An Injectable And Photocurable Methacrylate Modified Silk Fibroin Hydrogel Loaded BFGF For Spinal Cord Regeneration

Background:Spinal cord injury is a serious central nervous system disease.Intravenous or oral administration can improve functional after injury.It may also bring systemic side effects because of cannot exert the greatest pharmacological effect locally.Recently,biomaterials were used as targeted drug delivery and it can reduce the occurrence of systemic side effects.Purpose:We innovatively synthesized methacrylate-silk fibroin(Sil MA)hydrogels for bFGF delivery.And study the repairing function on spinal cord injury in rats from in vivo and in vitro.Methods:Part I:After degumming,silk fibroin was extracted from silkworm cocoons and dissolved with lithium bromide to reacted with glycidyl methacrylate to synthesize methacrylate-silk fibroin(Sil MA).The modification of Sil MA was determined by ~1H-NMR and FTIR.The pore structure with different concentrations was observed by scanning electron microscope.The mechanical properties were detected by rheology and stress-strain curve.The sustained release of bFGF was detected by ELISA.Part II:In vitro,the biocompatibility effect of Sil MA for undifferentiated PC12cells was detected by CCK-8 kit.Immunofluorescence was used to detect the function of induction with releasing of bFGF.Then the effect of Sil MA@bFGF on the function of mitochondria and the changes of ROS accumulation were observed through flow cytometer after primary neurons were hypoxic injury.Part III:10%Sil MA hydrogel was used for in vivo.The animals were divided into three groups,include injury group,Sil MA group and Sil MA@bFGF group.After the successful establishment of the spinal cord injury model,different hydrogels were treated respectively.BBB score was used to observe the improvement of lower limb function of rats.The expression of CD206 positive cells and NF-200 in neurons in the injured area were observed by immunofluorescence at 7 days after injury.H&E staining and Nissl staining were used to observe the structural changes at 28 days after injury.The protein expression of GFAP and MAP-2 were detected by immunofluorescence and western blot.Results:Part I:The synthesized methacrylate-silk fibroin can form hydrogel after irradiated with UV for 15 s under the action of the photoinitiator LAP.The ~1H-NMR results showed that GMA was successfully grafted with the lysine amino group at the end of SF.Scanning electron microscope showed that with the increase of Sil MA concentration,the porosity inside the hydrogel will become smaller.The results of rheometer show that the storage modulus of Sil MA is greater than the loss modulus.The compressive stress-strain curve shows that the compressive modulus of 10%Sil MA was lower than other concentrations.Different concentrations of hydrogel(10%,15%,20%)in PBS had significantly lower degradation rates than containing protease XIV.There is no difference in the degradation in PBS.The degradation rate of 10%Sil MA is the fastest in containing protease XIV.The bFGF rate of 10%Sil MA sustained-release was faster in the early stage,and then the sustained-release rate gradually decreased.Part II:The results of CCK-8 and live/dead staining of 3D cultured cells showed that 10%Sil MA hydrogel was better biocompatibility,while 15%and 20%Sil MA could reduce cell viability.In vitro,the results showed that 10%Sil MA@bFGF could induce the differentiation of undifferentiated PC12 cells and form axonal structures.The flow cytometry results of JC-1 and ROS showed that compared with injury group and the Sil MA group,10%Sil MA@bFGF could reduce the mitochondrial membrane potential changes and the production of ROS after injury in primary neurons.Part III:The BBB score was obtained until 28 days after injury.Compared with the injury group,Sil MA@bFGF hydrogel could significantly increase the functional score of the lower limbs.The number of CD206 positive cells with anti-inflammatory effect in the Sil MA group and the Sil MA@bFGF group was significantly increased at7 days after injury,compared with injury group.In addition,the expression of NF-200in nerve cells in the injured area in the Sil MA@bFGF group hydrogel was significantly increased compared with the other two groups.The hydrogels in the Sil MA group and the Sil MA@bFGF group could reduce the expression of GFAP and significantly increase the expression of MAP-2 protein at 28 days after injury,compared with injury group.H&E staining was used to observe the structure of important organs(heart,liver,spleen,lung,and kidney)in rats 28 days after surgery,indicating that Sil MA will not cause obvious organ damage during the degradation of Sil MA in vivo.Conclusion:The synthesized Sil MA hydrogel has the characteristics of injectability and fast photocuring.It can form hydrogel after irradiated with UV for 15 s and can be slowly released after loading bFGF.In vitro,the results showed that sustained release of bFGF could improve mitochondrial membrane potential and decrease ROS accumulation after injury.In vivo,shows that Sil MA can reduce local inflammatory response and protect damaged neurons in the early stage of injury.In the late stage of injury,Sil MA@bFGF can also promote neuronal axonal regeneration by inhibiting local gliosis,thereby improving the lower limb function of rats.