Fabrication Of FGLmx/NEPl-40Peptide Sustained Release System For Rat Spinal Cord Injury Repair

Part1Fabrication of FGLmx/NEP1-40peptide sustained release system and study on its properties and release profileObjective To investigate the physiochemical properties and release profile of FGLmx/NEP1-40.Methods Peptides of RADA16, RADA16-FGL and NEP1-40were synthesized, and were purified and tested by mass spectroscopy (MS) and high Performance liquid chromatography (HLPC). The FGLmx/NEPl-40were induced to self-assembly with basal media in vitro, and the self-assembling peptide hydrogel were morphologically observed. Atomic Force Microscope (AFM) was used to examine the nanostructures of FGLmx/NEP1-40, and circular dichroism (CD) was used to study the secondary structure of FGLmx. Subsequently, we studied the release profile of four different concentration of sustained release system (2%FGLmx/NEP1-40,1%FGLmx/NEP1-40,0.5%FGLmx/NEP1-40and1%RADA16/NEP1-40) and evaluated the second and tertiary structure of NEP1-40released from FGLmx/NEP1-40.Results Peptides of RADA16, RADA16-FGL and NEP1-40were successfully synthesized and were confirmed by MS and HLPC. With purity over98%, the molecular weight was1712.78,3458.74and4584.16, respectively. The nanofibers of FGLmx/NEP1-40were observed with AFM and typical β structure was detected by CD. The1%FGLmx/NEP1-40were the most efficient release system, the secondary and tertiary structure of NEP1-40released from1%FGLmx/NEP1-40did not change significantly.Conclusions FGLmx/NEP1-40with sustained release profile was fabricated successfully. Part2Biocompatibility and bioactivity of FGLmx/NEP1-40with spinal cord derived neural stem cellsObjective To evaluate the survival, proliferation, migration and differentiation of spinal cord derived neural stem cells (SC-NSCs) cultured on the FGLmx/NEP1-40sysetem.Methods SC-NSCs were primary isolated and seeded on the release system FGLmx/NEP1-40. Live/dead assay was performed to investigate the survival rate after cultured for3days, and BrdU incorporation was used for study the proliferation of SC-NSCs. The spontaneous differentiation of SC-NSCs on FGLmx/NEP1-40was evaluated10days after seeding by immunofluorescence after3days incubation, SC-NSCs seeded on each scaffold was stained by Calcein-AM and observed by using Laser scanning confocal microscope (LCM) to examine SC-NSCs migration.Results The viability of SC-NSCs on FGLmx/NEP1-40was equivalent to other scaffolds. BrdU incorporation assay exhibited that cells proliferation on each scaffold increased gradually over the culture time with significantly higher rate on FGLmx/NEP1-40. SC-NSCs could spontaneous differentiate into neurons and astrocytes, but with more neurons in FGLmx/NEP1-40and FGLmx. Furthermore, reconstructed image of LCM3D collections of SC-NSCs seeded on scaffolds show the SC-NSCs cultured on FGLmx/NEP1-40and FGLmx were migrated into the scaffold, while SC-NSCs attached on surface of RADA16gel did not penetrate into the scaffold.Conclusions We found that the release system FGLmx/NEP1-40had non-cytotoxic to neurons and be able to promote SC-NSCs proliferation and spontaneous differentiation in comparison to other two scaffold, while the migration ability into scaffold was equivalent to that on FGLmx. These results indicate that the release system FGLmx/NEP1-40could be useful in nerve tissue engineering. Part3Effect of FGLmx/NEP1-40on rat spinal cord injury repairObjective:To establish rats’model of spinal cord injury (SCI) and evaluate the effect of in vivo treatment of SCI with FGLmx/NEPl-40release systemMethods:Rats were anesthetized and treated with laminectomy at the T10vertebral segment. The SCI models were established by impact of spinal cord injury with Allen method. Then, the animals were respectively treated with injecting8μ1sucrose (control group),1%RADA16(RAD group),1%FGLmx (FGL group),1%FGLmx/NEP1-40(NEP group). Weekly after the treatment, the functional recovery of SCI rats was evaluated by the Basso, Beattie and Bresnahan Locomotor Rating Scale. After8weeks of treatment, immunofluorescence staining was performed to detect GFAP/Laminin, GFAP/Fibronectin and GAP-43/5-HT. Myelin sheath degeneration was evaluated with electron microscopy.Results:The rats SCI model was established successfully. The BBB scores of SCI rats increased gradually over time with higher score in group treated with1%FGLmx/NEP1-40than other groups. Furthermore, more intense fluorescence intensity of Laminin, Fibronectin and GAP-43/5-HT was found in the NEP group with less GFAP fluorescence intensity and myelin sheath degeneration.Conclusions:The1%FGLmx/NEP1-40release system could be a useful biomaterial in nerve tissue engineering for improving the function recovery of SCI rats, inhibiting the formation of astrogliosis, promoting the axnal regeneration and5-HT fibers growth, and reducing myelin degeneration with neuroprotection effect.