The Role And Mechanism Of Mechanosensitive Ion Channel Piezo1 In Osteogenesis And Angiogenesis Mediated By Triboelectric Nanogenerator

Part 1.The effect of triboelectric nanogenerator on the aged bone marrow mesenchymal stem cells Objective:To explore the regulating effect of TENG on osteogenic differentiation and promoting angiogenesis of aged BMSCs.Methods:A four-layer TENG was constructed with independent layer mode.The output current was measured at vibration frequencies of 0.5,1.0,1.5 and 2.0 Hz.The vibration frequency of 1.5Hz was set to detect cell proliferation under different current stimulation.Western blotting and RT-PCR were used to detect the expressions of Runx2,OCN and Col1 a in young BMSCs group,aged BMSCs group and aged BMSCs+TENG group.Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic differentiation ability of BMSCs in different groups.The HUVEC tubule formation experiment was used to detect the effect of TENG treated BMSCs on HUVEC angiogenesis.Results:TENG could maintain a stable output current of ?88 ?A under different vibration frequencies,and the cell proliferation was best under 30 ?A stimulation at1.5Hz vibration frequency.The expression levels of Col1 a,Runx-2,OCN and other genes in aging BMSCs were decreased,and TENG could up-regulate the expression levels of Col1 a,Runx-2,OCN and other genes in aged BMSCs.In aged BMSCs,calcium nodules alkaline phosphatase activity decreased.TENG increased calcium nodules and alkaline phosphatase activity in aged BMSCs.Aged BMSCs contributed to the decrease of tubule capacity,and TENG enhanced the ability of BMSCs to promote HUVEC tubule formation.Conclusion:TENG is able to enhance the ability of osteogenic differentiation of aged BMSCs,and enhance the ability of promoting HUVEC tubule formation of aged BMSCs,so as to restore the biological function of aged BMSCs.Part 2.The Function of mechanosensitive ion channel Piezo1 on triboelectric nanogenerator mediated osteogenesis and angiogenesis Objective:To investigate the mechanism of mechanosensitive ion channel Piezo1 in TENG mediated osteogenesis and angiogenesis,and the mechanism of TENG regulating the functional recovery of aged BMSCs.Methods:Western blotting and RT-PCR were used to detect the expression of Piezo1 and HIF-1? in BMSCs of young BMSCs group,aged BMSCs group and aged BMSCs+TENG group.The expression of EDN1 was detected by RT-PCR,and the secretion of EDN1 in different BMSCs groups was detected by ELISA.Ca²⁺ fluorescence probe FLUO-4AM was used to detect the difference of intracellular Ca²⁺ concentration of BMSCs in different groups.The expression of Piezo1 in different BMSCs groups was detected by immunofluorescence.Western blotting,RT-PCR and ELISA were used to detect the expression of genes related to HIF-1?,osteogenesis(COL1A,RUNX-2,and OCN)and promoting angiogenesis(EDN1)after si-Piezo1 interference.Western blotting,RT-PCR and ELISA were used to detect the expression of osteogenic genes(Col1a,Runx-2,and OCN)and promoting angiogenesis(EDN1)after si-HIF-1? interference,PIEZO1 agonist and TENG treatment.Results:The expressions of Piezo1 and HIF-1? in aged BMSCs were lower than those in the young group.The expression of Piezo1 and HIF-1? in aged BMSCs could be increased by TENG,and the secretion of EDN1 could be promoted.The intracellular Ca²⁺ concentration was low in aged BMSCs,and TENG increased the intracellular Ca²⁺ concentration in aged BMSCs.Immunofluorescence indicated that the expression of Piezo1 was low in aged BMSCs,and TENG promoted the expression of Piezo1.si-Piezo1 transfection could inhibit the expression of TENG-mediated HIF-1?,osteogenic genes and tube-promoting genes.si-HIF-1? transfection could inhibit the expression of TENG-mediated osteogenesis and tube-promoting genes,as well as Piezo1 agonist Yoda1-mediated osteogenesis and tube-promoting genes.Conclusion:TENG can not only promote the expression of Piezo1,but also activate the opening of Piezo1 channel proteins,and restore the osteogenesis and tube-promoting function of aging BMSCs by depending on PIEZO1.Part 3.The mechanosensitive ion channel Piezo1 