The Effects Of AFGF/ Sulfated Chito-oligosaccharide Thermo-sensitive Hydrogel In Spinal Cord Injury

Purpose:aFGF has been applied in the repair of central nerve injury for its neuroprotective effects.However,aFGF has a short half-life,and cannot pass through the blood-brain barrier or the blood-spinal cord barrier,thereby whose neurological applications are limited.We synthesized sulfonated chito-oligosaccharide?SCOS?,which has the similar effects of heparinoid and can replace heparin as a protective agent for aFGF.Meanwhile,SCOS is also a bioactive matrix material and can be used for the preparation of hydrogels.Therefore,in this study,SCOS was used as a drug-loaded material,supplemented with chitosan and poloxamer,to prepare a temperature-sensitive hydrogel,and the pharmacodynamic study of repair and treatment of spinal cord injury was performed to expand the application of aFGF.Methods:?1?Sodium hyposulfite?Na₂S₂O₄?induced RSC96 cell hypoxia-reoxygenation injury model was established,then MTT assay tested cell viability.The lactate dehydrogenase?LDH?amounts were determined to judge the damaged degree of RSC96.The protective effects of SCOS,aFGF,and their combination on the damaged RSC96 cells were investigated..?2?A temperature-sensitive hydrogel of a FGF was prepared by using the special reversible temperature-sensitive property of poloxamer.The internal structure of hydrogel was observed by scanning electron microscope.Enzyme-linked immunosorbent assay?ELISA?was used to determine the release amounts of aFGF in the aFGF/SCOS hydrogel.The in vivo biocompatibility test was performed in mice.?3?Established a spinal cord injury model in rats,and observed the recovery of hindlimb movements and the changes of spinal cord tissue structure through behavior and morphology.Western Blotting,immunohistochemistry,and immunofluorescence were used to study the changes in the proteins associated with motor function recovery.Morphological changes of myelin were observed by transmission electron microscopy.Microvascular regeneration was examined by micro-CT.Results:?1?The combination of aFGF and SCOS was better than aFGF alone,which indicated that SCOS can effectively enhance the protective effect of aFGF on Na₂S₂O₄-induced hypoxia-injured RSC96 cells;?2?A thermo-sensitive gel system was established successfully which was a liquid state at 4°C and a gelation state above 32°C.Scanning electron microscopy showed that the gel was porous and distributed in a three-dimensional structure.The ELISA results showed that aFGF were slowly released from aFGF/SCOS thermosensitive hydrogel,and the cumulative release quantities reached about 80%.aFGF/SCOS thermosensitive hydrogel was almost completely degraded in vivo for more than 45 days,which could ensure the retention time of aFGF in vivo and continued to exert its efficacy and no inflammatory reaction occurred.There was no significant morphological changes on main visceral organs in a FGF/SCOS thermosensitive hydrogel experiment groups,which indicated that the aFGF/SCOS thermosensitive hydrogel has good biocompatibility.?3?In all spinal cord injury repair studies in rats,the exercise ability of the model animals recovered significantly after aFGF treatment?P<0.05?,especially the a FGF/SCOS thermosensitive hydrogel groups were more effective than the aFGF alone group.Morphology showed that the spinal cord structure of the aFGF/SCOS thermosensitive hydrogel groups were relatively intact and the number of Nissl bodies increased.The results of GFAP showed that the aFGF/SCOS thermosensitive hydrogel groups significantly reduced the activation of astrocytes after 28d of administration.aFGF/SCOS thermosensitive hydrogel group significantly promoted the expressions of SYN,promoted the regeneration of axons and restored synaptic function.The aFGF/SCOS thermosensitive hydrogel groups could increase the number and thickness of myelin and the tightness.The expresses of CD31,VEGF and CT imaging of blood vessels showed that the aFGF/SCOS thermosensitive hydrogel group could promote the formation of blood vessels in the early stage and stabilize at the later stage,providing a good microenvironment for the repair and regeneration of neurons.Conclusion:SCOS has the similar effects of heparinoid and can replace heparin as a protective agent for aFGF.aFGF/SCOS thermo-sensitive hydrogel group was more effective than aFGF alone.The repaired mechanism of the aFGF/SCOS thermosensitive hydrogel maybe:1)inhibit the degradation of the tight junction protein to prevent the further destruction of the blood-medium barrier;2)inhibit the shedding of the myelin sheath,promote the increase of axons,and synapse function recovery;3)can significantly reduce the activation of astrocytes,4)can promote microvascular regeneration and recovery.This study not only provides a basis for the recovery of aFGF in spinal cord injury,but also provides a novel biomaterial system for the delivery of protein drugs.