Fabrication Of Functionalized Peptide Self-assembling Nanofiber Scaffolds Containing FGL Motif And Its Effect On Rat Spinal Cord Injury Repair

Part 1 Fabrication of functionalized peptide self-assembling nanofiber scaffolds containing FGL motif and Detection of the nanostructure and mechanics of FGL-NSObjective:To synthesize self-assembling peptide RADA-FGL with a sequence of AcN-RADARADARADARADAGGEVYVVAENQQGKSKA-CONH2 and investigate the condition of RADA-FGL self-assembly and detect the nanostructure and mechanics of FGL-NS.Methods:Both RADA-16 (AcN-RADARADARADARADA-CONH2) and RADA-FGL peptide were synthesized by using a peptide synthesizer, which were purified and tested by mass spectroscopy (MS) and high performance liquid chromatography (HPLC).1%(10 mg/ml) RADA-16,1% RADA-FGL peptide solution and 1% mixture of RADA-16 and RADA-FGL (FGL-NS) were induced to self-assembly with PBS in vitro, and the self-assembling peptide hydrogel were morphologically observed. Atomic Force Microscope (AFM) was used to examine the nanostructures of self-assembling peptide nanofibers. Rheological analysis of RADA-16, RADA-FGL and FGL-NS were performed by using a rheometer. Results:The RADA-16 and FGL-RADA peptides were successfully synthesized and were confirmed by MS and HLPC. The molecular weight of the RADA-FGL was 3458.74 Da with a purity of 95.87% and RADA-16 was 1712.78 Da with a purity of 98.16%. RADA-16 and RADA-FGL peptide were dissolved in distilled sterile water at a final concentration of 1%(10 mg/ml). Triggered with PBS, RADA-16 formed a hydrogel, but RADA-FGL remained as a non-viscous solution. However, when FGL-RADA is mixed 1:1 with RADA-16, it formed a self-supporting hydrogel. We studied the structures of 1% peptides of RADA-16, RADA-FGL and FGL-NS using AFM. There was no nanofiber formation in 1% of RADA-FGL, but the nanofibers were observed in FGL-NS peptide. The diameter of the fiber self-assembled by FGL-NS was 38.2±2.7 nm and the length of the fibers can be as long as several hundreds of nanometers, while the diameter of nanofibers of RADA-16 was 16.9±2.3 nm. Hydrogel formation of FGL-NS was further confirmed using rheological tests. Similar with RADA-16, the value of G' was greater than G", suggesting that FGL-NS behaved as an elastic solid. Furthermore, both G' and G" were independent of frequency and no crossover of G' and G" was observed, which is characteristic of cross-linked networks.Conclusions:Target self-assembling peptide RADA-FGL was synthesized successfully. Pure RADA-FGL could not self-assemble into nanofibers. However, the mixture of RADA-FGL and RADA-16 at a ratio of 1:1 could undergo self-assembly to form interwoven nanofibers that further form a hydrogel. Part 2Biocompatibility and bioactivity of functionalized self-assembling nanofiber scaffold containing FGL motif for rat dorsal root ganglion neurons and neural stem cellsObjective:To culture rat dorsal root ganglions neurons (DRGn) and neural stem cells (NSCs) in FGL-NS hydrogel in vitro and evaluate the biocompatibility and bioactivity of functionalized self-assembling nanofiber scaffold containing FGL motif for rat dorsal root ganglion neurons and neural stem cells.Methods:Both NSCs and dorsal root ganglion (DRG) explants were collected from newborn rats, and then DRG were dispersed into DRGn suspension by primary isolated culture methods. The DRG, DRGn and NSCs were divided into experimental (EG) and control group (CG). In EG:the cells were transplanted on FGL-NS. In CG:the cells were seeded on RADA-16. Growth of DRG and DRGn were observed under inverted microscope. The cytotoxicity of FGL-NS and its effect on attachment of DRGn and DRGn's viability were detected by live/dead assay kit and immunofluorescence staining combining with cell counting. Then, neurite length of DRG and DRGn was measured using Image Pro Plus Version 6.0 after 48 h of incubation. The survival rate of NSCs seeded on RADA-16 and FGL-NS was determined by a fluorescence microscope (FM) with fluorogenic ester Calcein-AM (CAM) and nucleic acid dye propidium iodide (PI) at 1 day and 7 day of incubation respectively. After 1,3,7,14 d of incubation respectively, FGL-NS/cells were taken out for cell proliferation assay by CCK-8. The differentiation of NSCs on FGL-NS