The Comparison Of Three Kinds Chemical Acellular Methods To Prepare Acellular Nerve And Preparation Of A New Nerve Scaffold

There are two Part of the experiment.Part One:The comparison of three kinds chemical acellular methods to prepare acellular nerveObjectThrough the comparison of three kinds chemical acellular methods to prepare acellular nerve to choose the most appropriate technique to prepare acellular nerve.Methods1. To deal with sciatic nerve of SD rat with lysophosphatidylcholine method, trinitrotol- uene and Sodium deoxycholate solution to in room temperature alternatively, as well as sodium azide, trinitrotoluene and sodium deoxycholate solution to in turn to prepare decell nerve separately.2. To estimate the morphological of the three kinds of decell material by olefin slices. Third, perform cytotoxicity and subcutaneous implant test to the materials.Results1. No changes occurred to the diameters and lengths of the three classes of decell nerve Scaffolds, soft texture, ductile, ivory white and translucent have been existed. Their elasticity was a slightly poorer and the tenacity was a little better than fresh nerve structures, the ductibility was better after be chemical extracted and can do conventional nerve anastomosis without strain.2.2. HE Staining the dissolved scraps of axon cell can be seen in the crosscut pieces obtained by Dumont method, which means the cells haven't been extracted thoroughly. Perineurium and outer membrane be dyed red appeared around the irregular round hollows formed by the endoneurium which has been dyed red. Endoneurium be dyed red arranged likes wave on the verticalcut pieces, as well as the columnar-tube like interspace which formed by the disappeared axon and myelin sheat. Big spaces appeared in the cavity of the endoneurium be dyed red on the crosscut and verticalcut pieces acquired by Sondell method. While the endoneurium tubes arranged uniformly on the pieces obtained by Haase method and no big spaces existed.3. Laminin immunohistochemical staining The SCBM dyed yellow around the empty endoneurium tube be kept on the pieces acquired by Dumont method. Normal neuromechanism disappeared on the veticalcut pieces while the SCBM dyed yellow has been reserved, arrayed likes wave. The neuromechanism myelin sheath disappeared on the crosscut pieces got by Sondel method while the SCBM dyed yellow formed irregular round hollows and a portion of the tube walls collapsed on the section of verticalcut pieces. Round and elliptic tube structures occurred on the acellular nerve on the crosscut pieces got by Haase method, while the myelin sheath disappeared and the SCBM dyed yellow formed regular round hollows. The tube walls on the section of verticalcut pieces arrayed continuously and no collapse appeared.4. Cell toxicity test Followed by MTT color comparison method, the cytotoxicity of the nerve Scaffold of the obtained acellular nerve by the different three series of chemical extraction were all zero grades. Direct contact method showed that the cell toxicity second grade of the Dumont method, first grade of Sondell and Haase method.5. Subcutaneous implantation experiment Followed by the reaction indexes of the inflammational cells, the inflammational reaction of Haase method is evidently lower than Dumont method and Sondell method in the first, second and forth weeks, while the Sondell method is superior than Dumont method. Index of fiber cavity formation showed the Haase method is superior than Sondell and the Sondell method is evidently superior than Dumont method.ConclusionAccording to the experiments above, it can come to the conclude that the Haase method is superior than Dumont and Sondell method . Part two:Preparation of a new nerve scaffoldObjectPreparation of a new type of nerve scaffold.Methods1.Acellular treatment the squid by Haase extraction method.2.Evaluate the morphology of the squid acellular material according to the olefin pieces.3.Test the cytotoxicity and subcutaneous implantation of the squid acellular material.ResultsBased on the comprehensive evaluation of morphology and compatibility, the good biological safety of the Scaffold has been confirmed and the material can be used of nerve transplantation in body.ConclusionThe new type of manual nerve Scaffold prepared by Haase method used squid nerve have better biological stability and compatibility, that means a material with potential application occurred in the area of nerve tissue engineering.