Functionalized Electrospun Scaffolds Loaded With Neural Stem Cells For Regeneration Therapy Of Spinal Cord Injury

Purpose:To prepare PGCL nanofibrous scaffolds with orientation.The scaffolds were modified them with immobilized growth factors DOPA-IGF1 and DOPA-NGF to have specific biological functions.Combining the functional scaffolds with neural stem cells to construct neural tissue engineering scaffolds for the repair of spinal cord injury and to explore its mechanism.Methods:Part I: First,based on electrospinning technology,nanofiber scaffolds with orientation were prepared by adjusting electrospinning process parameters such as solution concentration,distance between rolling receiver and spinneret,and voltage.The physicochemical properties(surface topography,hydrophobicity,mechanical properties,etc.)of the scaffolds were evaluated by different experimental methods.Select the best process parameters to prepare the materials required for subsequent experiments.Secondly,primary neural stem cells were extracted from the cerebral cortex of fetal rats(fetuses of SD rats at13 – 15 days of gestation)and expanded by suspension culture and identified using immunofluorescence staining for characteristic proteins.Finally,the biological safety of electrospun nanofiber scaffolds was evaluated by culturing cells with material extracts and cell live and dead staining.Part II: First,insulin-like growth factor(YKYKY-IGF1)and nerve growth factor(YKYKY-NGF)with precursor sequences of DOPA adhesion group composed of lysine and tyrosine were prepared by recombinant expression of fusion protein using genetic engineering technology,and immobilized recombinant growth factors DOPA-IGF1 and DOPA-NGF with DOPA domain were formed by tyrosine hydroxylase reaction.Proteins were characterized using Western blot(WB)and other modalities.Secondly,the adhesion and stability of immobilized growth factors were analyzed by enzyme linked immunosorbent assay(ELISA).Immobilized growth factors were used to modify the nanofiber scaffolds,and the materials before and after modification were comparatively characterized by contact angle,Fourier transform infrared spectroscopy,and XPS.Finally,the effects of different concentrations of growth factors on the proliferation and differentiation of neural stem cells were investigated by CCK-8 method,Western blot(WB)and immunofluorescence staining,and the optimal concentration was selected for subsequent experiments.Part III: First,four kinds of functionalized electrospinning scaffolds modified with immobilized growth factors were constructed and co-cultured with neural stem cells according to the previous experimental results.Then the effects of four bioactive scaffolds on the proliferation and differentiation of neural stem cells were investigated by Ed U cell proliferation assay,Western blot(WB)and immunofluorescence staining.Finally,the mechanism of behavioral regulation of neural stem cells by four bioactive scaffolds was investigated by transcriptomics.Part IV: First,a model of T9 spinal cord transection injury in SD rats was established,and then a neural tissue engineering scaffold was constructed and transplanted into the spinal cord injury of rats according to the previous experimental results,and postoperative observation and nursing were performed for 8 weeks.The recovery of motor function was analyzed by footprint test and BBB score.Finally,the spinal cord tissues of rats in each group were sampled,and the repair of spinal cord injury in rats was analyzed using HE staining,LFB staining and immunofluorescence staining to evaluate the cavity area,new neurons,glial scars,myelin regeneration,axonal growth,new blood vessels and local inflammatory reactions at the injury site.Results:Part I: The physical and chemical properties of nanofiber scaffolds were evaluated according to orthogonal experimental design.SEM results showed that nanofibrous scaffolds with good morphology could be prepared when polymer polymer materials with GA/CL of2/8,spinning solution concentration of 15 wt%,and spinning distance(distance between rolling receiver and spinneret)of 18 cm were used.The spinning diameter is uniform,the spinning is smooth and oriented,without breakage or beaded nodules,which is most conducive to cell adhesion and migration;the contact angle test shows that the contact angle of each group of samples is greater than 90 °,suggesting that the nanofiber scaffold belongs to hydrophobic materials as a whole;the mechanical performance test suggests that the scaffold has certain strength and toughness,and its elastic modulus and tensile strength increase with the increase of CL proportion;the extracted fetal rat primary neural stem cells are identified by immunofluorescence staining of the characteristic protein Nestin,and the results show that