The Effect Of Matrigel Used As Scaffold Material For Neural Stem Cell Transplantation In The Treament Of Acute Spinal Cord Injury

Objective To investigate the possibilities of Matrigel used as scaffold material for cell transplantation in repair of acute spinal cord injury.Main Methods 1.The primary neural stem cells were isolated from the hippocampus of SD rats newborn within 24 hours,and then were cultured in Serum-free neural stem cell culture medium,supplemented with 2%B27,1%penicillin/streptomycin,20 ng/ml EGF,20ng/ml FGF-2.Fetalbovine serum was added to make it naturally differentiated,and cellswere identified use immunofluorescence technique.NSCs infected with Ad-GFP adenovirus were seeded respectively in Matrigel,absorbale sponge,TCP/POC.Cell survival was observed at 2 h,1 d,3 d,7 d、14 din different scaffolds.The Matrigel / NSCs mixture was inoculated subcutaneously into nude miceand the growth and differentiationof transplanted NSCs in Matrigel were observed by histological analysis.2.Allen’s weight-drop method(25 g/cm)was used to produce acute spinal cord injury(SCI)at T10 segment.The animals were randomly divided into three groups: PBS control group(PBS was injected to the injured spinal cord),Matrigel group(Matrigel was injected to the injured spinal cord),and Matrigel/NSCs group(Matrigel/NSCs wasinjected to the injured spinal cord).Treament was performed 7 d after SCI.NSCs from passage 3(Cell density was 5×107/ml)labeled with PKH67 were mixed with Matrigel,and the mixture were then transplanted into injured spinal cord in Matrigel/NSCs group.Survival of tra nsplanted NSCs was observed at1,7,14,28 d post-transplanted with scanning confocal microscopy.BBB score was used to evaluate hind limb motor function for 8 weeks.The effect of Matrigel on injured spinal cord and transplanted NSCs was evaluated with H&E staining and immunohistochemical detection.Results 1.NSCs isolated from the hippocampus of neonatal rats had strong clonal ability and can form cell clusters,which presented irregularly spherical.Immunofluorescence results suggest that the Nestin antigen formed in the early stage was positive.Cellsgraduallymigrated from the neurospheres and cell morphology changed with addition of serum.Cell immunofluorescence results suggest that the differentiation of GFAP antigen and MAP2 antigen was positive.2.Compared with absorbale sponge and TCP / POC scaffolds,the survival rate and time of NSCs in Matrigel were better than those in the other two groups,which suggested NSCs could survive a long time in Matrigel.Immunofluorescence results suggested that NSCs could differentiate to β-tubulin 3 positive neurons in Matrigel.Nude mice subcutaneous tumorpathology test showed thatnerve cells grow wellandnew blood vessels were observed in NSCs/Matrigel group.Tissue immunological testing results suggested that transplanted cells could differentiate into β-tubulin 3 and MAP2 possitive neurons.3.NSCs labeld with PKH67 showed NSCs survived long in theinjured site.4.BBB score in NSCs/Matrigel group was the highest and had significant difference compared to the other two groups(P<0.05).There was no significant difference between PBS group and Matrigel group.At four weeks post-transplantion,H&E staining of spinal cord tissue showedliquefaction and necrosis of nerve tissue in PBS group.In Matrigel group,cavities in the spinal cord were filled with Matrigel and a few nerve cells survived.In NSCs/Matrigel group,neumours nerve cells grew well,and had little angiogenesis.Immunohistochemical results showed that there were no neun,NF200 and MAP2 positive neurons in PBS group.A large number of Nestin positive neural stem cells and GFAPpositive astrocytes were observed at the edge of the lesion.Positive expression of Nestin,neun,NF-200,MAP2,GFAP was observed both in Matrigel group and NSCs/Matrigel group,which was more in NSCs/Matrigel group than that in PBS group.The scaffolds were degraded to a certain extent at 8 weeks after implantation and the expression of neural cells marker decreased.Conclusion Neural stem cells can survive and differentiated into neurons in Matrigel.It is convenient and difficult to cause secondary damage to use Matrigel as scaffold for cell transplantation after acute spinal cord injury.