Preparation Of Muscle Basal Lamina With Neural Stem Cells Tissue Engineering Scaffolds

1. ObjectiveTo prepare the tissue engineering scaffold of Muscle Basal Lamina with Neural Stem Cells for the repair of spinal cord injury, which provides experimental basis for the tissue engineering scaffold transplantation to treat the spinal cord injury.2. Methodsa. Isolation, cultivation and identification of neural stem cells Neural stem cells were isolated from embryonic 14 days rat brain and cultivated by serum-free culture techniques within DMEM-F12 medium including B27, EGF and bFGF. While self-renewal capacity of the isolated cells was identified by anti-Nestin immunocytochemistry staining. After withdraw of EGF and bFGF from the medium, neural stem cells were induced into differentiation within medium including 10% of fetal bovine serum. Anti-NF and anti-GFAP immunocytochemistry staining were employed to identify neurons and astrocytes respectively.b. Preparation for the Muscle Basal Lamina Select fresh paravertebral muscles from both sides of spines, under the condition of 50rpm table classifier and 37℃water temperature , applying Chemical Acellularized Ppreparation of Muscle Basal Lamina in the process of distilled water for 48h, solution of 3%TritonX-100 for 48h,distilled water for 48h,solution of 1%SDS for 48h,PBS for 24h.c. Preparation for the tissue engineering scaffold of the Muscle Basal Lamina with Neural stem cellsDigesting Neural Stem Cells with Pancreatin of 0.05%, injecting them into the Muscle Basal Lamina and putting them in the 24-pore plate for culture. Seven days later, the process of fixation, dehydration, embedding, section-cutting and staining.d. transplantation of the tissue engineering scaffold of Muscle Basal Lamina with Neural Stem Cells into rat spinal cord injury Produce SCI animal model, transplating the well-prepared Muscle Basal Lamina with the Neural Stem Cells into the area lack of spinal cord. Four weeks later, fulfilling the process of perfusion, drawing material, embedding and section-cutting, and applying immunohistochemical technology.3. ResultsLarge amount of well-distributed Neural Stem Cells can be seen after 24h of culturing. Neurosphere can be seen after culture 5-7d. The expression of Nestin of the neural stem cells was detected by immunohistochemistry. these cells are plated in adhesive culture dishes and cultured in growth factor-free medium supplemented with serum,when positive cells of anti-NF, anti-GFAP can be found. It is certified that the cultured Neural Stem Cells have got the potential of multipotency. The milky Muscle Basal Lamina is under the chemical probation. The cellular component in the Muscle Basal Lamina has disappeared, and HE Staining showed that the elastic fibers and collagen fibers in Muscle Basal Lamina, with roughly paralled tubes in the frame, have grown into the effective channel for the axons to grow.Take Neural Stem Cells as seed cells, Muscle Basal Lamina as the scaffold to prepare the tissue engineering scaffold of the Muscle Basal Lamina with Neural stem cells.. By culturing Neural Stem Cells and Muscle Basal Lamina in vitro, their compatibility can be inspected. The result of the experiment shows that the Neural Stem Cells grow well with the neurobiological activity, which can firmly attach to Muscle Basal Lamina, and can be differentiated into neuron and astrocytes. This result indicates that the tissue engineering scaffold is a kind of good tissue engineering scaffold for the repair of spinal cord injury.The result by transplanting the complex of Neural Stem Cells-Muscle Basal Lamina into the body of rats for four weeks, HE staining has shown that Muscle Basal Lamina can be well connected with the normal spinal cord. Meanwhile, the surviving transplanted stem cells, as well as the dispersed neurons and star-shaped glial cells, could be visually seen, which proves that the transplanted Neural Stem Cells can be differentiated in vivo. By further inspection, the newborn nerve fibers were seen to go through the scaffold, which proved that the NSCs whose frames were transplanted may take the place of the damaged or died Neurons, to rebuild the Neuron Loop. The Muscle Basal Lamina can bridge the proximal and remote end on the damage position of spinal cord. The scaffold transplantation in Tissue Engineering can surely provide the promising treating plan for the recovering of spinal cord injury.4. ConclusionThe tissue engineering scaffold of Muscle Basal Lamina with Neural Stem Cells is manufactured successfully. It is a kind of good tissue engineering scaffold for the repair of spinal cord injury.