The Role And Mechanism Of The Mechanosensitive Ion Channel Protein Piezo1 In Fracture Healing

Part I Expression pattern of the mechanosensitive ion channel protein Piezo in fracture healingObjective:To investigate the cell types involved in fracture repair and the expression patterns of Piezo protein in various cells and callus tissues.Methods:Bone callus samples of mice at different healing stages were collected,and cell types were detected by 10 X single-cell RNA sequencing(sc-RNA seq),and the expression of Piezo protein in each cell type was analyzed.Clinical samples and mouse callus tissues were collected,and the expression pattern of Piezo protein in callus was studied by RT-PCR,western blot and immunohistochemical analysis.Results:The sc-RNA seq analysis showed that mouse callus tissues contained 15 cell clusters,including periosteal stem cells(PSCs),mesenchymal stem cells(MSCs),osteoblasts,chondrocytes,fibroblasts,myofibroblasts,osteoclasts,endothelial cells,and numerous immune cells(neutrophils,basophils,monocytes,dendritic cells,T cells,NK cells,and B cells).There are a large proportion of PSCs(14.7%),chondrocytes(5.4%)and osteoblasts(5.4%)in the callus of 10 days after fracture.There are a lot of immune cells in the callus at 20 days after fracture.Piezo1 was mainly expressed in PSCs,MSCs,osteoblasts and endothelial cells,while Piezo2 was only expressed in chondrocytes.We performed pseudotime analysis using RNA velocity and Monocle2 analysis,and found that PSCs are the ancestor of osteoblasts,chondrocytes,fibroblasts and myofibroblasts in the dataset.Interestingly,cells expressing Piezo 1 preferentially distributed at the beginning and middle of paths,while Piezo2 increased in expression levels at the middle of these paths.In human bone callus,the expression of Piezol was higher than in the matched bone tissue.In addition,Piezo1 expression level increased after fracture,peaked by day 14 and decreased by days 21 and 28 in fractured bones.Immunohistochemical analysis showed that Piezo1expressing positive cells were detected within all of the callus areas,including the cartilaginous callus and the intramembranous bone formation areas,and Piezo2 positive cells were only detected within the cartilaginous callus.Conclusion:The PSCs are the main cell source involved in fracture repair.Piezol is highly expressed in the process of fracture healing and may be as a major mechanosensor during fracture repair.Part Ⅱ The mechanosensitive ion channel protein Piezo1 mediates mechanotransduction of periosteal stem cells to regulate fracture healingObjective:To explore the role of Piezo1,a mechanosensitive ion channel protein,in mediating mechanical signal transduction of PSCs to regulate fracture healing.Methods:The expression of Piezo1 in bone callus was downregulated by Piezo1 shRNA.The Piezo1 specific agonist Yodal was injected intraperitoneally into mice to observe its effect on fracture healing.Next,we isolate and culture of PSCs from mice,the effect of Piezo1 activation on migration and differentiation of the PSCs was investigated by treatment of Yodal,and with or without Piezo1 shRNA and yes-associated protein(YAP)inhibitor.The role of YAP in Piezo1 activation was further investigated by intraperitoneally injection of YAP inhibitor in mice.Results:Downregulation of Piezo1 in callus leads to impaired fracture healing,while activation by its specific agonist,Yodal,promotes fracture healing through the stimulation of PSCs modulated chondrogenesis and osteogenesis,along with accelerated cartilage to bone transformation at late stage of fracture healing.Interestingly,vascular endothelial growth factor A(VEGFA),a key modulator of angiogenesis,is also upregulated after Yodal treated PSCs,indicating both direct and an indirect role of Piezo1 in angiogenesis.Mechanistically,activation of Piezo1 promotes expression of YAP and its nuclear localization in PSCs,which in turn increases the expression and nuclear localization of β-catenin.In detail,YAP directly interacts with β-catenin in the nucleus and forms a transcriptional YAP/β-catenin complex,which upregulates osteogenic,chondrogenic and angiogenic factors.These effects were significantly reduced when YAP inhibitor was introduced both in vitro and in vivo.Conclusion:Piezo1 plays an important role in fracture healing.Piezo1 can promote migration and differentiation of the PSCs by YAP/β-catenin pathway and indirectly promoted angiogenesis,which can be used as an effective target for fracture treatment.Part III Role of the mechanosensitive ion channel protein Piezo1 in delayed fracture unionObjective:To evaluate the therapeutic effect of Piezo1 specific agonist Yodal on delayed fracture union caused by mechanical unloading and it’s related mechanism.Methods:The tail suspension experiments were performed to mimic the mechanical unloading state of hind limbs,and intraperitoneal injection of Yodal was used to observe the improvement of fracture healing.The effects of Yodal on the migration of PSCs were studied by using the Glil-CreER;tdTomato mice.The effect of Yodal treatment on osteogenesis and angiogenesis was observed by immunohistochemical labeling OCN and Emcn.The expression of Piezo1 protein in different states was detected by Western blot analysis.Results:The results showed that the callus total volume(TV),bone volume(BV),BV/TV and bone mineral density(BMD)in the tail suspension group were significantly decreased compared with the ground group.Yodal enhanced the formation of mineralized bone in tail suspension,with higher BV,BV/TV and BMD in callus tissue.In the tail suspension group,the number of Td-positive cells,OCN-positive cells and blood vessels in callus tissue was significantly reduced at 14 days after fracture.Compared with the suspension group,there were more Td positive cells,OCN positive cells and blood vessels in the Yodal treament group.In addition,the expression of Piezo1 protein in the callus tissue was significantly reduced after 20 days of suspension,while the expression of Piezo1 protein was maintained by Yodal treatment.Conclusion:Piezo1 is an important mechanosensor in the process of fracture healing.Activation of Piezo1 not only promotes the fracture healing,but also improves the delayed union caused by mechanical unloading.Therefore,Piezo1 can be an effective target for preventing or treating the delayed union or nonunion caused by mechanical unloading.