Functionalized Self-assembling Nanofiber Hydrogel Peptide Scaffold RADAmx/FRM Promote The Regeneration Of The Contusion Spinal Cord Injury

Part 1 Fabrication of RADAmx/FRM self-assembled complexes and study on its properties and release profile OBJECTIVE: To establish the RADAmx/FRM polypeptide sustained release system,analyze the related physical and chemical properties and release law of this system,and establish the possibility of RADAmx/FRM as biomaterial.Methods: The polypeptide molecules RADA16,RADA16-FGL and FRM were synthesized by self-assembly technique,and the above three kinds of polypeptide molecules were analyzed and purified(mass spectrometer,high performance liquid chromatography).RADAmx self-assembly polypeptide was generated by using RADA16 and RADA16-FGL polypeptide molecules to form RADAmx/FRM selfassembly polypeptide molecule.The PBS was used to trigger the construction of RADAmx/FRM polypeptide sustained release system and then we can analyze the appearance of the gel.The ultramicrostructure of RADA16,RADAmx,RADAmx/FRM was analyzed using Atomic Force Microscope(AFM).The rheological characteristics of the RADA16?RADAmx?RADAmx/FRM system were analyzed using a rheometer.The regularity of FRM structural changes was evaluated by 0.5%RADAmx/FRM,1% RADAmx/FRM,2% RADAmx/FRM and 1% RADA16/FRM.We used the circular dichroism and fluorescence spectrum techniques to analize the secondary structure and the tertiary structure of protein of the FRM released from the RADAmx/FRM polypeptide sustained release system.Results:.Our team successfully synthesized the Peptides of RADA16,RADA16-FGL and FRM with the purity over 98%,and the molecular weight of them was 1712.78 Da,3458.74 Da and 1432.46 Da respectively.We observed the micro structure of the nanoFabrial Reticular structure of the RADAmx/FRM polypeptide sustained release system with the AFM.We observed no significant changes has happed for the secondary structure and the tertiary structure of protein of the FRM released from the RADAmx/FRM polypeptide sustained release system.Conclusion: The construction of RADAmx/FRM sustained release system was successfully carried out,and the identification of FRM related structure and its regulation of sustained release were also completed.Part 2 Biocompatibility and bioactivity of RADAmx/FRM complexs for neuronsOBJECTIVE: To study the biocompatibility of RADAmx/FRM sustained release system as a biomolecule in spinal cord injury biomarkers.The effects of RADAmx/FRM on the proliferation,survival and differentiation of neural stem cells(NSCs)and the growth of the axon.Methods: Neural stem cells were isolated from neonatal mice and cultured.The expression of Nestin in neural stem cells was identified by immunofluorescence staining.The neural stem cells were identified.The neural stem cells were inoculated with 1% RADAmx/FRM sustained release system.The survival rate of neural stem cells was detected by MTT assay.The differentiation of neural stem cells was analyzed by immunofluorescence.The axon growth of the neural stem cells were analyzed by the fluorescence microscope after cultured with the RADAmx/FRM Sustained Release System.Results: Nestin protein was strongly expressed by immunostaining of the neural cells from the mice,and the isolated cells were identified as neural stem cells.At the same time,the number of viable neural stem cells and the axon growth in the 1% RADAmx/FRM sustained release system was significantly higher than that in the other groups.Conclusion: RADAmx/FRM has good biomaterial properties,which can not only improve the survival rate and proliferation of neural stem cells,but also induce the differentiation of neural stem cells into neurons.It could also enhance the growth of the axon.These results indicated the RADAmx/FRM sustained release system could be very benefical in the application of neural tissue engineering for the repair of the SCI?Part 3 Repair of Spinal Cord Injury by RADAmx/FRM Peptide Sustained Release SystemOBJECTIVE: To investigate the efficacy and related biological mechanism of RADAmx/FRM in the repair of spinal cord injury by transplanting the RADAmx/FRM peptide sustained-release system into the core injury area by constructing the rat model of spinal cord injury in vitro.Methods: To use SPF SD rats,the rats were exposed to T10 spinal cord and combined with Allen to prepare SCI animal models.Wound area to build the model of rats given different intervention measures,so as to build the sham operation group,the spinal cord injury group,the 1% RADAmx/FRM group,and after the intervention measures for sports ability score(BBB method,1 time/week);To evaluate the hollow status of the core damage area of rat model in 8 weeks after surgery.Using immunofluorescence method analysis the GFAP/NF2000 protein expression changes of differrent groups in the damaged region,to assess different intervention measures in the regeneration of axons,the formation of scar tissue in the role?Results: The spinal cord injury model was established in this study.The exercise ability score of each group was improved gradually with the prolongation of time.At the beginning of the fifth week,the athletic ability score of RADAmx/FRM group was significantly higher than that of other groups,and the difference was significant(P <0.05),and thus RADAmx/FRM can promote the recovery of exercise capacity in rats.The area of voids in spinal cord tissue of RADAmx/FRM group was significantly smaller than that of blank control group(p <0.05)by HE staining.The fluorescence intensity of NF2000 protein in spinal cord tissue of RADAmx/FRM group was significantly higher than that of other groups by immunofluorescence staining.The fluorescence intensity of GFAP protein was significantly lower than that of other groups The difference was statistically significant(p <0.05)..Conclusion: RADAmx/FRM can significantly improve the motor ability of rats with spinal cord injury and can inhibit the formation of voids and glial scar in spinal cord injury.It can promote the regeneration of axons.It can be used as the biomaterial of spinal cord injury As a conclusion,the RADAmx/FRM could promote the repair of spinal cord injury.