A Neuroprotective Sericin Hydrogel As An Effective Neuronal Cell Carrier For The Repair Of Ischemic Stroke

Part 1.A neuro-protective sericin hydrogel explored as a cell carrier for the repair of strokeObjective:Fabricate a neuro-protective sericin hydrogel for the repair of stroke and evaluate its performance.Methods:We obtained the sericin solution from the cocoons by the LiBr extraction method.The genipin was selected as a crosslinker,and the genipin crosslinked sericin hydrogel was fabricated through a covalent cross-linking reaction.A serial of properties were evaluated,including the secondary structure,the mechanical strength,the swelling ratio,the microstructure and the degradation behaviors.The cytoskeleton staining and the Live&Dead staining were performed to study the cyto-compatibility of the sericin hydrogel.The neuro-protective property was assessed by the OGD model and the neurotrophic effect was assessed by the primary cortical neuronal axon outgrowth experiment.The underlying molecular mechanism was explored by the western blotting and the signal pathway inhibition assay.The in vivo transplantation was also performed to evaluate its performance as a cell carrier.Results:The sericin solution formed a stable and light blue hydrogel.It exhibits the lower mechanical strength,the stable swelling ratio,the porous microstructure and the good degradation behavior.The F-actin staining and Live&Dead staining have confirmed its excellent cyto-compatibility.The OGD assay has found the neuro-protective property of the sericin solution and its degraded products.Under 0.05mg/mL sericin solution,the outgrowth of neuronal axon was enhanced.The western blotting assay confirmed that the Bcl-2/Bax signaling pathway play a key role in the neuro-protective behavior.The Nuak-1 inhibitor assay showed the enhanced outgrowth of neuronal axon benefits from the Lkb-1/Nuak-1 pathway.The in vivo transplantation experiment indicated that this sericin hydrogel have the powerful potential in the neural tissue engineering.Conclusion:Our fabricated genipin crosslinked sericin hydrogel could be utilized as a promising biomaterial in the neural tissue engineering for the stroke treatment.Part 2.A carbon nano-tube hybrid sericin scaffold with injectability and conductivity characteristics applied in neural repairing field.Objective:Design a conductive sericin hydrogel with injectability and utilize it in neural tissue repair fieldMethods:The gentle LiBr extraction method was used to obtain the pure sericin solution.The carbon nano-tubes were mixed in the genipin crosslinked sericin hydrogels by the ultrasonic method.The physical and chemical characteristics of the carbon nano-tube hybrid sericin scaffold were tested,including the porous micro-structure,the mechanical modulus and the swelling behaviors.The confocal analysis,the CCK-8 assay and the flow cytometry were performed to evaluate its cyto-compatibility.The injectability property of the hybrid sericin scaffold was evaluated by the in vitro and in vivo injection assay.The electrical property was study by the impedance measurement experiment.The immune-fluorescence staining,the Real-time quantitative PCR and the western blotting experiments were performed to study the pro-differentiation characteristics of this hybrid sericin scaffold.Results:The CNTs hybrid sericin scaffold was successfully synthesized and the CNTs were homogeneously distributed in the scaffold.The SEM images confirmed that the scaffold possessed the interconnected porous microstructures.We also found that the mechanical modulus of the scaffold was controllable through adjusting the content of the CNTs during the synthesis process.The swelling assay showed us that the scaffold had the stable swelling ratio after it was immersed in PBS for 48 hours.In the cyto-toxicity assay,the scaffolds with 0.1-0.5mg/mL CNTs exhibited the excellent cyto-compatibility.In the in vitro and in vivo injection experiments,we found and confirmed that the scaffold possessed the injectable property.Benefiting from the incorporated CNTs,the impedance of the scaffold was significantly decreased.In the Real-time quantitative PCR,the Tuj-1 immune-fluorescence staining and the western blotting experiments,we found that the scaffold with CNTs can promote the BMSCs differentiated into neural cells.Conclusion:This CNTs hybrid sericin scaffold with injectability and pro-differentiation properties has the potential in the neural tissue repair field.