Preparation of acellular nerve grafts with triton X-100

Jinbao Han; Juwen Chen; Baohui Zhao; Jichun Zhang; Dehu Tian; Jiuhui Han

摘要：BACKGROUND: The source of nerve allograft enriches.We may choose expediently nerve allograft to repair injured nerve and the structure of choice nerve homlolgy or similar with the injured nerve,but the immunological rejiction limits the climical application of nerve allograft.The ideal substitute of autograft never is reaearching.OBJECTIVE: In this experiment,Triton X-100 was used to extract the Schwann cells and myelin sheaths of allograft nerve and obtain the inartiflcial and eliminated antigenicity nerve-transplanter(nerve grafts).DESIGN: Controlled experiment.SETTING: Department of Hand Surgery,the Third Affiliated Hospital of Hebei Medical University;Second Department of Orthopedics,Fourth Center Hospital of Tianjin.MATERIALS: Thirty heath New Zealand big ear white rabbit .of either sex(gender).weighing 2000-3000 g,were provided by the Center of Experimental Animal of Hebei Medical University.Triton X-100 was offered by SIGMA Company.METHODS: The experiment was carried out at the Central Laboratory of the Third Affiliated Hospital of Hebei Medical University from December 2003 to December 2004.Sixty pieces of sciatic nerves.10-mm-long nerve segment,which were taken from 30 rabbits,were incised.They were randomly divided into chemical extraction group(n=50)and control group(n=10).In the chemical extraction groups,the nerves were put into 3% Triton X-100 solution.They were treated with Triton X-100 for 12 hours,24 hours,48 hours,96 hours and 1 week.respectively.They were examined in every period.The control groups did not treated with anything. ①Respectively two segments of nerve by 2 mm length were taken from each nerve in the every periods.②The laminin immunohistochemical stained sections were performed with image acquisition and analyzed with multicolor pathological image analysis system.Measured the laminin antibody reaction part of each section and computed laminin average gray degrees of the unit area.All dates were analyzed by SPSS 10.0 software.MAIN OUTCOME MEASURES:①General observation and histological observation in two groups;②Compared with laminin average gray degrees of the unit area in each section.RESULTS:①General observation:In the control groups,fresh nerve was polish,rigidity and elasticity.After the nerves were chemical extracted,the floccules was seen at two ends and around of the nerves.The nerves being extraction presented ivory and lackluster. Its diameter and length compared reduced,tenderness and tenacity with the fresh nerve.0bserved by light microscope,Schwann cells,myelin sheaths and basement membrane distribute uniformly in control groups.After the nerves were extracted,Schwann cells and myelin sheaths disappeared. Basement membrane presented barrier array in longitudinal sections. Between the membranes was the basement membrane tube.Observed with scanning electron microscope,the basement membrane tubes composed by collagen fibers were remained and collagen fibers maintained their former position,form and structure.Further,the structure of membrane was seen in the tubes.It was Schwann cells basement membrane.②In chemical extraction groups,laminin average gray degrees of the unit area were 140.1±3.41(12 hours),142.1±3.14(24 hours),142.1±3.14(48 hours),140.4±4.03(96 hours),141.7±2.62(1 week).In the control groups,laminin average gray degree of the unit area was 142.7±7.24.There were not significant differences among the groups (P＞0.05).CONCLUSION:The method of chemical extraction by using of Triton X-100 may be an ideal measure for preparing tissue-engineered nerve-transplanter and reserved the live of laminin in the basement membrane.