Bioinspired Electrospun Hydrogel Fiber Scaffold For Spinal Cord Regeneration

Part 1.Fabrication and related investigation of GelMA hydrogel scaffold[Objective]Fabricate the aligned Gelatin and GelMA hydrogel scaffolds and investigate their physical characteristics.[Methods]Firstly,GelMA was synthesized,then aligned hydrogel fibers were achieved by electrospinning and ultraviolet(UV)light crosslinking.After a few repeated processes,fiber bundles can begot.Electrospinning technique was applied to make Gelatin and GelMA fibers be orientally,then oriented Gelatin fiber membrane were crosslinked bythe glutaraldehyde,and crosslinked GelMA fibers were obtained with the exposure to UV light.A certain thickness of fiber film can beobtained by adding the membranes over and over again on a slide,andthen a cylindrical fiber bundle can be obtained by rewinding the film.Scanning electron microscope(SEM)was used to observe the morphology of these fibers,and related results were analyzed by NanoMesurer software in the terms of diameter distribution.The swelling ratio and degradation rate of fibers were explored by weighting them at different time points.The mechanical properties were examined by biomechanical instrument.[Results]As shown in the results of SEM and NanoMesurer analysis,oriented fibers with smooth surface was successfully fabricated.The diameter of GelMA fibers was 1.1±0.13 μm,and 1.4±0.13 μm for Gelatin.Certain capability in stretching could be observed in both groups,the maximum stretching length of GelMA was 4.54 times the original length,and 1.94 times for that of Gelatin scaffolds.In the terms of Young,s modulus,GelMA was 0.7 Kpa which is significantly lower than 1.5 Kpa for Gelatin ones.According to the results of repeated stress-strain experiments,the slope between stress and strain of GelMA fibers was more stable comparing with that of Gelatin.In swelling experiments,the swelling ratio of GelMA was 600%,and 530%for Gelatin fibers on the first day(p<0.05).However,the swelling rate was decreasing with the time,meanwhile,the swelling rate of GelMA was obvious higher than that of Gelatin.As shown in the degradation investigation,after three days,94.8±0.84%GelMA was remined in weight,and 95.2±0.84%for Gelatin(p<0.05).35 days later,only 19±1.58%of GelMA scaffolds was remined,the Gelatin fibers still had 40±1.58%(p<0.05).[Conclusion]GelMA hydrogel fibers with excellent directional arrangement and mechanical properties were fabricated,at the same time,satisfactory capabilities in maintaining water and degradation could be obtained.Part 2 The investigation on the proliferation and margination of BMSCs and hippocampal neurons promoted by GelMA hydrogel scaffold[Objective]Investigating the ability of directed Gelatin and GelMA hydrogel scaffolds in promoting the migration,adhesion and proliferation of rat BMSCs and hippocampal neurons.[Methods]BMSCs were implanted on Gelatin and GelMA scaffolds.After one day’s culture,the cells implanted on the scaffold were dehydrated by alcohol,and the adhesion and migration of cells were observed by SEM after lyophilization process,BMSCs was implanted on these two scaffolds,respectively.After one day and three days of culture,the cell proliferation rate was measured by CCK-8 assay.The ability of these scaffolds in promoting cell adhesion was detected by the method of Phalloidin staining.The biocompatibility of the scaffold was examined by live/dead cell staining.The effects of fiber scaffolds on the morphology and axon growth of hippocampal neurons were observed by fluorescence staining.After 1,2,and 3 days of incubation,the depth of cells migrated into Gelatin and GelMA scaffolds were observed with confocal microscope combined by Phalloidin staining.[Results]BMSCs cultured on Gelatin and GelMA scaffoldsshowed satisfied morphology and adhesion statue,and cells migrated deeply into GelMA scaffold.The results indicated that BMSCs can adhere on both scaffolds.The cells in the control group showing irregular shape,while the cells growing on Gelatin and GelMA scaffolds were slender and orientated.The results of live/dead staining showed that most of the cells co-incubated on Gelatin and GelMA scaffolds were alive.CCK-8 showed that the cells proliferated successfully on the 1st and 3rd day of culture.GelMA scaffold significantly promoted the proliferation of cells.Comparing with the other two groups,there was significant statistical significance(p<0.05).The axon staining of hippocampal neurons showed that there was no extension of axons in the control group.While relatively long extension can be found in Gelatin and GelMA scaffold,among which the axon extension of GelMA scaffold was the best.The cells on the GelMA scaffold were gradually moving deeply down with time passed.