修复神经节段性缺损的人工神经桥接物微观空间结构特征

背景:神经组织再生的特殊性导致始终没有一种成熟和完整的系统来解决神经组织节段性损伤后的修复问题。利用组织工程的方法去实现这个目标具有极大的难度与挑战性。目的:研制一种可用于临床神经损伤修复的人工神经桥接物,并对其进行微观空间结构的研究。设计:开放性实验研究。单位:解放军第四军医大学西京医院全军骨科研究所。材料:实验于2001-11/2003-01在解放军第四军医大学西京医院全军骨科研究所完成。Ⅰ型胶原蛋白、Ⅳ型胶原蛋白、明胶购于美国Sigma-aldrich公司。方法:制备桥接材料:分别将Ⅰ型胶原蛋白和Ⅳ型胶原蛋白放入0.05 mol/L醋酸溶液中高速搅拌制成悬浊液,混合两种悬浊液保持4℃恒温搅拌,制成胶原蛋白和硫酸类肝素的悬浊液,抽真空静置后注入内径为3 mm的硅胶管中密封两端,分别以5种不同速度(10×10-5 m/s,5×10-5 m/s,2.5×10-5 m/s,1.0×10-5 m/s,1.0×10-6 m/s)进行冷淋、赋型。行大体观察。并将以不同速度制成的各组材料切成横截面、纵截面以及45°斜截面以备光学显微镜观察。同时在扫描电镜下进行内部微管结构的观察,并进行材料内部的微管直径的计算,实际孔径=(放大率×微管面积)÷(标尺长度×孔周长)。主要观察指标:①桥接材料的大体观察结果。②桥接材料的显微镜观察结果。③桥接材料的扫描电镜观察结果以及材料内部的微管直径。结果:①大体观察:制备出的材料均为规则的圆柱体,且外型均匀。柔韧性较好,质地均匀,弹性较强。②光学显微镜结果:材料外表面为全封闭结构,无孔裂;表面光滑平整,连续性好。③电镜观察结果:材料的外表面为叠瓦状,内部的微管结构均匀,走行一致,且基本相互平行;纵向走行的微管之间相互独立,且呈封闭状态,无互通的桥连管道相连接,与生物体神经的纤维束的走行特点完全相同。材料内部微管的横截面基本为圆型,形状较为规则,直径大小较为均匀。材料内部的微管连续性好,无中断或横隔,且微管的小梁壁连续性好,表面光滑,无褶皱。材料内部的微管直径为197.3～258.8μm。结论:利用生物相容性较好且可降解的胶原和明胶经混合溶解及冷淋后形成具有单一纵向微管的神经桥接物,具有与正常神经高度仿生的微结构,可用作基础研究与临床神经损伤修复的替代物。

Characteristics of micro-spatial structure of artifical nerve bridging substance for repairing nervous segmental defect

BACKGROUND: There is no any mature and complete system to repair nerve tissue after segmental injury due to particularity of nerve tissue regeneration. Using tissue-engineering technique to solve this problem is difficult and challenged.OBJECTIVE: To research an artificial nerve bridging substance in application of repairing nerve injury on clinic and obverse its microcosmic spatial structure at the same time.DESIGN: Open experiment.SETTING: Orthopaedic Research Institute, Xijing Hospital of the Fourth Military Medical University of Chinese PLA.MATERTALS: The experiment was carried out in the Orthopaedic Research Institute, Xijing Hospital of the Fourth Military Medical University of Chinese PLA from November 2001 to January 2003. CollagenⅠ, collagen Ⅳ and gelatin were provided by Sigma-aldrich Company, USA.METHODS: Establishment of bridging substance: Collagen Ⅰ (65.53%) and collagen Ⅳ (32.77%) were added into 0.05 mol/L acetic acid solution, respectively; and then, two suspensions were mixed together and stirred at 4℃. The suspension of collagen and heparin sulfate was pumped, held still and poured into silica gel tube with bore of 3 mm.Continuously, two ends of tube were enclosed and suffered from cold drench and mold at five various speeds. General observation was done. Composites were intercepted cross section, vertical section and 45° section for observation by optic microscope at different speeds of 10×10-5 m/s, 5×10-5 m/s, 2.5×10-5 m/s, 1.0×10-5 m/s and 1.0×10-6 m/s; meanwhile, interior structure of microtubule was observed with scanning electron microscope. Interior diameter of microtubule was calculated as the following formula: actual aperture = (magnification × area of microtubule) ÷ (length of guage × perimeter of aperture).MAIN OUTCOME MEASURES: ① General observation; ② observation with microscope; ③ observation with scanning electron microscope and interior diameter of microtubule.RESULTS: ① General observation: Models were regular cylinders and had symmetrical features. Flexility was wonderful,texture was well-proportioned, and elasticity was strong. ② Results of optic microscope: Structure of external surface was completely blocking without any poricidal dehiscence. Surface was smooth and had good continuity. ③ Results of electron microscope: External surface shaped like terrace tile; asides, interior structure of microtubule was average and its courser was coincident and parallel to each other. Microtubules with longitudinal courser were independence on each other. The structure was blocking and did not connect to each other. This was as the same as courser of nerve fibrous bands of organis. Cross section of interior microtubule was general round and regular, and the diameter was average. Interior microtubule had groat continuity without breaks or transection. Trabeculae of microtubule also had groat continuity,and its surface was smooth. Interior diameter of microtubule ranged from 197.3 μm to 258.8 μm.CONCLUSION: Nerve bridging substance is made of collagen and gelatin which are characterized by groat compatibility and degradation after mixture, lysis and cold drench, It has singly longitudinal microtubule and microstructure of high imitation by normal nerves, so nerve bridging substance can be used in basic researches and repair nerve injury on clinic.