was evaluated 7 days after seeding by double and single immunostaining. After 24 h of incubation, NSCs seeded on FGL-NS and RADA-FGL was stained by CAM and observed by using Laser confocal microscopy in order to examine NSCs migration.Results:The viability of DRGn and NSCs on the FGL-NS hydrogel was equivalent to that on RADA-16 surface after 1 d and 7 d of incubation respectively. The number of DRGn attached on FGL-NS (42.3±3.23) was significantly higher (p<0.05) than that on RADA-16 (29.9±2.13) after incubation for 4 h. Phase-contrast photomicrographs show that the morphology of DRG explants and DRGn varied greatly on FGL-NS and RADA-16 gels. By day 3 in culture, there were only small networks formed by a few neurons on RADA-16 samples. Additionally, few and short neurites extended from DRG explants were present on RADA-16. In contrast, the network connections were formed by DRGn seeding on FGL-NS and DRG explants on the FGL-NS have longer neurites. After 24 h and 48 h of culture, the percentage of cells with neurites was compared between RADA-16 and FGL-NS hydrogels. After 24 h, the percentage was 49.3±1.91% on RADA-16,57.6±2.47 % on FGL-NS. After 48 h, the percentage was 66.1±2.67% on RADA-16,81.2±2.80% on FGL-NS. The level of neurite growth was significantly higher (p<0.05) on FGL-NS. Furthermore, neurite outgrowth was quantified by measuring the longest neurite of DRGn and DRG explants on day 2 after culturing. Average neurite length of DRGn was 78.9±5.18μm on RADA-16,136.27±10.27μm on FGL-NS. Maximal DRG neurite length was 291.13±31.02μm on RADA-16,573.33±34.43μm on FGL-NS. Cell Proliferation assay of NSCs exhibited that the A value increased gradually over the culture time and A value on FGL-NS is significantly higher than that on RADA-16. But, there is no significant difference of the percentage of neurons and astrocytes between FGL-NS and RADA-16. Furthermore, reconstructed image of LCM 3D collections of NSCs seeded on gels show the NSCs cultured on FGL-NS were migrated into the scaffold, while NSCs attached on the surface of RADA-16 gel did not penetrate into the scaffold.Conclusion:We found that the functionalized self-assembling peptide scaffold was non-cytotoxic to neurons and able to promote adhesion and neurite sprouting of neurons. Furthermore, FGL-NS could improve NSCs proliferation and migration into scaffold in comparison to pure RADA-16 peptide scaffold, but have no effect on the differentiation of NSCs. These results indicate that the novel designer peptide scaffold containing FGL motif could be useful in nerve tissue engineering. Part 3Effect of functionalized peptide self-assembling nanofiber scaffolds containing FGL motif on rat spinal cord injury repairObjective:To establish rats' model of spinal cord injury and evaluate the effect of in vivo treatment of SCI with FGL-NS peptide that could self-assemble in vivo into functionalized nanofiber scaffold.Methods:The rats were anesthetized and treated with laminectomy at the T10 vertebral segment. Then, the SCI models were established by compressing the spinal cord with a 24 g modified aneurysm clip. After 1 d of SCI, the animals were respectively treated with injecting 2.5μL 580μM solution of glucose,1% RADA-16 and 1% FGL-NS peptide solution into the injured zone. After 3 d,1 w,3 w,5 w,7 w and 9 w of treatment, the functional recovery of SCI rats was evaluated by the Basso, Beattie and Bresnahan Locomotor Rating Scale. After 9 w of treatment, the apoptotic cell death, the number of neurons with neurite and astrocyte were detected by Caspase-3, NF-200 and GFAP immunohistochemistry.Results:The model of spinal cord injury rats was established successfully. The BBB scores of SCI rats treated with FGL-NS were higher than those of RADA-16 group after 5 w of treatment. Furthermore, in vivo treatment with FGL-NS reduced cell death, increased the number of neurons with neurite and inhibited the formation of astrogila at the site of injury.Conclusion:Functionalized peptide self-assembling nanofiber scaffolds containing FGL motif could be a useful biomaterial in nerve tissue engineering for improving the functional recovery of SCI rats, inhibiting the formation of astrogila, reducing the cell death and increasing the number of neurons in site of injury.