Study On The Role And Mechanism Of Piezo1 Channel In Fracture Healing

ObjectiveThe Cre-LoxP system was used to generate mice with endothelial cell-specific deletion of Piezo1 to examine the role of Piezo1 in fracture healing.MethodsImmunofluorescence technique was used to examine the expression and distribution of Piezo1 in the bone vasculature;Endothelial cells were isolated from bone vasculature using flow cytometry,the m RNA and protein levels of endothelial Piezo1 in Piezo1^fl/fl mice and Piezo1^ΔEC mice were examine using qRT-PCR and WB.A unilateral open transverse femur fracture model was generated and the mice were subjected to radiographic examination every week using Micro-CT to investigate the effect of knockout of endothelial Piezo1 on fracture repair.The expression level of EMCN,RUNX2 and OSX in fracture site of Piezo1^fl/fl mice and Piezo1^ΔEC mice was examine using laser confocal.Microangiography was used to examine the role of Piezo1 in angiogenesis during bone fracture healing.Three-point bending test was used to examine the bone mechanical properties.Calpain activity assay was used to evaluate the effect of knockout of endothelial Piezo1 on calpain activity during vascularization.QRT-PCR and WB were used to determine the effect of Piezo1 on PI3K-AKT phosphorylation level and Notch signal.ResultsThe results of immunostaining showed that Piezo1 was expressed in the endothelial cells of bone vasculature.Micro-CT results of fractures in Piezo1^fl/fl mice revealed the complete formation of external callus at the fracture site at 21 days post facture（DPF）and the completion of bone remodeling at 42 DPF.Although Piezo1^ΔEC mice exhibited callus formation along the periosteum extending away from the fracture line,the bridging of callus was not observed at 21 DPF.Moreover,radiographic analysis revealed a clear radiolucent gap between the broken ends at 42 DPF.Compared with those in Piezo1^fl/flmice,trabecular bone volume fraction（BV/TV）,trabecular thickness（Tb.Th）,and trabecular number（Tb.N）were significantly lower,and trabecular separation（Tb.Sp）was higher in Piezo1^ΔEC mice at 21 and 42 DPF.The expression level of EMCN,RUNX2 and OSX in Piezo1^ΔEC mice was significantly lower than that in Piezo1^fl/fl mice at 21 and42DPF.The number of new blood vessels around the fracture site in Piezo1^fl/fl mice was higher than that around the fracture site in Piezo1^ΔEC mice at 21 DPF.Furthermore,the formation of blood vessels was complete in Piezo1^fl/fl mice at 42 DPF but was impaired in Piezo1^ΔEC mice.The results of three-point bending test showed that yield load and bending stiffness of the femur at 21 and 42 DPF in Piezo1^ΔEC mice was significantly lower than that in Piezo1^fl/fl mice.The calpain activity in Piezo1^ΔEC mice was significantly lower than that in Piezo1^fl/fl mice at 21 and 42 DPF.The phosphorylation of PI3K and AKT in Piezo1^ΔECmice was significantly lower than that in Piezo1^fl/fl mice at the fracture lesions at 42 DPF.The expression levels of CD31 and Notch1 intracellular domain（N1ICD）in Piezo1^ΔECmice were downregulated when compared with those in Piezo1^fl/fl mice.ConclusionThe deletion of endothelial Piezo1 resulted in impaired bone fracture repair,down-regulation of calpain activity during vascularization,down-regulation of phosphorylated PI3K-AKT,and impaired Notch signaling during bone fracture union.These findings indicated that Piezo1 protein is a potential target for enhancing bone regeneration and treating delayed or nonunion bone fractures.