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Objective:To investigate the effect and mechanism of exosomes derived from human umbilical cord mesenchymal stem cells (hUC-MSC) in repair of tendon injury in rats.Methods:The hUC-MSC were cultured and the surface markers were identified by flow cytometry. The cells were induced to differentiate into osteoblasts, chondroblasts and adipocytes using a specific media. Meanwhile, the exosomes were isolated from the cell supernatant using exosome separation columns, and were identified by transmission electron microscopy, PKH67 staining and Western blot. A total of 40 Wistar rats were used to establish the Achilles tendon injury model by surgical resection. The rats were divided into hUC-MSC group (Group A) (with 100 μg exosome injected at the injured site) and control group (Group B) (with 250 μl normal saline injected at the injured site) according to the random number table, with 20 rats per group. The expressions of transforming growth factor β (TGF-β), bone morphogenetic protein (BMP-2), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the tendon tissues of both groups were detected using q-PCR, Western blot and immunofluorescence assay at 4 weeks following injection. The expression of collagen III in the injured tissues of both groups was detected by immunohiestochemistry.Results:The isolated and cultured hUC-MSC presented fusiform under an inverted microscope. After osteogenic differentiation, the cells exhibited a cube nodular structure, and the Alizarin red staining was positive. After adipogenic differentiation, the fat was observed inside the cells, which was red by oil red O staining. After chondroblast differentiation, the cells secreted a large amount of glycosaminoglycans, and a strong positive was revealed by Alisin blue staining. The hUC-MSC-derived exosomes showed round disc shape with a depressed internal structure under a transmission electron microscope, which was verified via PKH67 staining. The Western blot analysis showed high expressions of motility-related protein-1 (CD9) and lysosomal associated membrane protein 3 (CD63). The q-PCR test revealed that the mRNA expressions of TGF-β (4.887±0.767), BMP-2 (3.079±0.150), VEGF (3.108±0.508) and FGF-2 (4.211±0.522) in Group A were markedly higher than those in Group B (1.000±0.062, 0.918±0.129, 1.004±0.103, 1.010±0.169, respectively) ( P<0.01), and that the mRNA expression of IL-1β (0.697±0.037) and TNF-α (0.793±0.021) in Group A was markedly lower than those in Group B (1.004±0.089 and 1.006±0.015, respectively) ( P<0.01). The Western blot analysis revealed that the protein expressions of TGF-β (1.434±0.041), BMP-2 (1.798±0.177), VEGF (1.552±0.113) and FGF-2 (1.357±0.039) in Group A were markedly higher than those in Group B (1.002±0.032, 0.992±0.068, 1.007±0.070, 0.994±0.051) ( P<0.01), and that the protein expressions of IL-1β (0.705±0.016) and TNF-α (0.840±0.045) in Group A was markedly lower than those in Group B (1.000±0.016, 1.003±0.040) ( P<0.01). The immunofluorescence revealed that the positive expression rates of TGF-β and VEGF in Group A were not significantly different from those in Group B ( P>0.05). However, the positive expression rates of BMP-2 (2.278±0.208) and FGF-2 (4.656±0.106) in Group A were markedly higher than those in Group B (0.315±0.101, 1.661±0.110) ( P<0.05 or 0.01), and the positive expression rates of IL-1β (1.677±0.947) and TNF-α (1.520±0.088) in Group A were greatly lower than those in Group B (4.296±0.291, 2.373±0.273, respectively) ( P<0.01). In Group A, the tendon collagen fibers were arranged regularly and tightly, with relatively significant expression of collagen III; while the tendon collagen fibers in Group B were distributed loosely, accompanying broken scarlike healing. Conclusion:The hUC-MSC-derived exosomes can prompt the repair of the injured tendon tissues, which may be associated with the function in up-regulating the expressions of growth factors including TGF-β, BMP-2, VEGF and FGF-2, enhancing the expression of collagen III and inhibiting the expression of the inflammatory cytokines including IL-1β and TNF-α.
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Objective To explore the effect of intensive upper limb therapy assisted by a hand robot on motor function after stroke and performance in the activities of daily living.Methods Thirty-two hemiplegic stroke survivors were randomly divided into a conventional rehabilitation group (group A,n =10),a robot-assisted therapy group (group B,n=10),and a robot-assisted intensive therapy group (group C,n=12).The three groups all received routine drug therapy and daily 40 min sessions of conventional rehabilitation training 5 days a week for 4 weeks.Those in groups B and C were additionally provided with 20 min of robot-assisted therapy or 40 min of more intensive robot-assisted intensive therapy respectively.Before and after the intervention,the 3 groups were assessed using the Fugl-Meyer assessment for the upper extremities (FMA-UE),the action research arm test (ARAT) and the modified Barthel Index (MBI).Results No significant differences were observed among the 3 groups in any of the measurements before the treatment.In each group the average FMA-UE,MBI and ARAT scores had increased significantly after four weeks of treatment.The improvements in the average FMA-UE and ARAT scores were more significant in group B than in group A,while the FMA,MBI and ARAT scores suggested a significantly greater improvement in group C than in group B.Conclusion Robot assistance can help to improve upper extremity motor function after a stroke.It also has an obvious effect on improving performance in the activities of daily living.
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Objective To verify the anti-inflammatory effects of intra-articular injection of botulinum toxin type A (BoNT/A) on adjuvant-induced arthritis using a rat model.Methods A murine model of chronic ankle arthritis was established in 90 Wistar rats by injection of 0.1 ml of complete Freund adjuvant (CFA) into the pads of their left paws.They were then randomly divided into a BoNT group (n =30) which received an intra-articular injection of 0.1 ml (20 IU) of BoNT/A,an NS group (n=30) which received intra-articular injection of0.1 ml of normal saline solution and a sham group (n =30) which were punctured without any injection.In addition,30 normal rats formed a control group.Infrared thermal imaging was performed and an index of arthritis was evaluated every three days.The infrared thermal imaging revealed the expression of interleukin-1β (IL-1β) through hematoxy-eosin (HE) staining.Results The arthritis index began to increase 3 days after the injection of CFA and it had increased significantly after 10 days,reaching a peak value of 18,24 days after the injection.The infrared thermal imaging showed that the temperature in the right paw increased greatly after the injection.Following the development of arthritis,the temperature declined gradually,arriving at a steady temperature of between 37.5 and 38.0 ℃ in both ankles 20 days after the injection.The average temperature in both paws of the BoNT group had decreased significantly more by 7 and 14 days after the injection than in the NS and sham groups.The expression of IL-1β in the synovium of the ankle joint also had decreased significantly more in the BoNT group after 7 and 14 days.HE scoring showed an obvious histopathologic change in the hypertrophic synovium,inflamnatory cell infiltration,cartilage destruction and exposure of subchondral bone after 7 and 14 days compared with right after the injection in all groups except the control group.Moreover,the average HE scores of the BoNT group rats after 7 and 14 days were significantly lower than those seen in the NS and sham groups at the same time points.Conclusion Intra-articular injection of botulinum toxin type A has an anti-inflammatory effect on arthritis induced by complete Freund adjuvant,at least in rats.