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Tissue Engineering Technology Research In Neural Stem Cells Repairing Spinal Cord Injury

Posted on:2003-12-18Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y H ChengFull Text:PDF
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OBJECTIVE: To develop and research the possibilities of the complex of neural stem cells and chitosan in the repair of spinal cord injury, hoping to provide a new idea and further a new method for the cure of spinal cord injury by tissue engineering technology.METHODS: 1. Cells from the embryonic Sprague-Dawley rat (SD rat) cerebral cortices were mechanically dissociated and then cultured and expanded in the medium of DMEM/F12, B27, and bFGF, these fast expanding and neurosphere-forming cells were identified by immunocytochemical procedures, and other characteristics, such as clone forming ability and multipotential differentiation of these cells were tested . 2. These neural stem cells were cocultured with chitosan in the above medium and in the medium supplemented by 1%NBS (Newborn Bovine Serum, NBS) for one week. The proliferation and differentiation of these cells on the chitosan were observed by inverted microscope, and then these cells were stained by anti-nestin and other immunocytochemical procedures, to observe the influence of chitosan on these cells and examined by scanning electron microscope, to explore the biocompatibility of them. 3. Transplantation of the complex of neural stem cells and the tube of the sponge-like chitosan into the injured adult rat thoracic spinal cord. Adult female SD rat spinal cords were transected at the T8 cord level and a 4-5mmsegment caudal to the transection was removed, and then the complex was positioned into the gap between the rostral and caudal stumps of the spinal cord. Animals were followed over time for signs of functional recovery and rated by the combined behavioral score (CBS), and the general conditions of the animals were observed about the wound healing, complications, body weight etc. The electrophysiological integrity of the rat spinal cord was assessed at the sacral, lumber, thoracic, lower cervical and cortical levels by Somatosensory Evoked Potentials(SEP) and Motor Evoked Potentials(MEP). Two weeks and one or two month later, some rats were sacrificed, to test the efficacy of the complex to serve as bridges for curing these gaps. The spinal cord regions rostral and caudal to the graft and the graft itself were prepared for light and electron microscopy, immunohistochemistry, to inspect the growth, proliferation, migration and differentiation of these neural stem cells, their influence and effect on the propriospinal neural cells and the regeneration of axon, and the change of the chitosan, and others received injections of wheat-germ agglutinin conjugated to horseradish peroxidase for anterograde tracing, to learn the axonal growth into and further leave the graft.RESULTS: 1. Neural stem cells were successfully cultured from embryonic SD rat cerebral cortices, and they formed typical floating neurospheres in the medium, and were stained by anti-nestin. They have the ability of proliferation, clonality and can differentiate into neurons and neuroglia cells. 2. When cocultured with chitosan in different conditions, neural stem cells can grow very well on the chitosan membrane and retain the characteristics of stem cells, and can further differentiate into neurons and neuroglia cells when the medium was supplemented with 1% NBS. 3. When transplantedinto spinal gaps, grafts made of neural stem cells and chitosan can bridge the spinal gaps in appearance. SEP and MEP recordings show that there are some degrees of electrophysiological recovery after transplantation. And neural stem cells can survive, migrate and differentiate in the bridge, and axons extended into the grafts are observed in experimental animals. CONCLUSIONS: 1. Neural stem cells can be cultured and isolated from embryonic SD rat cerebral cortices. 2. Biocompatibility between neural stem cells and chitosan is very good. 3. The complex of neural stem cells and chitosan can bridge the gap in the spinal cords of adult SD rats, and can promote function recovery of the spinal cord after injury.
Keywords/Search Tags:Neural Stem Cell, Chitosan, Spinal Cord Injury, Engineering Tissue Technology
PDF Full Text Request
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