| BACKGROUND: Nonunion is one of the clinical complications in traumatic orthopedics.At present,the cure rate of clinical main treatment methods such as autologous bone transplantation is unsatisfactory.Some patients need to perform two or even multiple operations,which seriously affects the life of patients.quality.In recent years,with the deepening of tissue engineering technology research,stem cell therapy has received more and more attention,but there are still risks such as ethics and teratogenicity.Exosomes are vesicles secreted by cells,containing nucleic acids,proteins and other substances to complete the transmission of information between cells.Studies have confirmed that exosomes can play a similar role to the source cells.In this study,a hydrogel(piGEL)with specific affinity to bone tissue was used as a carrier,and a exosome derived from urinary stem cells(USC-Exos)was used to form a gel exosomes complex(USC-Exos-piGEL)The therapeutic effect of the complex on nonunion was verified by in vitro and in vivo experiments.METHODS: This study used the experimental technique established in the previous research group to extract and culture urine-derived stem cells(USC)from human urine,and extract the secreted exosomes(USC-exos)by ultrafiltration.(1)The morphology and size of USC-exos were observed by TEM,and the exosome particle size and exosome suspension concentration were determined by qNANO technique.(2)The proliferation of human umbilical vein endothelial cells HuVECs was verified by CCK-8 cell proliferation assay in vitro.(3)In vitro experiments were conducted to verify the migration of HuVECs by USC-exos by scratch test and transwell experiment.(4)The effect of USC-exos on the promotion of HuVECs into tubes was verified in vitro by tube-forming experiments.(5)The USC-Exos-piGEL complex was constructed in vitro and the amount and rate of exosomes released from the complex were determined in PBS buffer.(6)The animal model of femur nonunion was constructed,and the USC-Exos-piGEL complex was implanted into the nonunion of the bone.At the 4th and 8th week after surgery,X-ray,micro-CT,vascular perfusion,Methods such as tissue sectioning were used to assess the healing of nonunion and the regeneration of blood vessels around the nonunion.RESULTS:(1)USC-Exos was obtained by ultrafiltration combined with ultracentrifugation.The morphology was spherical and had a complete membrane structure with a size of 90-160 nm and a concentration of exosomes of about 1.97×1010 cells/ml.(2)In the scratch,proliferation and tube formation experiments,the USC-exos group showed a good promotion effect and was positively correlated with the content of USC-exos,and the results were statistically significant(P<0.05).(3)Photoinimide gel has good compatibility with exosomes and can be slowly released in vitro for more than 1 week.(4)Rats at 4 weeks and 8 weeks after femoral nonunion,X-ray,micro-CT and tissue sections showed that the bones in the experimental group were significantly healed and the bone mass increased.The vascular perfusion and tissue sections showed immunofluorescence detection.Significant angiogenesis in the nonunion of the group.Conclusion: Urine-derived stem cell-derived exosomes(USC-Exos)can promote the proliferation,migration and tube formation of HuVECs in vitro.The USC-Exos-piGEL complex implanted in vivo can effectively repair nonunion.The effect is related to the increase of blood vessel regeneration around the bone. |
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