the extraction is successful;the extraction test and live and dead staining of cells on the material surface show that the material has good biosafety.Part II: Y-IGF1 and Y-NGF were successfully expressed and prepared by genetic engineering technology,and DOPA-IGF1 and DOPA-NGF,which have strong adhesion,were generated by tyrosine hydroxylase reaction.Adhesion and stability were tested by enzyme-linked immunosorbent assay(ELISA).The results showed that the recombinant growth factor had strong adhesion,and the high concentration growth factor combined closely with the material,had strong stability,and was not easy to release.The comparison of contact angle,Fourier transform infrared spectroscopy,XPS and other methods before and after modification suggests that the recombinant growth factor successfully modifies the nanofiber scaffold,and the hydrophilicity increases greatly,which is conducive to cell adhesion and growth.Using the amount of immobilized growth factor per unit area as the unit of measurement,we obtained that D-IGF1 and D-NGF at different concentrations in different concentration groups promoted the proliferation of neural stem cells and neuronal orientation differentiation by CCK-8 assay,Western blot(WB),and immunofluorescence staining,with the best effect at 50 ng/cm2,so we selected this concentration for subsequent experiments.Part III: Four kinds of functionalized electrospinning scaffolds modified with immobilized growth factor(Control,D-IGF1,D-NGF,D-IGF1&D-NGF)were successfully constructed and neural stem cells were co-cultured with them.Ed U cell proliferation assay showed that neural stem cells in the double immobilized growth factor group had the strongest proliferation activity.Western blot(WB)and immunofluorescence staining results showed that D-NGF group could differentiate more neural stem cells into neurons and promote axonal growth compared with D-IGF1 group,while the double factor group had the strongest differentiation effect on neural stem cells into neurons and promoted axonal growth.Transcriptomic analysis suggests that D-IGF1 or D-NGF alone or in combination with two recombinant growth factors can regulate the gene expression and cellular behavior of neural stem cells.When the two growth factors are used in combination,the gene expression of cells is most significantly regulated,and D-NGF may play a dominant role in the gene expression regulation of neural stem cells.The addition of D-IGF1 may change the regulation mode of D-NGF,and the mutual influence of the two may have a synergistic and additive effect,thus increasing the differential gene expression of INN VS N group and producing a stronger biological effect,and finally has a certain effect on the paracrine secretion,cell survival,differentiation,migration,angiogenesis,axonal growth and inflammation-mediated functions of cells,which may involve the signaling pathways including: MAPK/ERK pathway,PI3K/Akt pathway,etc.Part IV: A neural tissue engineering scaffold equipped with double immobilized growth factors was constructed and implanted into the injured site of rats undergoing total spinal cord transection.Behavioral assessments showed that rats implanted with neural tissue engineered scaffolds had the best recovery of motor function compared with other experimental groups.Histological evaluation such as HE staining,LFB staining,and immunofluorescence staining showed that neural tissue engineering scaffolds equipped with double immobilized growth factors could effectively promote neuronal regeneration,axonal growth,and vascular regeneration at the injured site,while also having a certain inhibitory effect on scar formation and local inflammatory response.Conclusion:(1)PGCL nanofiber scaffolds with certain morphological characteristics,mechanical properties and degradation properties can be prepared by adjusting different process parameters using electrospinning technology,and have good biosafety.(2)The immobilized recombinant growth factors D-IGF1 and D-NGF were successfully prepared by genetic engineering technology,which have high adhesion,stability and biological activity.They can be used for surface modification of biomaterials,which can make biomaterials have certain biological functions.(3)Nanofiber scaffolds modified with D-NGF were more effective than D-IGF1 in promoting neural stem cell differentiation into neurons and promoting axonal growth,while the combination of the two growth factors had a stronger biological effect.(4)PGCL nanofiber scaffolds modified with double immobilized growth factors loaded with neural stem cells can effectively promote neuronal regeneration,axonal growth,and angiogenesis at the injured site in rats with spinal cord injury in vivo,and also have a certain inhibitory effect on scar formation and local inflammatory response.