regulates the biological functions of aged bone marrow mesenchymal stem cells Objective:To investigate the effect and mechanism of the mechanosensitive ion channel Piezo1 in regulating the biological function of aged bone marrow mesenchymal stem cells.Methods:According to different treatment methods,aged BMSCs were divided into three groups,namely aged group,aged+Yoda1 group and aged+TENG+GsMTx4.Western blotting,RT-PCR and ELISA were used to detect the expression levels of RUNX-2,OCN,COL1 A and HIF1? in aged BMSCs,as well as the secretion of vascular factor EDN1.Fluo-4AM,a Ca²⁺ fluorescent probe,was used to detect the difference of Ca²⁺ concentration in the cytoplasm of aged BMSCs.Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic differentiation ability of aged BMSCs in different groups.HIF-1? expression in aged BMSCs in different groups was also detected by immunofluorescence.HUVEC tubule formation experiment was used to detect the effect of aged BMSCs in different treatment groups on HUVEC angiogenesis.Results:Piezo1 agonist Yoda1 promoted the expression of HIF-1?,osteogenesis(Col1a,Runx-2 and OCN)and the secretion of promoting tubule formation factor(EDN1)in aged BMSCs.Piezo1 inhibitor GsMTx4 can inhibit the function of osteogenesis and promoting angiogenesis of aged BMSCs mediated by TENG.Yoda1 increased intracellular Ca²⁺ concentration in aged BMSCs,while GsMTx4 inhibited the opening of Piezo1 channel protein in TENG stimulated aged BMSCs,and no significant changes in intracellular Ca²⁺ concentration was observed.Yoda1 increased the calcium nodules and alkaline phosphatase activity of aged BMSCs,and GsMTx4 inhibited the osteogenic differentiation of aged BMSCs mediated by TENG.Yoda1 increased the expression of HIF-1? in aged BMSCs,while GsMTx4 inhibited the expression of HIF-1? in TENG stimulated aged BMSCs.Yoda1 enhanced the ability of BMSCs to promote HUVEC tubule formation,while GsMTx4 inhibited the TENG stimulated aged BMSCs and enhanced the tubule formation.Conclusion:Piezo1 plays a key role in restoring the biological functions of aged BMSCs,and activation of PIEZO1 can restore the biological functions of aged BMSCs.The enhanced function of TENG-mediated aged BMSCs was further verified by activation of the mechanosensitive ion channel Piezo1.Part 4.Role of mechanosensitive ion channel Piezo1 in bone defect repair mediated by triboelectric nanogenerator Objective:To investigate the role of TENG-mediated aged BMSCs in bone defect repair in vivo,and the mechanism of mechanosensitive ion channel Piezo1 in TENG-mediated bone defect repair.Methods:According to different treatment methods,in vivo experiments were divided into four groups: young group,aged group,aged+TENG group and aged+TENG+ siPiezo1 group.The rat skull bone defect animal model was established,and BMSCs loaded by sodium alginate gel were used to repair the skull bone defect.At 4 and 8 weeks after surgery,micro-CT was used to detect the osteogenesis at the skull defect site,including the bone volume/total volume,bone mineral density,and trabecular number,and hematoxylin-eosin staining was used to observe the new bone tissue.CD31 staining was used to observe the neovascularization of the skull defect in rats.Results:The bone defect repair ability of aged BMSCs was lower than that of the young group.TENG could enhance the bone defect repair ability of aged BMSCs,and siPiezo1 inhibited the bone defect repair function of aged BMSCs mediated by TENG.In the aged BMSCs group,the new bone was less than that in the young group.The new bone tissue in the aged BMSCs group was increased by TENG,and siPiezo1 inhibited the new bone tissue in the aged BMSCs group mediated by TENG.In the aged BMSCs group,the neovascularization was less than that in the young group.TENG increased the neovascularization of aged BMSCs,while SIPIEZO1 inhibited the neovascularization of aged BMSCs mediated by TENG.Conclusion:The bone defect repair ability of aged BMSCs is decreased,and TENG can promote the bone defect repair ability of aged BMSCs,depending on the activation of mechanosensitive ion channel Piezo1.