| Objective: Polydopamine(PDA)is an adhesive polymer that can functionalize the surface of any material.It has strong adhesion and good biocompatibility,which makes PDA not only give materials good hydrophilic properties,adhesion and biocompatibility,but also can be used as a platform for secondary modification,grafting various active molecules on the surface of the substrate.Based on its unique surface modification function,this study aimed to construct a Polydopamine-mediated surface modification of fibrin glue scaffold adhering sonic hedeghog,and explored its effect on the differentiation of neural stem cell(NSCs).Methods:(1)According to thematerials coated on the surface of FG,the protein scaffold were divided into three groups: FG control group(without any coating),FG-PD group(coating with polydopamine),FG-PD-SHH group(coating with polydopamine and SHH).First,the FG scaffold was obtained after fibrin glue freeze-drying.Then the FG-PD scaffold was prepared by using the natural adhesion characteristics of PDA.Finally,the FG-PD scaffold was grafted with the active factor SHH to obtain a FG-PD-SHH scaffold.Scanning electron microscope was used to observe the microstructure of the three groups of scaffolds,and the ELISA was used to measure the SHH adhesion and sustained release effect of FG-PD-SHH scaffolds.The microstructure was measured by scanning electron microscope(SEM)and the sustained release effect of SHH in the FG-PD-SHH group was observed by ELISA.(2)Neurospheres were cultured from fetal mice brain and then characterized,and the neural stem cells were cultured with the FG group(control),FG-PD group(coating with polydopamine)and FG-PD-SHH scaffold(coating with polydopamine and SHH)respectively.(3)To measure the effect of the different scaffolds on the growth and differentiation of rat NSCs;The third generation of rat NSCs were transplanted on the FG scaffolds,FG-PD scaffolds,and FG-PD-SHH scaffolds respectively,and cultivated by serum neurons medium.The effect of scaffolds on NSCs proliferation was determined by MTT.Scanning electron microscope was used to observe the growth of NSCs on the surface of scaffolds.After co-cultivation for 14 days,Immunofluorescence and western blotting were used to detect the differentiation of NSCs(GAP43,MBP,GFAP).(4)Sixty Sprague-Dawley rats were randomly assigned for the spinal cord injury model and randomly divided into four groups: spinal cord injury group(SCI);FG group;FG-PD group;FG-PD-SHH group.The function of the hind limbs was respectively evaluated by the BBB scores.Results:(1)Scanning electron microscopy(SEM)showed: fibrin glue scaffold had a sponge-like network structure after lyophilization,dopamine and SHH particles adhered to the network structurally.The ELISA results showed that the FG-PD scaffold had strong SHH adhesion,the cross-linking rate of SHH was high within 24 hours;At the same time,FG-PD-SHH scaffold had a good performance of sustainedrelease of SHH and could be maintained for about 19 days.(2)Rat NSCs were observed to aggregate from lots of suspended single cells into spheres by microscope,which had highly expressed nestin.(3)MTT showed that the FG-PD-SHH scaffold promoted the proliferation of rat NSCs.Scanning electron microscopy(SEM)showed that the NSCs in FG-PD-SHH group had the better growth and differentiation.Immunofluorescence and Western Blot results showed that the expression of GAP43,MBP in the FG-PD-SHH group was higher than that in FG and FG-PD group(P<0.05),while GFAP was lowly expressed(P<0.05).(4)The BBB scores in the FG,FG-PD,and FG-PD-SHH groups were higher than those in the SCI group from the 2nd week,the BBB scores of FG-PD-SHH group increased significantly compared with the other groups(P <0.05).Conclusion: The FG-PD-SHH scaffold had a good effect on SHH adhesion and slow-release,it could promote the proliferation and differentiation of NSCs in vitro and promote the repair of spinal cord injury in rats. |
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