| Background: Neural regeneration after spinal cord injury had always been a hot spot in biomedical research. In recent years, many pilot studies had proved that injured spine cord still retains a certain ability of regeneration. Although some experiments had showed encourageous and promising results, repairing of injured spine cord in adult mammal is still very difficult. As tissue engineering approaching the stage of ripeness gradually, a therapeutic mode of"ideal seed cell + suitable scaffold material + appropriate cytokine"offers a promising approach to repair spinal cord injury. Suitable scaffold material can fill the defective incomplete architecture, inhibit invasion of cicatricle, and offer anchoring sites and three-dimensional architecture, promote growth of seed cells in a prefabricated morphous; absorb a great deal of molecules which are important in promoting functional recovery, create eligible microenvironment, help transplanted cells acquire sufficient nutrient substance, so that cell proliferation and metabolism can progress steadily; temporarily fill the cava caused by spine cord injury, accompanied with degradation, absorption and vanishing of scaffold material, neogenetic tissue will fill the injured region completely, complete the reconstruction of succession of spine cord. Accordingly, scaffold material plays a vital role in functional remodeling of injured spine cord.Scaffold materials used in spine cord tissue engineering can be classified into two categories by the source, one is native material and the other is artificial material. The best quality of degradable native material is superordinary biocompatibility; degradation products will be easily absorbed and will not bring about inflammatory reaction. But the mechanical performance of native material is not satisfactory; furthermore, the degradation progression cannot be regulated readily. The degradation speed and intension of degradable synthetic macromolecular material can be readily regulated. This kind of material can be easily molded and construct three-dimensional scaffold with high porosity. However, degradation products are harmful to neural stem cells.In this study, a kind of native material collagen will be used to construct scaffold , and the feature of this scaffold will be modified by chemical cross-linking and physical cross-linking methods. Native scaffold material collagen will be compared with artificial synthetic material PGA and PLGA in performance test and biocompatibility of scaffold materials, and then the advantages and disadvantages of different kind of scaffold materials will be identified. Neural stem cells will be inoculated in the scaffold constructed by selected scaffold material, and then fetal spinal cord extracts will be administered as cytokines to imitate embryonal microenvironment suitable for development of neural stem cells, provide neurochemical factors, which are essential in growth and differentiation of neural stem cells, promote the differentiation of neural stem cells into neuron phenotype. Compound nervous tissue with certain tissue morphous will be constructed in vitro, which consists of neuron, glial cell and other components. This compound nervous tissue will be transplanted to repair spine cord injury in adults.Objective: To Select suitable scaffold material, construct tissue engineering spine cord by tissue engineering techniques, and study the effect of tissue engineering spine cord on repairing of injured spine cord in rats.Methods: 1. culture and induced differentiation of neural stem cell. Neural stem cells were isolated from embryonic 14 days spinal cord of Sprague-Dawley rats. After clonal expansion, NSC were identified by anti-Nestin antibody immunofluorescence, and bionomics of these cells were tested. Embryo spine cord extract was prepared. The extract was added to the cultural medium of neural stem cells to induce differentiation of neural stem cells. after differentiation of NSC for 3 or 7 days, NSC were identified by immunofluorescence. We observed phenotypes of differentiated cells and calculated the percentage of neuron-like cells. Neural stem cells in control group were induced by medium containing 10% fetal bovine serum. 2. select spine cord tissue engineering scaffold material. Rat tail-derived collagen sponge scaffold was prepared. The feature of the scaffold was modified by chemical cross-linking and physical cross-linking methods. PGA and PLGA scaffold were constructed. After tests of mechanical performance, interval porosity, degradation speed and biocompatibility, advantages and disadvantages of different scaffold materials were identified, and then the best scaffold material was selected. 3. construction of tissue engineering spine cord in vitro. NSC were inoculated in the scaffold, embryo spine cord extract was administered to induce differentiation of NSC for 3 days. The tissue architecture of tissue engineering spine cord were revealed by HE stain, immunofluorescence and scanning electron microscope. 