Experimental Study On Fibrin Scaffold Transplantion Repairing The Spinal Cord Injury

Objective:To investigate the effects of the fibrin scaffold on the differentiation and the proliferation of neural stem cells and astrocytes and evaluate the biocompatibility of the scaffold with the spinal cord tissue.Methods:1.Fibrinogen was mixed with medium,then added the fresh rat blood serum. The reticular structured fibrin scaffolds were prepared.The surface and internal structures of the scaffolds were observed by electron microscopy.2.The spinal cord derived neural stem cells and the gliocytes were cultured in vitro respectively.The purified neural stem cells or gliocytes were seeded separately onto the fibrin scaffolds as experimental group and the glass slides modified with poly-L-lysine(PLL) as control group.At different time in culture(3d,7d and 14d) the neural stem cells were immunofluorescence stained with antibodies against the marker of neurons ie.neurofilament (NF).The length of NF-positive neuritis was masured and the average value was calculated in the culture well(n=4).The gliocytes were immunofluorescence stained with antibodies against the marker of astrocytes ie.glial fibrillary acidic protein(GFAP) at 14 days in vitro.The total cells and the GFAP-positive cells were counted from 5 different fields of vision in the culture well(n=4),then the average ratio of GFAP-positive cells was calculated.The differentiation of neural stem cells,the extension of neurites and the proliferation of astrocytes on the fibrin scaffolds were compared with those on the slides.The protein of GFAP was detected by Western blotting to analyse the mature degree of astrocytes.3.The rats were divided into two groups at random,the scaffold transplantation group and the spinal cord injury control group.The rats in two groups were suffered from spinal cord injury.In the scaffold transplantation group,the cylindrical fibrin scaffolds were implanted into the injured spinal cord.The rats of each group were sacrificed respectively at different times after operation,and the spinal segments or the scaffolds were removed out.The tissue reconstruction of the scaffold in the injured spinal cord was observed with electron microscope, in order to evaluate the scaffold histocompatibility.The regeneration of the nerve fibers and the proliferation of the gliacytes were analysed with immunofluorescence and immunoblotting methods,in order to invesgate the effect of fibrin scaffold transplantation on the regeneration of nerve fiber and the formation of glial scar after spinal cord injury in rats.4.The rats of each group were respectively estimated by system of the BBB locomotion score in 4 weeks,8 weeks and 12 weeks after operation.Comparison of hindlimb movements recovery in rats of different groups,in order to appraise the effect and feasibility of this scaffold to repair the injured spinal cord.Results:1.The profile of fibrin scaffold appears translucent and posseses soft like spongy. The light microscope image of the longitudinal section of the cylindrical scaffold stained with Eosin,showed the reticular structure.SEM and TEM images showed that the scaffold formed a three-dimension(3-D) porous network structure.2.The comparison of average length of the NF-positive neurites at different time points in vitro.lmmunofluorescence staining showed that the NF-positive neurites in the fibrin scaffold group were longer than those in the control group,(at 3 days and at 7 days,P＜0.05.at 14 days, P＜0.01).whereas GFAP-positive cells were fewer than those in the control group(P＜0.05).The comparison of percentage of GFAP-positive cells cultured after 14 days.The percentage of cells was 23.5%in the scaffold group,and 69.1%in the control group,respectively.The expression of GFAP in the cells on the scaffold was lower than that in the control group.3.The adhesions around the transplanted segment were separated carefully,then,we removed out the whole spinal cord from the experimental group 12 weeks after operation.The implanted scaffold was fused with the spinal cord and the appearance of spinal cord was plump. A large number of glial scars were formed and adhered to the dorsal connective tissue in the control group 12 weeks after operation.Majority of the injured spinal cord was healed,and there were collapses in the injured segment.The spinal cord was thinner.The spinal cord specimens and TEM images of the cross-sections of the implanted scaffold / injured spinal cord 12 weeks after operation.The TEM image of the cross-section of the implanted scaffold/spinal cord of experimental group,there were myelinated nerve fibers and the capillary in the area.The TEM image of the cross-section of the injuredsegment of the spinal cord in the control group,there were necrotic cavities with the thick wall,Asterisks showed the plasma cells in the cavities.4.From 4 weeks to 12 weeks after operation,the regenerating nerve fibers could grow into the scaffold then become more and more in case of scaffold transplantation,but there were fewer gliocytes in the scaffold.In the control group,the necrotic cavities were formed after spinal cord injury.There were a lot of gliacytes around the cavity.The results of Western blotting were that the contents of the neurofilament(NF) were higher than the glial fibrilliary acidic protein(GFAP) in the scaffold transplantation group.However,the results of the control group were on the contrary.There were significant differences between the scaffold group and the control group(P＜0.05).5.The control group rats of hindlimb movements recovery were lower than scaffold transplantation group after spinal cord injury.From 4 weeks to 12 weeks,the BBB locomotion scores of scaffold transplantation group were improved significantly contrasting to that of control group(P＜0.05).Conclusion:1.The profile of fibrin scaffold appears translucent and posseses flexibility. It could promote differentiation of the neural stem cells to neurones and extension of the neurites.Meanwhile,the scaffold could inhibit proliferation and mature of the astrocytes.2.The fibrin scaffold possessed a 3-D porous network structure.It has exhibit satisfactory biodegradation and histocompatibility in vivo.With regard to the biological functions,this scaffold could not only promote the regeneration and extension of nerve fiber,but also inhibit the formation of the glial scar.3.The fibrin scaffolds implanted into rats' spinal cords could promote the recovery of hindlimb movements.Fibrinogen as a biomaterial has a bright perspective in construction of the tissue engineering scaffold to repair the injured spinal cord.