Transplantation of NT-3 delivery acellular optic nerve scaffold to improve motor function in transected rat spinal cord
【摘要】：Objective To generate a bioactive scaffold with line-shaped tubes in vitro and investigate whether the scaffold transplanted into the injury site could promote nerve fiber regeneration and functional recovery in rat spinal cord transected completely. Methods By using aseptic decellular, DNAase hydrolysis and freeze-drying techniques, natural optic nerve was removed all cell component and coated with neurotrophin-3(NT-3)/fibroin complex at the surface to fabricate a NT-3 delivery acellular optic nerve scaffold. Then, dorsal root ganglion(DRG) was seeded on the surface of NT-3 delivery acellular optic nerve scaffold in vitro to observe the effect of the scaffold on DRG neurite growth, and the scaffold were transplanted into the injury site of spinal cord injury of rat. Results Fresh normal optic nerve of longitudinal section was stained by HE showing high-density tubes with optic nerve fibers. By using aseptic decellular technique and freeze-drying technique, the NT-3 delivery acellular optic nerve scaffold still maintained its natural 3 dimension(3 D) structure of extracellular matrix. Circle-shaped pipelines made of perineurium-like extracellular matrix in optic nerve of transverse section could be obseved and there were line-shaped tubes with interconnections in optic nerve of longitudinal section after all cell component was removed. These results were further conformed under a scanning electron microscope. The present study proved that cell component had been removed by the results below;1.Cell nuclei were invisible by Hoechst33342 staining. 2. DNA electrophoresis showed that residual DNA fragments were less than 200 bp. 3. DNA quantification showed 50 ng per mg scaffold dry weight. Therefore, cells seeded in the scaffold could grow without nucleic acid residue effect. Then, after DRG was seeded on the scaffold in vitro, we found that DRG derived NF200 positive neurites could grow a long distance in the surface of the scaffold. Furthermore, two months later after the scaffold transplanted, a lot of regenerating nerve fibers were observed in the injury/graft site of spinal cord and the motor function of paralysis hindlimbs was improved with higher BBB score and favorable cortical motor evoked potential. Conclusion The present findings suggest that NT-3 delivery acellular optic nerve scaffold can promote long distance growth of DRG neurite in vitro and may improve motor function of spinal cord transected completely by regenerating nerve fibers in vivo.