| Objective: spinal cord injury(SCI)is a traumatic central nervous system disease with highly rate of disability and serious consequence.In recent years,with the continuous development of the transportation and construction industry,the incidence of spinal cord injury is also rising.Sensor and motor dysfunction caused by spinal cord injury is often permanent and it brings great economic and psychological burden to the patients and families.Therefore,it is an important study in the research and treatment of spinal cord injury in medical field,and the scientific community has been exploring various methods to improve the therapeutic effect of SCI.A large number of experimental studies have confirmed that tissue engineering has a broad prospect in the treatment of SCI,and the basic model of tissue engineering for the treatment of SCI is the biological scaffold with seeded cells.In this experiment,we base on the reported literature and the previous studies of our lab.A 3-D printer was used for bionic spinal cord scaffold.Neuro D1-overexpression was studied in NSCs survival and differentiation.To evaluate the effect of the 3-D scaffold combined to NeuroD1-modified neural stem cells for spinal cord injury.Methods:1 After pregnant 14 d SD rat was anesthetized and disinfected,the brain hippocampus of the fetal rat was isolated.Then the hippocampus was grinded and digested.The cells were cultured after centrifugation and counting.The growth of NSCs was observed under phase-contrast microscope.When the diameter of neurosphere was 100-150 μm,the NSCs were passaged and cultured after digestion and counting.The third generation of NSCs was used to detect the purity by NSCs specific protein(Nestin)and DAPI immunofluorescence.After digestion and counting,the third generation of NSCs was cultured on polylysine plate.7 days later,the differentiation potential of NSCs was identified by neuron specific protein(MAP2),astrocytes specific protein(GFAP)and oligodendrocytes specific protein(MBP)immunofluorescence.2 The Retrovirus with NeuroD1 gene was infected with the neural stem cells,and the effect on the survival and differentiation of neural stem cells were studied.3 Collagen-heparin sulfate hydrogel was prepared firstly,and a new 3-D bio-printer was used to make bionic spinal cord scaffold.The structure was observed to measure its porosity.The scaffold was immersed in simulated body fluid to observe the quality change.The experiment was divided into two groups,Group A:the scaffold was co-cultured with rat neural stem cells for 7 days to observe cell adhesion and morphological changes;Group B: neural stem cells were cultured in polylysine in 24 wells culture plate.MTT assay was used to detect the cell viability.Immunofluorescence staining was used to identify the differentiation of NSCs.4 To evaluate the effect of the 3-D scaffold combined to Neuro D1-modified neural stem cells for spinal cord injury.The experiment was divided into four groups: A group is the control group,B is simple channel group,C group is channel + NSCs-GFP group,and D group is channel +NSCs-NeuroD1 group.Results:1 The NSCs grew well and proliferated rapidly observed under phase-contrast microscope.After 7 days,the NSCs can form a diameter of about 150 μm neurosphere and the neurospheres have good transmittance.Nestin and DAPI double staining was more than 95% by immunofluorescence.The identification test showed MAP2,GFAP and MBP positive cells and they can be labeled by DAPI.2 The cells were green on the fluorescence microscope,cells were transfected GFP successfully.The cells were transfected GFP successfully by PCR and Western blot.The group NSCs-Neuro D1 is the highest on NeuroD1 mRNA expression by PCR,and it had significant difference with the other two groups(P<0.05).The group NSCs-NeuroD1 is the highest on MAP2 mRNA expression by PCR,and it had significant difference with the other two groups(P<0.05).PCR results showed that MAP2 mRNA in group NSCs-NeuroD1 expression is the highest,and the other two groups had significant difference(P<0.05).The neurite length of the NSCs-NeuroD1 group was increased,and it had significant difference with the other two groups(P<0.05).MTT results showed that The proliferation of NSCs-NeuroD1 group was increased by MTT,and it had significant difference with the other two groups(P<0.05).3 Bionic spinal cord scaffold was fabricated by 3-D printer successfully.Electron microscopy revealed the micro porous structure with parallel and longitudinal structure.In vitro,the value of pH is not changed dramatically.After 8 weeks,the scaffold was completely degraded,and it meeted the requirements of tissue engineering scaffolds.MTT showed that there was no significant difference between two groups(P>0.05).Cultured with neural stem cells,it can significantly increase the cell adhesion rate,promote cell proliferation and differentiation.Quantitative analysis showed that there was significant difference on the differentiation rate of neuron between group A and group B(P<0.05).4 The BBB score of channel +NSCs-NeuroD1 group was the highest,and it had significant difference compared with the other three groups(P<0.05).In week 8,the Tarlov and Rivlin score of channel+NSCs-NeuroD1 group was the highest,and it had significant difference compared with the other three groups(P<0.05)Electrophysiological results show that the latency of movement and sensory of channel+NSCs-NeuroD1 was the lowset,and it had significant difference compared with the other three groups(P<0.05);the amplitude of movement and sensory of channel+NSCs-NeuroD1 was the highest,and it had significant difference compared with the other three groups(P<0.05).Immunofluorescence staining showed that there were GFP cells,differentiated into neurons in the channel+NSCs-NeuroD1,and it had significant difference compared with the other three groups(P<0.05).Conclusions:1 The method of primary cell culture of NSCs from embryonic SD rat was established successfully.The NSCs have great growth condition and high purity.The NSCs have multiple differentiation potentials and can differentiate into neurons,astrocytes and oligodendrocytes.We provide experimental basis for cell replacement therapy for the repair of nervous system injury and degenerative disease.2 NeuroD1 can promote the differentiation of neural stem cells into neurons,the length of neurite length,and it has a promoting effect on the proliferation of neural stem cells.3 The 3-D scaffold has good biocompatibility and biological properties.It can promote the proliferation and differentiation of neural stem cells,and it is a neural tissue engineering scaffold with great value of research and application.4 The 3-D scaffold combined to NeuroD1-modified neural stem cells can promote the nerve loop of spinal cord injury rats,and it can promote the recovery of motor and sensory function. |
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