Study On Chondrogenic Differentiation Of SPIO-labeled BMSCs By Pulsed Magnetic Field

Objective:Explore the pulse magnetic field enhanced superparamagnetic iron oxide labeled bone marrow mesenchymal stem cells proliferation and induced chondrocyte differentiation and repair of cartilage defects in vivo repair therapy.Methods:Synthesis and characterization of superparamagnetitic iron oxide(SPIO):1 mmol of Fe(acac)3,2.5 mmol of 1,2-hexadecanediol,1mmol of oleic acid,and 3 mmol of oleylamine were added into 10 mL of benzyl ether,followed by magnetic stirring.The resulting mixture was heated to 200°C for 15 min and then to 300°C for 1 h of refluxing.The obtained SPIO precipitation was further modified with low-molecular-weight amphiphilic alkylated PEI2000.The morphology and size of the obtained SPIO were investigated using a TecaniTM analytical transmission electron microscope(TEM)with an operating voltage of 200 kV.The hydrodynamic size was measured using a particle size analyzer.Pulsed magnetic field enhances SPIO-labeled bone marrow mesenchymal stem cell proliferation and chondrocyte differentiation ability and exploration of TGF-?/SMAD signaling pathway:Extraction of bone marrow mesenchymal stem cells from SD rats for 3-7 days is passaged in vitro in the third generation,MTT was used to examine the effect of different concentrations of SPIO on the toxicity of bone marrow mesenchymal stem cells.The Prussian blue staining was used to observe the number of bone marrow mesenchymal stem cells positive cells labeled with different concentrations of SPIO.The experimental groups were:control group,SPIO group,PMF group,PMF-SPIO group.The MTT screens out the best duration in a pulsed magnetic field.Annexin V+/PI-was used to verify the effects of various groups on the apoptosis of bone marrow mesenchymal stem cells under different conditions.MTT was used to verify the effect of pulsed magnetic fields on the proliferation of BMSCs on different days 1,2,3,4,and 5 days.Bone marrow mesenchymal stem cells were induced into groups without TGF-?~-group(B group,BP group,BS group,BSP group)and TGF-?~+group(BT group,BTP group,BTS group,BTSP group).Bone marrow mesenchymal stem cells were cultured for 7 days,14 days,and 21 days for GAG/DNA detection,and the expression of the special marker genes Acan,Col2a1,and Sox9 of chondrogenic differentiation were detected.The effect of TGF-?/SMAD signaling pathway-associated protein expression in the differentiation of bone marrow mesenchymal stem cells into chondrogenic cells under the condition of pulsed magnetic field stimulation was tested in various experimental groups by immunoblot(WB),and the expression of relevant proteins was examined.Change levels to verify the impact of pulsed magnetic fields at the cellular level.Verification of regeneration of cartilage defect in bone marrow mesenchymal stem cells stimulated by SPIO stimulated by pulsed magnetic field:MRI was used to detect T2 signal value of SPIO-labeled bone marrow mesenchymal stem cells and SPIO-labeled bone marrow mesenchymal stem cells in the joint cavity T2 signal value.The articular cartilage defect model was established.Bone marrow mesenchymal stem cells from different groups(B group,BP group,BS group,and BSP group)were injected into the joint cavity to repair the defect.After the eighth week,materials were taken and the joints were grossly scored to observe their repair status.The fixed tissue sections were stained with HE,stained with Safranine green,and immunohistochemically stained to observe the repair effect between different groups.Results:The SPIO nanoparticles were successfully prepared.In morphology,they are spheres of uniform size.The MTT assay verified that there was no effect on cell viability compared to the control group at SPIO concentrations of 10?g/mL,20?g/mL,30?g/mL,and 40?g/mL.Prussian blue staining showed that as the concentration of SPIO increased,the blue iron particles in the cytoplasm increased significantly.In the pulsed magnetic field conditions,the optimal duration of action was 30min.Flow-through results showed that SPIO and pulsed magnetic fields had no effect on the apoptosis of BMSCs.In addition,a significant increase in the proliferation rate of SPIO-labeled bone marrow mesenchymal stem cells under pulsed magnetic field stimulation was also verified.Under the condition of pulsed magnetic field,the secretion of GAG from bone marrow mesenchymal stem cells labeled with SPIO and the expression of related cartilage genes were significantly increased.At the protein level,it was verified that the pulsed magnetic field can enhance the TGF-?/SMAD signal pathway of SPIO-labeled bone marrow mesenchymal stem cells during differentiation.In vivo experiments,through observation of tissue and related histological staining,verified that under the condition of pulsed magnetic field,SPIO-labeled bone marrow mesenchymal stem cells could repair the defects of cartilage defects.Conclusion:In the present study,the pulsed magnetic field represents a non-invasive method to enhance the cartilage-specific gene expression of bone marrow mesenchymal stem cells,which improves chondrogenic differentiation through exposure to pulsed magnetic field SPIO-labeled bone marrow mesenchymal stem cells,providing continuous and Stable stimulation overcomes the use of previously used barrier growth factors in vivo.And provide effective clues for cartilage regeneration.Our findings may represent a realistic therapeutic strategy based on the treatment of damaged cartilage by cells.