiation in LIPUS+2-APB group was significantly lower than thatof LIPUS+DMSO group.Overexpression and siRNA interference with adenoviral vectors of mouse TRPM7 were successfully constructed,and WB verified the interference efficiency of TRPM7 overexpression and siRNA.ALP staining and alizarin red staining showed that the positive staining of Ad-RFP+LIPUS group and Ad-TRPM7+LIPUS group was significantly increased compared with the control group,but the positive staining of Ad-TRPM7+LIPUS group was significantly more than that of Ad-RFP,the Ad-siTRPM7+LIPUS group was significantly lower than the Ad-RFP+LIPUS group;the ALP reading in the Ad-TRPM7+LIPUS group was significantly higher than that in the Ad-RFP+LIPUS group(*P<0.05),the ALP readings of the Ad-siTRPM7+LIPUS group were lower than those of the Ad-RFP+LIPUS group(**P<0.01).WB assay showed that the expression of bone differentiation-related gene protein in Ad-siTRPM7+LIPUS was significantly decreased compared with Ad-RFP+LIPUS group(*P<0.05,**P<0.01,***P<0.001).Conclusion: LIPUS treatment can significantly up-regulate the expression of TRPM7 in BMSCs and DPSCs.TRPM7 plays an important regulatory role in the process of osteogenic differentiation of BMSCs and DPSCs by LIPUS.Part III Preliminary study on the role of TRPM7 in regulation of intracellular calcium oscillation in LIPUS-induced microfilament depolymerizationObjective: To investigate the effects of LIPUS on intracellular calcium concentration and cytoskeleton microfilaments,and the role of TRPM7 in this process.Methods: BMSCs were cultured and divided into control group and LIPUS group.Fluo-4 reagent and flow cytometry were used to detect intracellular calcium concentration in the two groups.Then they were divided into three groups: LIPUS group,LIPUS + 2-APB(2-APB inhibits the release of calcium from endoplasmic reticulum as intracellular calcium pool)group and LIPUS + calcium-free medium group.Subsequently,the experiment was divided into two groups:Ad-RFP+LIPUS group and Ad-siTRPM7+LIPUS group,intracellular calcium oscillation was observed in both groups.BMSCs were divided into control group,LIPUS group and 2-APB+LIPUS group.The changes of F-actin morphology of BMSCs cytoskeleton were observed by confocal laser scanning.Results: BMSCs were obtained in vitro.Flow cytometry showed that the intracellular calcium concentration in LIPUS group was significantly higher than that in control group.After 2-APB andcalcium-free medium were used,the changes of intracellular calcium concentration induced by LIPUS were significantly reduced.After TRPM7 silencing,the changes of intracellular calcium concentration induced by LIPUS were also significantly inhibited.Laser confocal imaging showed that there were more bundles of F-actin in the control cells,and a large number of stress fibers were formed in the cells.After LIPUS treatment,F-actin gradually depolymerized,and the formation of stress fibers decreased.The microfilament depolymerization was inhibited by 2-APB.BMSCs of rats were cultured in vitro,immunofluorescence microscopy showed that microfilaments were depolymerized after LIPUS treatment,while microfilament depolymerization induced by LIPUS was inhibited after 2-APB treatment.Conclusion: After LIPUS treatment,intracellular Ca2+concentration increased,both endoplasmic reticulum Ca2+ pool and extracellular Ca2+ might be involved,which could be partially regulated by TRPM7.LIPUS,as a mechanical stress,could promote the depolymerization of cytoskeletal microfilaments,which may be related to TRPM7 and intracellular Ca2+ release,and further study is needed.