ObjectiveTo explore the potential therapeutic effects of endothelial progenitor cells derived small extracellular vesicles (EPCs-sEVs) on spinal cord injury in mice.MethodsEPCs 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 T10 lamina in sham group, and prepared T10 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 1×109 and 1×1010cells/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 T10 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.ResultsAfter 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.ConclusionEPCs-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 investigate the effect of M2-like macrophage/microglia-derived mitochondria transplantation in treatment of mouse spinal cord injury (SCI). Methods BV2 cells were classified into M1 (LPS treatment), M2 (IL-4 treatment), and M0 (no treatment) groups. After receiving M1 and M2 polarization, BV2 cells received microscopic observation, immunofluorescence staining [Arginase-1 (Arg-1)] and flow cytometry [inducible nitric oxide synthase (iNOS), Arg-1] to determine the result of polarization. MitoSox Red and 2, 7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) stainings were used to evaluate mitochondrial function difference. Mitochondria was isolated from M2-like BV2 cells through differential velocity centrifugation for following transplantation. Then Western blot was used to measure the expression levels of the relevant complexes (complexes Ⅱ, Ⅲ, Ⅳ, and Ⅴ) in the oxidative phosphorylation (OXPHOS), and compared with M2-like BV2 cells to evaluate whether the mitochondria were obtained. Thirty-six female C57BL/6 mice were randomly divided into 3 groups (n=12). Mice from sham group were only received the T10 laminectomy. After the T10 spinal cord injury (SCI) model was prepared in the SCI group and mitochondria transplantation (MT) group, mitochondrial storage solution and mitochondria (100 μg) derived from M2-like BV2 cells were injected into the injured segment, respectively. After operation, the Basso Mouse Scale (BMS) score was performed to evaluate the motor function recovery. And immunofluorescence staining, lycopersicon esculentum agglutinin (LEA)-FITC staining, and ELISA [vascular endothelial growth factor A (VEGFA)] were also performed. Results After polarization induction, BV2 cells in M1 and M2 groups showed specific morphological changes of M1-like and M2-like macrophages, respectively. Immunofluorescence staining showed that the positive expression of M2-like macrophages marker (Arg-1) was significantly higher in M2 group than in M0 group and M1 group (P<0.05). Flow cytometry showed that the expression of M1-like macrophage marker (iNOS) was significantly higher in M1 group than in M0 group and M2 group (P<0.05), and the expression of Arg-1 was significantly higher in M2 group than in M0 group and M1 group (P<0.05). MitoSox Red and DCFH-DA stainings showed that the fluorescence intensity of the M2 group was significantly lower than that of the M1 group (P<0.05), and there was no significant difference with the M0 group (P>0.05). The M2-like BV2 cells-derived mitochondria was identified through Western blot assay. Animal experiments showed that the BMS scores of MT group at 21 and 28 days after operation were significantly higher than those of SCI group (P<0.05). At 14 days after operation, the number of iNOS-positive cells in MT group was significantly lower than that in SCI group (P<0.05), but still higher than that in sham group (P<0.05); the number of LEA-positive cells and the expression of VEGFA in MT group were significantly more than those in the other two groups (P<0.05). Conclusion M2-like macrophage/microglia-derived mitochondria transplantation can promote angiogenesis and inhibit inflammatory M1-like macrophage/microglia polarization after mouse SCI to improve function recovery.
ObjectiveTo investigate clinical application of the free peroneal artery perforator flap in soft tissue defect of foot and ankle.MethodsThe clinical data of 18 patients with soft tissue defects of foot and ankle who were repaired with free peroneal artery perforator flaps between March 2019 and March 2020 were retrospectively analyzed. Among them, there were 11 males and 7 females; the age ranged from 21 to 58 years, with an average age of 45 years. The defect was located in the ankle in 2 cases, in the hindfoot in 4 cases, in the midfoot in 5 cases, and in the forefoot in 7 cases. The causes of injury included 11 cases of traffic accident, 4 cases of machine injuries, 3 cases of infection and necrosis after internal fixation. The time from injury to flap repair was 12-48 days, with an average of 24 days. The range of wound was 3 cm×3 cm to 15 cm×8 cm, and the range of skin flap was 4 cm×3 cm to 16 cm×9 cm. The flap harvesting time, operation time, intraoperative blood loss, and complications were recorded; the flap survival and patient satisfaction were observed during follow-up; and the American Orthopaedic Foot and Ankle Society (AOFAS) foot function score was used to evaluate the foot function.ResultsThe flap harvesting time was 15-33 minutes (mean, 22 minutes); the operation time was 120-160 minutes (mean, 150 minutes); the intraoperative blood loss was 90-180 mL (mean, 120 mL). There were 3 cases of vascular crisis after operation, including 2 cases of arterial crisis, which survived after vascular exploration and vein graft repair; 1 case of venous crisis, partial necrosis of the skin flap, and skin grafting to cover the wound after repeated debridement. The remaining 15 skin flaps survived completely. All patients were followed up 6 months. The skin flaps were in good shape without obvious bloat. According to the AOFAS foot function score, 5 cases were excellent, 10 cases were good, and 3 cases were fair. The excellent and good rate was 83.3%.ConclusionThe free peroneal artery perforator flap is easy to harvest, the shape and size of the flap are easy to design, and it does not damage the main blood vessels of the limb. The appearance and function of the limbs are satisfactory after operation. It can be widely used in the repair of soft tissue defects of the foot and ankle.