(38.9±4.92 um on the first day,127.55±14.54 um on the second day,and 197.5±18.1um on the third day).While the cells were migrated lower on the Gelatin scaffold(24±4.35um on the first day,41.87±6.28um on the second day,and 60.6±8.4um on the third day).Quantitative data showed that there was significant difference between GelMA group and Gelatin group(p<0.05).[Conclusion]GelMA scaffold has good biocompatibility,which can promote cell adhesion and proliferation on the scaffold and accelerate neuronal cells’ axon elongation in vitro.Part 3 The study on the spinalcordinjury by GelMA hydrogel scaffold[Objective]Investigating the ability of GelMA hydrogel scaffold in promoting spinal cord injury repair.[Methods]Spinal cord injury model was established.Followed by saline irrigation,theGelMA or Gelatin fiber scaffolds were placed in the defect area,whilethe control group did not implant anything.The Basso,Beattie,andBresnahan(BBB)locomotor rating scale was applied to investigate thefunctional recovery of hindlimbs during open field locomotion.At 12 weeks after operation,the spinal cord whichcontained the lesion/graft site was sectioned for immunohistochemical staining(including neural stem cell staining,neuronal cell staining,nerve axon staining,glial scar staining,astrocyte staining and vascular endothelial cell staining).[Results]The motor funetion of hindlimbs in rats was evaluated with BBB score at each time pointafter surgery.After the surgery,the locomotor function of thelower limbs of the rats in the control group and Gelatin grouprecover slowly and remain limited over the period of observation,while rats in the GelMA group exhibit signifcant betterfunctional recovery.At 12 weeks,the score of GelMA groupreached 12.4+1.67,which was significantly higher than thatof blank(6.6+1.52)and Gelatin(8+1.23)groups(p<0.05).In this study,spinal cord nerve regeneration was observed by immunohistochemical staining.Quantitative measurement of optical density revealed that the number of neural stem cells in the spinal cord in the GelMA group was significantly higher than that in the Gelatin group and control group(p<0.05),It is proved that the GelMA hydrogel scaffold is more suitable for the migration and survival of the sur:rounding NSCs into the hydrogel scaffold.The neuronal cells were the cellular basis for the recovery of spinal cord function.Similarly,the tuj-1 labeled neurons differentiated by NSCs increased more significantly in the GelMA scaffold than that in the Gelatin group and the control group(p<0.05).The positive signals of synaptophysin in GelMA hydrogel scaffold were significantly higher than those in Gelatin group and control group(p<0.05),which proved that there was more axon formation in GelMA hydrogel scaffold.The results of astrocyte and glial scar staining showed that there were a large number of glial cell proliferation and glial scar formation in Gelatin group and control group,while significantly less in GelMA group(p<0.05).By comparing images of vascular endothelial cells labeled with CD31,the GelMA group had more fluorescence than the other two groups.The quantitative results also showed that there was more vascular regeneration in GelMA hydrogel scaffold than in Gelatin hydrogel scaffold and groups.[Conclusion]The soft biomimetic scaffolds constructed by electrospun gel fibers not only promote the migration of neural stem cells,induce them to differentiate into nerve cells,but also inhibit the formation of glial scar and promote angiogenesis.In addition,the scaffold has high elasticity and can resist deformation without protecting the bony spinal canal.Bioinspired hydrogel microfibers have been shovn to be effective and versatile in triggering spinal functional regeneration.