Effect And Mechanism Of Mechanical Stress On Tendon-bone Healing And Cell Proliferation,Apoptosis And Differentiation

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Effect of Mechanical Loading on Tendon-Bone Healing and Proliferation and Matrix Formation of co-culture of Mesenchymal Stem Cells and Tendon Cells ObjectiveThis study investigated the effect of mechanical loading on tendon-bone healing in a rabbit anterior cruciate ligament(ACL)reconstruction model as well as cell proliferation and matrix formation in co-culture of bone-marrow mesenchymal stem cells(BMSCs)and tendon cells(TCs).MethodsThe effect of continuous passive motion(CPM)therapy on tendon-bone healing in a rabbit ACL reconstruction model was evaluated by histological analysis,biomechanical testing and gene expressions at the tendon-bone interface 6 and 12 weeks postoperatively.Furthermore,in vitro experiments,BMSCs and TCs were isolated and expanded.Then the two sources of cells were co-cultured in a ratio of 1: 1 and exposed to mechanical stretch.Alarma Blue assay was used to detect changes in cell proliferation and RT-PCR assay was used to detect matrix synthesis in BMSC/TC co-culture system.ResultsPostoperative CPM therapy significantly enhanced tendon-bone healing,as evidenced by increased amount of fibrocartilage,elevated ultimate load to failure levels,and up-regulated gene expressions of Collagen?,alkaline phosphatase,osteopontin,Tenascin C and tenomodulin at the tendon-bone junction.In addition,BMSC/TC co-culture treated with mechanical stretch showed a higher rate of cell proliferation and enhanced expressions of Collagen?,Collagen ?,alkaline phosphatase,osteopontin,Tenascin C and tenomodulin than that of controls.ConclusionThese results demonstrated that proliferation and differentiation of local precursor cells could be enhanced by mechanical stimulation,which results in enhanced regenerative potential of BMSCs and TCs in tendon-bone healing.Section? Effect and Mechanism of Cyclic Compressive Stress on Rat Osteoblasts Apoptosis ObjectiveTo investigate the effect of cyclic compressive stress on rat osteoblasts apoptosis.MethodWe used a novel liquid drop method to generate mechanical stress on osteoblast monolayers.After treatment with different levels of mechanical stress,apoptosis of osteoblasts were examined using TUNEL staining and Caspase-3 activity assay.After treatment with representative mechanical stress,activations of mitogenactivated protein kinases(MAPKs)and PI3-kinase(PI3K)/Akt signaling pathways were investigated by Western blot.Osteoblasts apoptosis was observed using Flow cytometric analysis after treated with specific inhibitors prior to large-magnitude mechanical stimulation.Protein levels of Bax/Bcl-2/caspase-3 signaling were determined using Western blot with or without inhibitors of PI3K/Akt and phosphorylation of c-jun N-terminal kinase(JNK)MAPK after treatment with large-magnitude mechanical stimulation.ResultsMechanical stimulation led to osteoblasts apoptosis in a dose-dependent manner,as small-magnitude stress inhibited cell apoptosis while large-magnitude stress led to increased cell apoptosis.Mechanical stimulation led to a remarkable activation of MAPKs and PI3K/Akt signaling pathways.However,the phosphorylation of ERK and Akt began with increasing and then decreased,while the phosphorylation of JNK and p38 showed an increasing trend continuously.Mechanical loading of 0.6Hz×8cm significantly induced apoptosis compared with control cells(P<0.05),Inhibition of the PI3K/Akt pathway caused a significant increase in mechanical stimulation-induced apoptosis compared with the mechanical loading group(P<0.05).In addition,Inhibition of the JNK pathway partly eliminated the apoptotic effect of mechanical stimulation on cell apoptosis(P<0.05).Large-magnitude mechanical stress induced osteoblasts apoptosis by regulating Bad,Bcl-2,Bax and activation of Caspases-3.Blocking JNK activity could down-regulate the expression of Bax/ Bcl-2 and Caspase-3 under mechanical stress.However,PI3K/Akt inhibitor partly blocked the anti-apoptotic properties through up-regulating the expression of Bax/ Bcl-2 and Caspase-3.ConclusionIn summary,activation of JNK and Akt by mechanical stress independently resulted in an opposite effect on cell apoptosis,resulting in inhibition of apoptosis upon small-magnitude stress and increased apoptosis upon large-magnitude stress.Small-magnitude stress led to activation of PI3K/Akt which protected against apoptosis,whereas large-magnitude stress led to activation of JNK MAPK which increased apoptosis.Section ? Effect and Mechanism of Mechanical Stress on Adipogenesis and Osteogenesis of Rat Mesenchymal Stem Cells ObjectiveTo investigate effect and mechanism of different levels of cyclic mechanical stress on adipogenesis and osteogenesis of rat BMSCs.MethodsRat BMSCs were exposed to different levels of cyclic mechanical stress generated by liquid drops and cultured in general medium or adipogenic medium.Markers of osteogenic(Runx2 and Collagen I)and adipogenic(C/EBP?,PPAR? and lipid droplets)differentiation were detected using Western blot and histological staining.The protein levels of members of the phosphatidylinositol 3-kinase(PI3K)/Akt/glycogen synthase kinase 3?(GSK-3?)/?-catenin signaling pathway were also examined.Cells were incubated with Li Cl and then cultured in adipogenic medium and effects of osteogenic and adipogenic differentiations were detected.Activation of PI3K/Akt/GSK-3?/?-catenin signaling by the treatment with LY294002 and mechanical stress was detected using Western blot.ResultsSmall-magnitude stress significantly up-regulated Runx2 and Collagen I and down-regulated PPAR? and C/EBP? expression in BMSCs cultured in adipogenic medium,while large-magnitude stress reversed the effect when compared with unloading groups(P<0.05).The PI3K/Akt signaling pathway could be strongly activated by mechanical stimulation;however,large-magnitude stress led to decreased activation of Akt and ?-catenin and increased activation of GSK-3? when compared with small-magnitude stress.Activation of ?-catenin with Li Cl led to increased expression of Runx2 and Collagen I and reduction of C/EBP? and PPAR? expression in BMSCs(P<0.05).Inhibition of PI3K/Akt signaling with LY294002 partially blocked the activation of Akt and expression of ?-catenin.ConclusionsOur results indicate that mechanical stress-regulated osteogenesis and adipogenesis of rat BMSCs is mediated,at least in part,by the PI3K/Akt/GSK-3?/?-catenin signaling pathway.Small-magnitude stress could enhance osteogenic differentiation and inhibit adipogenic differentiation of BMSCs while large-magnitude stress exerted an opposite effect.