4. Transplantation of tissue engineering spine cord to repair acute spine cord injury in adult rats. Rat models of spine cord hemi transsection were prepared. Models among group A were transplanted with tissue engineering spine cord, models among group B were transplanted with NSC and scaffold, models among group C were transplanted with scaffold material, models among group D was blank control group. After operation for 3 or 8 weeks, differentiation and survival of transplanted NSC in different groups were observed by histological method. Motor and sensory function of models were assessed by improved standard BBB score and SEP examination, and then the effect of tissue engineering spine cord on repairing of acute spine cord injury in adult rats was evaluated.Results:1. Specific marker Nestin was expressed by both primarily cultured NSC and subcultured NSC. The growth of NSC was divided into three different phases, namely lap phase of growth, exponential phase of growth and plateau phase of growth. After differentiation of NSC induced by embryonic spine cord extract for 3 to 7 days, 16.37% and 18.14% of NSC differentiated into neurons respectively, the percentages of groups induced by embryonic spine cord extract were significantly higher than the percentages of control groups (6.77% and 7.69%).2. The biocompatibilities of collagen scaffold modified by EDC and DHT showed no significant difference to that of native collagen.. They biocompatibilities of collagen scaffold were better than the of PGA and PLGA. The mechanical property of EDC corss-linking collagen was bettle than that of DHT corss-linking collagen and native collagen, moreover, the EDC corss-linking collagen significantly inhibited degradation of collagen in vitro.3. The biocompatibilities of collagen scaffold between modified by EDC and DHT showed no significant difference. They were both better than the biocompatibilities of PGA and PLGA. EDC and DHT significantly inhibited degradation of collagen in vitro. Collagen scaffold modified by EDC did not inhibit differentiation of NSC induced by embryonic spine cord extract in vitro. Tissue engineering spine cord consisted of NF200 positive cells, GFAP positive cells and MBP positive cells, the percentage of NSC differentiated into neurons still occupied 30%.4. The experiment started 2 weeks after the operation and ended 8 weeks after the operation. Function scores of group A and B were significantly higher than those of group C and D. Functional recovery of group A was more significant than group B. From 2 weeks after operation to 6 weeks after operation, BBB score of both group A and B recovered quickly. 6 weeks later, functional recovery reached plateau phase. 6 weeks after operation, the latent periods of P1 and N1 of SEP of models among group A were significantly longer than control group, but significantly shorter than group B, C and D. Histological experiments performed 3 weeks or 8 weeks after operation showed that most of labeled cells in the graft of group A survived, and the number of cells was significantly larger than that of group B. A great deal of NF200 positive cells appeared in group A, 8 weeks after operation, GFAP positive cells formed network structure similar to network structure of normal spine cord tissue. Although some transplanted NSC in group B differentiated into neurons, the network structure formed by GFAP positive cells lacked regularity, showed more disorderly than the network structure formed in group A. although infiltration of inflammatory cells was found in group C, Excessive hyperplasia of glial scar was not found in boundary zoneConclusion:1. Embryonic spine cord extract could promote survival of NSC and induce NSC to differentiate into neurons, astrocytes, and oligodendrocytes.2. Collagen sponge modified by EDC crosslinking retained the natural biocompatibility of collagen, did not collapse in cultural medium, retained the intensity to some extent , maintained the shape and porousness and significantly inhibited degradation of collagen, so Collagen sponge modified by EDC crosslinking can be chose as scaffold material for spine cord tissue engineering.3. Tissue-engineered spinal cord constructed with NSC, collagen scaffold modified by EDC cross-linking and embryonic spine cord extract survived and differentiated to mature in vitro. It possessed distinct tissue structure, which consisted of neurons, astrocytes, oligodendrocytes and other components. These cells were well-distributed in three dimensional scaffold. Some newborn neuron formed fiber contact and the axons from glia cells formed a relative regular structure. 4. After the tissue engineering spinal cord built transplanted in to the animal's inuried spinal cord, NSC could survive and differentiate into functional neurons which mabey form axon contact. The axons of glia cells formed a regular structure similar to that of normal spinal cord at a certain extent. The modified collagen scaffold tissue engineered spinal cord might benefit to behavioral improvement and anatomic reconstruction to certain degrees for adult rat spinal cord injury.
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