The Experimental Study On Repairing Spinal Cord Injury In Rat With Fibrin Glue Scaffold Incorporated SHH/Chitosan Microspheres

Objective: This study aimed to fabricate a fibrin glue(FG)scaffold incorporated sonic hedeghog(SHH)-loaded chitosan(CS)microspheres,and investigate its effect on the proliferation and differentiation of ectomesenchymal stem cells(EMSCs).Establishing a rat model of Spinal Cord Injury(SCI),transplanting EMSCs and composite fibrin scaffolds(FG-CS-SHH)to the spinal cord injury and evaluating the effect of SCI repair effect.Methods:(1)Firstly,The SHH-containing chitosan microspheres were prepared by via ionic gel method and mixed with fibrin glue.The fibrin glue scaffold embedded with SHH-loaded CS microspheres were obtained after freeze-drying(Fg-CS-SHH).The structure of composite scaffold was observed with scanning electron microscope(SEM)and the ELISA kit was employed to study the SHH releasing behavior,meanwhile,the SHH /chitosan scaffold(CS-SHH)and the SHH/fibrin scaffold(FG-SHH)were prepared as control.(2)The primary generation EMSCs were isolated from nasal mucosa and cultured.The neurosphere-derived EMSCs were cultured from the third-generation EMSCs by suspension culture methods.The surface marker protein was identified by immunofluorescence.(3)The neurosphere-derived EMSCs were transplanted on the above scaffolds and poly-L-lysine(PLL)-coated glass respectively.The compatibility of the scaffolds with the cell was determined by MTT.After co-cultured for 14 days,the differentiation of EMSCs were investigated with immunofluorescence staining(?3-Tubulin,MAP-2and MBP)and Western blot.(4)60 Sprague-Dawley host rats were randomly assigned into four groups:transection of spinal cord group(SCI);fibrin scaffold group(FG-SHH);chitosan scaffold group(CS-SHH)and composite fibrin scaffold group(Fg-CS-SHH).The rats of each group were respectively estimated by the BBB scores every week after operation.The spinal cord tissues were removed 12 weeks after surgery.Immunohistochemical staining and Western Blot were used to observe theexpression of protein(GAP43,GFAP and NF200)in spinal cord injury.Results:(1)SEM showed that Fg-CS-SHH scaffold after lyophilization had reticular structure,which looked like sponge,and CS-SHH microspheres were dispersed uniformly.ELISA showed that the release of SHH from Fg-CS-SHH scaffold was relatively gentler than the control groups and could last for a longer time to form a more stable and sustained release system.(2)EMSCs highly expressed stem cell markers of nestin,vimentin and S100.The neurosphere-derived EMSCs highly expressed stem cell markers of nestin and CD133.(3)MTT showed that FG-CS-SHH had good biocompatibility;Immunofluorescence staining indicated that the differentiated EMSCs could highly expressed nerve-related proteins(GAP43,GFAP and NF200)after co-cultured with FG-CS-SHH scaffold.Western blotting indicated that the expression of nerve-related protein in FG-CS-SHH scaffold was better than control groups(P<0.05).(4)The BBB scores of FG-CS-SHH group increased significantly from the postoperative period to 12 weeks compared with the control groups(P<0.05).Spinal cord tissue was removed 12 weeks after surgery.Immunohistochemical staining and Western Blot indicated that the expression of GAP43 and NF200 were significantly increased in the FG-CS-SHH group near the spinal cord injury,while the expression of GFAP was decreased.Conclusion: The FG-CS-SHH scaffold exhibited a sustained release effect and had good biocompatibility.It could induce the differentiation of EMSCs into neuron-like cells and significantly promoted the repair of rat spinal cord transection injury.