| Objective: To construct aptamer(Apt19s)modified acellular spinal cord hydrogel,observe its internal structure,and evaluate its physicochemical and biological properties.The Spinal cord model of rats was constructed and the composite hydrogel was implanted to explore the repair effect of hydrogel on Spinal cord injury(SCI).Methods:(1)Preparation and characterization of the composite hydrogel: The aptamer was connected to the acellular spinal cord of rats in the form of chemical reaction grafting coupling(the aptamer was modified on the surface of the acellular spinal cord of rats),and the aptamer modified acellular spinal cord was further compounded with gelatin based hydrogel to prepare the aptamer modified acellular spinal cord hydrogel;The microstructure of the hydrogel was observed by scanning electron microscope.The water content rate of the hydrogel was evaluated by water absorption weighing method.The degradation rate of scaffolds was determined by phosphate buffer(PBS)solution.(2)Biocompatibility test of composite hydrogel: Live/dead cell staining was used to detect the activity of neural stem cells after co-incubation with composite hydrogel.(3)Determination of binding affinity between Apt19 s and different cells and determination of adhesion ability of composite hydrogel to neural stem cells: Fluorescence spectrophotometer was used to detect binding affinity between Apt19 s and different cells,and fluorescence microscope was used to observe the adhesion effect of composite hydrogel on neural stem cells.(4)The repair effect of composite hydrogel on SCI rats was investigated in vivo: adult healthy male SD rats(300-350 g,n=36)were selected and randomly divided into four groups:Sham group(the operation lasted until only the lamina of the spinal canal was opened,the spinal cord was exposed without any other treatment of the spinal cord,i.e.the sham group,n=9),Pure SCI group(only spinal cord semi-resection without hydrogel transplantation,i.e.Blank group,n=9),SCI+ASC/ hydrogel group(spinal cord semi-resection was performed on experimental animals,and acellular spinal cord/ hydrogel without aptamer modification was implanted after surgery,i.e.Pure hydrogel implantation group,n=9),SCI+Apt19s modified ASC/ hydrogel group(spinal cord semi-resection was performed on experimental animals,and acellular spinal cord/hydrogel functionalized by aptamer Apt19 s were implanted after surgery,i.e.Apt19 s hydrogel implantation group,n=9);A half-cut model of spinal cord injury was constructed,and hydrogel were transplanted to the spinal cord defect site of rats in the corresponding groups.BBB exercise scores were performed 1-4 weeks after surgery to evaluate the recovery of lower limb motor function.The gross morphology of the injured spinal cord was observed by naked eye at the 4th week after surgery.HE staining was used to observe the tissue repair of the injured site.The number of new neurons was observed by Nissl staining.The expression levels of neural stem cell marker Nestin,early neuron markerβIII-Tublin,mature neuron marker MAP2 and astrocyte marker GFAP were detected by immunofluorescence staining.To evaluate the repair effect of this hydrogel on spinal cord injury in rats.Results:(1)Aptamer modified acellular spinal cord hydrogel have three-dimensional porous structure with uniform size,good water retention,degradability and appropriate degradation time,and can cover the acute subacute stage and part of chronic stage of spinal cord injury repair.The introduction of aptamer does not change the internal structural characteristics of the hydrogel such as pore size and porosity,nor does it affect the swelling and degradation characteristics of the hydrogel.(2)Aptamer modified acellular spinal cord hydrogel has good biocompatibility and can support cell adhesion and survival on it.The binding affinity between Apt19 s and neural stem cells was the highest in adult neurons and mouse macrophages.Compared with the acellular spinal cord/hydrogel without aptamer modification,the acellular spinal cord hydrogel modified by aptamer Apt19 s showed stronger adhesion to neural stem cells.(3)At 4 weeks after surgery,BBB exercise scores in Apt19 s modified ASC/ hydrogel implantation group were better than those in SCI and ASC/ hydrogel implantation group.Compared with the other two groups except Sham group,HE staining showed that the aptamer modified acellular spinal cord hydrogel implantation group had the most proliferative cells in the lesion of injured spinal cord and had the best tissue integrity.The results of Nissl staining showed that the aptamer modified acellular spinal cord hydrogel implantation group had the largest number of Nissl bodies in the injured spinal cord lesions,the distribution was more uniform,and the morphology was the most regular.Immunofluorescence assay results of Nestin,βIII-Tublin,MAP2 and GFAP showed that: At 4 weeks after surgery,compared with the other two groups except Sham group,the aptamer modified acellular spinal cord hydrogel implantation group had the most Nestin,βIII-Tublin,MAP2 positive particles and the least GFAP positive particles in the lesion of injured spinal cord,and the difference was statistically significant.Conclusions: The acellular spinal cord hydrogel modified by Apt19 s with three-dimensional porous structure has good water retention,degradability and biocompatibility,which can promote the adhesion of neural stem cells,recruit neural stem cells in the acute stage of spinal cord injury,increase the number of parenchymal cells in the injured area,reduce the composition of interstitial cells,and reduce the formation of glial scar after SCI,and promote the recovery of nerve function. |
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