| Background:Spinal Cord Injury (SCI) is a common trauma in the field of orthopedic and results in severe neurological dysfunction and disability, which it can affect the individual physical and mental health, but also a heavy burden on families and society. With the developing of transportation, mining, sports and the growing construction industry, The SCI incidence of rising trendy year by year. In 2004, the American Spinal Injury Statistical Center reports:Each year, there are more than 11,000 cases of new cases in the United States and the incidence of SCI is 15-40 cases/1 million in the worldwide.SCI was considered impossible to cure disease in the past. The treatment of paraplegia after SCI has been pessimistic since a long time. In recent years, with the basic research of SCI, which has deepened the understanding of the relation of the factors after the restoration, and Molecular biology, Cell biology and other disciplines, provide a new means for the treatment of SCI and bring new hope for the successful treatment of human SCI, at same time, there are always new treatments for spinal cord injury applied to clinical trials, such as peripheral nerve transplantation, embryo spinal cord transplants, neural stem cell transplantation, bone marrow-derived mesenchymal stem cells, olfactory ensheathing cell transplantation and a variety of experimental drug treatment, however, the treatment of spinal cord injury in a variety of domestic and international programs were not to achieve a satisfactory clinical efficacy. Countries around the world to spinal cord injury, as a problem with the research focus on how to promote spinal cord regeneration and functional recovery.With the rise of spinal cord tissue engineering, the repair and functional recovery after SCI has been brought new hope. Tissue engineered spinal cord is the seed cells in vitro cultivation in the stent material which the excellent biocompatibility and can be degraded gradually in the body. It is important for us to select tissue-engineered three-factor:seed cells, scaffold materials and the appropriate cell neurotrophic factor to use tissue-engineering technique to repair tissue damage.In this study, bone marrow-derived mesenchymal stem cells as seed cells (BMSCs), We Select the natural biodegradable materials:chitosan, alginate, then through cross-linking the two outside exposure, freeze-dried preparation of multi-channel bracket material, BMSCs were grown in the composite scaffold for tissue engineering in the spinal cord to explore its role in the repair of spinal cord injury, in order to discovery of a new method of treatment for spinal cord injury.Objective To investigate tissue engineered spinal cord which was constructed of bone marrow mesenchymal stem cells (BMSCs) seeded on the Chitosan-Alginate scaffolds bridging the both stumps of hemi-transection spinal cord injury (SCI) in rats to repair the acute SCI.Methods BMSCs were separated and cultured from adult male SD rat. Chitosan-alginate scaffold was produced via freeze drying, of which the structure was observed by scanning electron microscope (SEM) and the toxicity was determined through leaching liquor test. Tissue engineered spinal cord was constructed by seeding second passage BMSCs on the chitosan-alginate scaffolds (1×106/ml) in vitro and its biocompatibility was observed under SEM at 1,3, and 5 days. Moreover,40 adult female SD rats were made SCI models by hemi-transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). Tissue engineered spinal cord or chitosan-alginate scaffolds or BMSCs were implanted in groups A, B, and C, respectively. Group D was blank control whose spinal dura mater was sutured directly. After 1,2,4, and 6 weeks of surgery, the functional recovery of the hindlimbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. Other indexes were tested by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing, HE staining and immunofluorescence staining after 6 weeks of surgery.Results Chitosan-alginate scaffold showed three-dimensional porous sponge structure under SEM. The cells adhered to and grew on the surface of scaffold, arranging in a directional manner after 3 days of co-culture. The cytotoxicity of chitosan-alginate scaffold was in grade 0-1. At 2,4, and 6 weeks after operation, the BBB score was higher in group A than in other groups and was lower in group D than in other groups; showing significant differences (P< 0.05). At 4 and 6 weeks, the BBB score was higher in group B than in group C (P< 0.05). After 6 weeks of operation, WGA-HRP retrograde tracing indicated that there was no regenerated nerve fiber through the both stumps of SCI in each group. HE and immunofluorescence staining revealed that host spinal cord and tissue engineering spinal cord linked much compactly, no scar tissue grew, and a large number of neurofilament 200 (NF-200) positive fibers and neuron specitic enolase (NSE) positive cells were detected in the lesioned area in group A. In group B, a small quantity of scar tissue intruded into non-degradative chitosan-alginate scaffold at the lesion area edge, and a few of NSE flourescence or NF-200 flourescence was observed at the junctional zone. The both stumps of SCI in group C or group D were filled with a large number of scar tissue, and NSE positive cells or NF-200 positive cells were not detected. Otherwise, there were obviously porosis at the SCI of group D.Conclusion The tissue engineered spinal cord constructed by multi-channel chitosan-alginate bioscaffolds and BMSCs would repair the acute SCI of rat. It would be widely applied as the matrix material in the future.Key words Tissue engineered spinal cord Chitosan Alginate Bone marrow mesenchymal stem cells Acute spinal cord injury RatFoundation item:Medical Science and Technique Foundation of Chinese PLA during Eleventh Five-Year Plan Period (06MA081)... |
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