Junqing Lin1, Tengli Huang1, Tao Gao1, Xianyou Zheng1. 1. Department of Orthopedic Surgery, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai, 200233, P.R.China.
Abstract
OBJECTIVE: To explore the potential therapeutic effects of endothelial progenitor cells derived small extracellular vesicles (EPCs-sEVs) on spinal cord injury in mice. METHODS: EPCs were separated from femur and tibia bone marrow of 20 C57BL/6 male mice, and identified by double fluorescence staining and flow cytometry. Then the EPCs were passaged and the cell supernatants from P2-P4 generations EPCs were collected; the EPCs-sEVs were extracted by ultracentrifugation and identified by transmission electron microscopy, nanoflow cytometry, and Western blot. Forty C57BL/6 female mice were randomly divided into 4 groups ( n=10). The mice were only removed T 10 lamina in sham group, and prepared T 10 spinal cord injury models in the model group and the low and high concentration intervention groups. After 30 minutes, 3 days, and 7 days of operation, the mice in low and high concentration intervention groups were injected with EPCs-sEVs at concentrations of 1×10 9 and 1×10 10cells/mL through the tail vein, respectively. The behavioral examinations [Basso Mouse Scale (BMS) score, inclined plate test, Von Frey test] , and the gross, HE staining, and immunohistochemical staining were performed to observe the structural changes of the spinal cord at 4 weeks after operation. Another 3 C57BL/6 female mice were taken to prepare T 10 spinal cord injury models, and DiR-labeled EPCs- sEVs were injected through the tail vein. After 30 minutes, in vivo imaging was used to observe whether the EPCs-sEVs reached the spinal cord injury site. RESULTS: After identification, EPCs and EPCs-sEVs derived from mouse bone marrow were successfully obtained. In vivo imaging of the spinal cord showed that EPCs-sEVs were recruited to the spinal cord injury site within 30 minutes after injection. There was no significant difference in BMS scores and the maximum angle of the inclined plate test between two intervention groups and the model group within 2 weeks after operation ( P>0.05), while both were significantly better than the model group ( P<0.05) after 2 weeks. The Von Frey test showed that the mechanical pain threshold of the two intervention groups were significantly higher than that of model group and lower than that of sham group ( P<0.05); there was no significant difference between two intervention groups ( P>0.05). Compared with the model group, the injured segment of the two intervention groups had smaller spinal cord tissue defects, less mononuclear cells infiltration, more obvious tissue structure recovery, and more angiogenesis, and these differences were significant ( P<0.05); there was no significant difference between the two intervention groups. CONCLUSION: EPCs-sEVs can promote the repair of spinal cord injury in mice and provide a new plan for the biological treatment of spinal cord injury.
OBJECTIVE: To explore the potential therapeutic effects of endothelial progenitor cells derived small extracellular vesicles (EPCs-sEVs) on spinal cord injury in mice. METHODS: EPCs were separated from femur and tibia bone marrow of 20 C57BL/6 male mice, and identified by double fluorescence staining and flow cytometry. Then the EPCs were passaged and the cell supernatants from P2-P4 generations EPCs were collected; the EPCs-sEVs were extracted by ultracentrifugation and identified by transmission electron microscopy, nanoflow cytometry, and Western blot. Forty C57BL/6 female mice were randomly divided into 4 groups ( n=10). The mice were only removed T 10 lamina in sham group, and prepared T 10 spinal cord injury models in the model group and the low and high concentration intervention groups. After 30 minutes, 3 days, and 7 days of operation, the mice in low and high concentration intervention groups were injected with EPCs-sEVs at concentrations of 1×10 9 and 1×10 10cells/mL through the tail vein, respectively. The behavioral examinations [Basso Mouse Scale (BMS) score, inclined plate test, Von Frey test] , and the gross, HE staining, and immunohistochemical staining were performed to observe the structural changes of the spinal cord at 4 weeks after operation. Another 3 C57BL/6 female mice were taken to prepare T 10 spinal cord injury models, and DiR-labeled EPCs- sEVs were injected through the tail vein. After 30 minutes, in vivo imaging was used to observe whether the EPCs-sEVs reached the spinal cord injury site. RESULTS: After identification, EPCs and EPCs-sEVs derived from mouse bone marrow were successfully obtained. In vivo imaging of the spinal cord showed that EPCs-sEVs were recruited to the spinal cord injury site within 30 minutes after injection. There was no significant difference in BMS scores and the maximum angle of the inclined plate test between two intervention groups and the model group within 2 weeks after operation ( P>0.05), while both were significantly better than the model group ( P<0.05) after 2 weeks. The Von Frey test showed that the mechanical pain threshold of the two intervention groups were significantly higher than that of model group and lower than that of sham group ( P<0.05); there was no significant difference between two intervention groups ( P>0.05). Compared with the model group, the injured segment of the two intervention groups had smaller spinal cord tissue defects, less mononuclear cells infiltration, more obvious tissue structure recovery, and more angiogenesis, and these differences were significant ( P<0.05); there was no significant difference between the two intervention groups. CONCLUSION: EPCs-sEVs can promote the repair of spinal cord injury in mice and provide a new plan for the biological treatment of spinal cord injury.
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