Construction Of GelMA/PLGA-lysoGM1 Coaxial Electrospinning Scaffold And Its Effection In Traumatic Brain Injury Repair

Traumatic brain injury（TBI）caused brain tissue was defected,neural cells connection was distrupted and brain function was disabled,it has high rate of disability and mortality,furthermore,secondary damaged microenvirment was developed and caused complex structure of central neuronal system was broken,axon growth suppressed and lost its direction,balance of neurotransmitter deliver was broken caused function of brain was severly disabled in finally.Nowdays,it is a significant way that scaffolds plant into injury site to promote injured tissue recovery.In neuroal tissue structure,axon is an aligned arrange structure that promote its development,growth and biological function.Extracellular matrix（ECM）can be constructed by electrospunging technology,and regulate the directional arrangement by adjusting spinning parameters and receiver morphology,which is easy to provide physical and biochemical signals for axon formation and extension of nerve cells in structure and function.Poly（lactic-co-glycolic acid,PLGA）because of its good degradability and biocompatibility was widely used in tissue engineering,but its function and structure is very simple,lack cells recogined bioactive site on its surface,it has severly hydrophobicity that not benefit with cells adhsion and so on.Therefore,it is a new ideal that change its charactertion or surface modification for PLGA fibrous scaffold.Monosialotetrahexosylganglioside（GM1）was used to protects neurons and promotes the neurons repair,and used to treat diseases of nervous system.Arg-Gly-Asp（RGD）polypeptide sequence was founed in Gelatin methacrylate（GelMA）,which benefits for cells adhesion and migration,its hydrophilicity group such as–OH could be enhance hydrophilicity ability of scaffold.Base on this,in this article,GelMA/PLGA-lysoGM1coaxial eletronspun fiber was constructed to provide new strategy and approach by neuronal cells culuture in vitro and TBI mouse model in vivo experiments.The conclusion of this article in the following:（1）The ¹H NMR result of GelMA hydrogel have two peaks at ppm 5.6 and 5.4,it is-CH=CH₂ group of methacrylate.At peak of ppm 2.8 is weaker,it is lysine residue–NH₂of gelatin reacts with methacrylate,peak at ppm1.8 is more poignant,it is-NH-CO group in GelMA hydrogel,it indicates that GelMA hydrogel successfully synthesis.The FTIR of PLGA-lysoGM1 indicates a characteristic peak in 1671cm^-1 wavelength,it means amido peak.The ¹H NMR of PLGA-lysoGM1 shows a characteristic peak at ppm 8.0,it meas a peak of–NH-CO.The above results demonstrate that–NH-CO bond was breaken in GM1 to form lysoGM1 by hydrosis reaction of sphingolipid ceramide N-deacylase（SCDase）enzyme.PLGA-lysoGM1 was produced when lysoGM1 its–NH₂ group occurs condensation reaction with–COOH group of PLGA under the amine condensation reagent of 1-Hydroxybenzotriazole（HoBt）/N,N′-dicyclohexy lcarbodiimide（DCC）.（2）When collecter speed is 700 rpm/min,volate is 15 KV,collect distance is 10cm,pump speed is 1 m L/h,scanning electron microscope（SEM）images show that the fibers present a fully shape,a uniform diameter,no adhesion with other fibers and no break fibers,most of fibers angle is 0°-180°in the graphy,but when the collecter speed is 1400 rpm/min,the fibers present a anligned tendency,most of fibers are distributed at angle 100°-160°,Transmission electron microscope（TEM）and laser confocal microscope results tell that the structure of electrospuning fiber is shell-core.Results of water sorption and permeability point out GelMA/PLGA-lysoGM1 shows higher sorption and great permeability,futhermore,water sorption and permeability also decreased along with GelMA hydrogel concentration.Young’s modulus results demonstrates that Yong’s modulus decreasing along GelMA hydrogel.Water contact angle analysis demostrates that water contact angle of GelMA/PLGA-lysoGM1 coaxial eletrospuning fiber scaffold is aroud 0°,it shows that GelMA/PLGA-lysoGM1 scaffold with high hydrophilicity,it more benefit for neuronal cells adhesion,migration,migration and growth.（3）In vitro cells exprements indicate that the results of CCK-8 and Calcein-AM/PI live/dead assay show GelMA/PLGA-lysoGM1 eletrospuning fibers scaffold with better biological compatibility and cells growth function compare with PLGA and PLGA-lysoGM1 eletrospuning fiber scaffold.The consequence of western blot of N-cadherin demonstrate that GelMA/PLGA-lysoGM1 eletrospuning fiber is better for cells adhesion,migration and the development of synapsis.Immunofluorescence ofβⅢ-tubulin found that neuronal cells exrepressβⅢ-tubulin protein after retinoic acid（RA）introduced.The statistical results of axon length showed that lysoGM1 was beneficial to the extension and formation of axons in nerve cells.When GelMA hydrogel concentration is 12%,the length of axon is 73.28±12.22μm,it could be because of high GelMA concentration changed the stiffness of scaffold.（4）In vivo,wire grip socore shows that there is signifancance difference（P<0.05）between control mouse and GelMA/PLGA-lysoGM1 scaffold TBI model after 4 days,but no difference with PLGA-lysoGM1 group,it indicates that GelMA/PLGA-lysoGM1 scaffold coaxial electrospuning could promote the recovery of brain function.The results of H&E stain explain that control group with no ability of tissue healing till to 6^thweeks,PLGA-lysoGM1 TBI model group found that cells is hard to invasive in scaffolds to healing.But,cells is more easily invasive in GelMA/PLGA-lysoGM1 fibers scaffolds,also recovery condition of defected tissue is better than others groups.The results of Ki67,βⅢ-tubulin and GFAP immunofluorescence show that GelMA/PLGA-lysoGM1 electrosppun scaffold promote injury brain tissue repair compare with control and PLGA-lysoGM1 group at 6 weeks.GelMA and PLGA-lysoGM1 were successfully synthesized,and GelMA/PLGA-lysoGM1 coaxial electrospinning scaffold was successfully constructed in this paper.In vitro cell experiments,neuro-2a cells were induced to differentiate into nerve cells by retinoic acid.CCK-8,Calcein-AM/PI cells living/dead assay,N-cadherin protein western blotting andβⅢ-tubulin immunofluorescence staining confirmed GelMA/PLGA-lysoGM1 coaxial electrospinning scaffold has good promote nerve cell proliferation,good biocompatibility,promote cell adhesion and migration,and promote the axon extension.In animal experiments,hole size of d=3 mm,Φ=2 mm TBI model was created in C57 mouse,Though the grasp line test score,H&E staining,Ki67,βⅢ-tubulin and GFAP experimental methods,such as immunofluorescence staining showed that GelMA/PLGA-lysoGM1 electrospinning scaffold compared with control and PLGA-lyso-GM1 electrospun scaffold promoted the defects of the brain tissue repair.