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The Role of Low Dose Ultrashortwave in Repairing Rat Sciatic Nerve Lesions by Using Acellular Allogenic Nerve Scaffold Implanted with Bone Mesenchymal Stem Cells

Author: PangChaoJian
Tutor: ZuoXiaoJie
School: China Medical University
Course: Human Anatomy and Embryology
Keywords: Acellular nerve Ultrashort wave therapy Peripheral nerve regeneration Schwann cells Bone marrow mesenchymal stem cells
CLC: R651.3
Type: Master's thesis
Year: 2009
Downloads: 54
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Abstract

Purpose of ultrashort wave therapy has efficacy eliminate inflammation, reduce edema, promote vasodilation, improving blood circulation and nutrition of tissues and organs, and enhance tissue metabolism, ultrashort wave on the repair of damaged tissue has been proven in the field of rehabilitation science and has been widely used. Studies have shown that played a role of accelerated nerve growth speed off cell stents and ultrashort wave therapy combined repair sciatic nerve injury treatment. In recent years, with the continuous development of tissue engineering, the application with the seed cell scaffold for repair of peripheral nerve injury has matured. Allogenic off cellular neural extracellular matrix material (acellular extracellular matrix.AECM) as the biological activity of nerve substitute good biomimetic and tissue compatibility, provide a suitable microenvironment for axonal regeneration after transplantation can effectively promote nerve regeneration. In recent years, with to prove that it has a high plasticity and low immunogenicity, easy to obtain in vitro rapid amplification and long-term survival of bone marrow mesenchymal stem cells (Bone marrow stromal cells, BMSCs) differentiate into Schwann cell research deepening characteristics, these conditions make repair peripheral nerve injury ideal seed cells. This experiment is to observe the small doses of ultrashort wave of planting BMSC decellularized scaffolds repair sciatic nerve injury role, provided the experimental basis for clinical nerve repair and rehabilitation. Materials and Methods l off cellular neural graft preparation wistar rats anesthetized remove both sides of the sciatic nerve, acellular method using chemical extraction prepare acellular nerve matrix scaffold. 2, BMSCs Isolation, culture and identification of male Wistar rats 25-30 days after the Acceptance of BMSCs isolated from the tibia and femur, purified adherent cells and cultured to the third generation of alternate. The third generation of BMSCs respectively Osteogenic culture fluid and into a fat-induced culture liquid culture to observe its morphology changes. Madder prime red staining and Oil Red O staining were elucidated BMSCs induced osteogenic and fat cells. Experimental animals and grouping male Wistar rats 48, weighing 200-250g, experimental animals by the China Medical University, were divided into three groups, namely Total Burke's modified Eagle's medium (Dulbecco's ModifiedEagle's stent injection Medium, DMEM), stent injecting BMSCs group and after the injection of BMSCs application of ultrashort wave treatment group were sacrificed after 2, 4, 8 and 12 weeks were in transplant 4. 4, the acellular nerve bracket transplantation the third generation BMSCs were the micro syringe according to the inoculation concentration of 1 × 10 6 cells / ml is injected into the the prepared stent and placed in a co-cultured in the incubator for three days; under sterile conditions under the joint training of the neural stent suture to neurological defects. 5, ultrashort wave treatment of postoperative day that small doses of ultrashort wave nerve defects at the treatment of transplant animals. Observe the indicators and detection methods (1) the AECM the morphology of light and electron microscopy structure. (2) BMSCs cell growth was observed under an inverted microscope. (3) footprint assay of rat sciatic functional index (sciatic function index, SFI) tracking to observe the recovery of neurological function. (4) The number of regenerating nerve fibers, axon diameter, transmission electron microscopy detection regenerating nerve myelin thickness toluidine blue staining. (5) immunofluorescence staining analysis of the regeneration of nerve within the S-100, glial fibrillary acidic protein (Glialfibrillary Irregularity in the protein, GFAP) and vascular endothelial growth factor (Vascular endothelial growthfactor, VEGF) expression. (6) RT-PCR detection of regeneration the nerve within the S-100mRNA and VEGFmRNA expression. 7, the statistical analysis. The experimental results, light and electron microscopy to the use of The chemical extraction acellular prepared AECM retain the integrity of the basement membrane tube consisting essentially three-dimensional structure. 2, observed under an inverted microscope to adherent BMSC cells were round, the fibroblast-like, or spindle-like, after 12 days to achieve 90% confluence. 3, respectively, with the the madder prime red and oil red 0 BMSCs induced to osteoblasts and fat cells staining found each to the induction of cell differentiation, namely red bone matrix deposition and lipid droplets. 4, the experimental animals walking experiment proved faster as time grows the SFI gradually rising, rising ultrashort wave therapy group SFI. 5, the regenerating nerve toluidine blue staining and transmission electron microscopy detection: growth with the time of transplant recipients, ultrashort wave therapy group therapy group regenerated nerve myelin thickness increases, the diameter increases and an increase in the number of nerve fibers. 6, immunofluorescence results found the S-100 protein and GFAP positive expression in nerve regeneration have-dependent growth and time into the expression of ultrashort wave treatment group is better than the other two groups. VEGF expression peaked in the first four weeks and then gradually decline. 7, RT-PCR detection of nerve regeneration the S-100mRNA expression time proportional to the growth the inside the S-100mRNA and VEGFmRNA expression ultrashort wave treatment group increased significantly. VEGFmRNA expression in the first four weeks after. Conclusion l, the role better than pure decellularized scaffolds of of injected BMSCs decellularized scaffolds on nerve injury recovery, recovery of neurological function after application of ultrashort wave therapy sound and ultrashort wave therapy group. 2, S-100 cells within the graft and the expression of GFAP positive proof of decellularized scaffolds can promote BMSC to Schwann cell differentiation or migration, ultrashort wave therapy can play accelerated differentiation or migration speed. Graft a high level of VEGFmRNA and protein inferred ultrashort wave therapy may promote the regeneration of nerves around the blood supply increase to its raise more nutrients.

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