壳聚糖导管复合自体骨髓间充质干细胞修复大鼠13mm坐骨神经缺损

Chitosan conduits combined with autologous mesenchymal stem cells to repair 13-mm sciatic nerve defects

目的:观察壳聚糖神经导管复合自体骨髓间充质干细胞,构建组织工程化人工神经,修复大鼠13mm坐骨神经缺损的效果。方法:实验于2002-10/2004-08在北京军事医学科学院基础医学研究所九室及河北大学附属医院中心实验室完成。实验分组:Wistar大鼠30只按随机数字表法分成3组,实验组、细胞外基质凝胶组和生理盐水对照组,每组10只。壳聚糖神经导管桥接大鼠右侧13mm坐骨神经缺损。实验组无菌条件下抽取股骨髓腔内骨髓组织,贴壁分离法纯化、增殖骨髓间充质干细胞,按1×109L-1细胞浓度与细胞外基质凝胶混合,植入自体神经再生室内;细胞外基质凝胶组神经再生室内植入细胞外基质凝胶,生理盐水对照组神经再生室内植入生理盐水。实验评估:术后12周观察以下指标:①大体观察:观察再生神经。②坐骨神经功能指数测定:选择印迹清晰的足印分别测量正常足(N)和伤侧足(E)的3个指标:足印长度(PL):足尖到足跟的最大距离;足趾宽度(TS):第1～5趾的距离;中间足趾宽度(IT):第2～4趾的距离。结果精确到0.1mm。坐骨神经指数=-38.3[(EPL-NPL)/NPL]+109.5[(ETS-NTS)/NTS]]+13.3[(EIT-NIT)/NIT]-8.8。坐骨神经指数值为0～-11%表示神经功能完全正常,-100%表示神经功能完全丧失,-11%～-100%表示部分神经功能恢复。③电生理检测:检测患侧小腿三头肌肌电图,检测再生神经的运动传导速度和波幅。④腓肠肌湿质量恢复率:腓肠肌湿质量恢复率(%)=手术侧腓肠肌湿质量/对侧正常腓肠肌湿质量×100%。⑤神经组织学检查:苏木精-伊红及Loyez苏木精髓鞘染色,光镜下观察再生神经横断面再生神经髓鞘的形成;Loyez苏木精髓鞘染色及Bielschowsky改良镀银染色,观察纵向切片再生神经神经纤维;神经细丝蛋白免疫组化染色,观察再生的神经轴突染色。随机选取部分正常坐骨神经作为正常对照。⑥透射电镜观察:再生神经的超微结构。⑦再生神经纤维图像分析:观察再生神经有效神经截面积、有髓神经纤维数目、有髓神经纤维密度、有髓神经纤维直径、髓鞘厚度。结果:30只大鼠全部进入结果分析。①大体观察:术后12周,实验组及细胞外基质凝胶组导管内均有再生神经生成,外形似正常神经,直径较正常神经细,生理盐水对照组导管内无再生神经通过间隙。②坐骨神经指数:术后8,12周实验组坐骨神经指数高于细胞外基质凝胶组[分别为(-72.18±3.11)%,(-76.85±2.76)%;(-62.91±2.87)%,(-69.63±2.52)%],差异有显著性意义(F=7.85,P<0.01)。③电生理检查:术后12周实验组运动神经传导速度及波幅明显高于细胞外基质凝胶组[分别为(41.29±3.83),(32.64±3.52)m/s;(3.21±0.34),(2.85±0.22)mV],差异有显著性意义(F=6.39,P<0.01)。实验组、细胞外基质凝胶组腓肠肌肌电图呈部分失神经电位表现,生理盐水对照组则为完全失神经电位。④腓肠肌湿质量恢复率:实验组、细胞外基质凝胶组腓肠肌湿质量恢复率明显高于生理盐水对照组[分别为(69.32±2.65)%,(66.72±1.75)%,(53.41±1.97)%],差异有非常显著性意义(F=9.32,P<0.01),实验组与细胞外基质凝胶组比较差异有显著性意义(F=6.25,P<0.05)。⑤组织学检查:术后12周实验组、细胞外基质凝胶组再生神经纤维排列整齐、密集,神经导管交界处无瘢痕,实验组再生神经纤维多且直径较粗大,排列更为规则。⑥透射电镜观察:实验组和细胞外基质凝胶组均见再生的有髓神经纤维。⑦再生神经纤维图像分析:术后12周实验组有髓神经纤维密度、神经纤维有效面积、有髓神经纤维数量、直径及髓鞘厚度均优于细胞外基质凝胶组。结论:壳聚糖神经导管复合自体骨髓间充质干细胞能够促进周围神经的再生并可以作为种子细胞构建人工神经应用于周围神经缺损的修复。

AIM： To evaluate the effectiveness of chitcoan conduits seeded with autologous mesenchymal stem cells to bridge 13-mm sciatic nerve defect by tissue engineering technique in rats. METHODS： The experiment was completed in the Ninth Room, Institute of Basic Medical Sciences, the Academy of Military Medical Sciences and the Central Laboratory of Hebei University Affiliated Hospital from October 2002 to August 2004. Totally 30 adult Wistar rats were randomly divided into 3 groups： experimental group, extracellular matrix （ECM） group and saline control group, each group contained 10 rats. A 13-mm-long nerve defect was caused in the right sciatic nerve and then was bridged by chitosan conduit. The thigh bone marrow was taken out from animals of experimental group under sterile condition, mesenchymal stem cells were purified using adherence method, cultivated and proliferated, then grafted into nerve regenerating room by 1 ×10^9 L^-1 cell density after mixed with ECM; In ECM and control groups, ECM and physiological saline were grafted into nerve regenerating room, respectively. Twelve weeks after operation, the experimental evaluation was made, including：（1）gross observation for regenerative nerve.（2）Sciatic nerve function index （SFI）： Clear footprint of both normal feet （N） and experimental feet （E） were measured in three indices： podogram length （PL）： the maximal distance from tiptoe to heel; toe spreadn （TS）： the distance between the first and the frith toes; inter-toes distance （IT）： the distance between the second and the fourth toes. All the results were calculated with the fidelity of 0.1 mm. SFI=-38.3[（EPL-NPL）/NPL]＋109.5[（ETS-NTS）/NTS]]＋13.3[（EIT-NIT）/NIT]-8.8. And SFI score of 0 to -11% stood for normal functional nerve; -100% for complete loss of nerve function; -11% to -100% SFI for partly recovery of nerve function. （3）Electrophysiological detection： The motor nerve conduction velocity and wave amplitude were determined using triceps surae electromyogram. （4）Gastrocnemius wet weight recovery rate （%）-wet weight of experimental gastrocnemius/wet weight of normal gastrocnemius×100%.（5）Hisotological staining： Hematoxylin-eosin staining and Loyez myelin staining were used to observe the myelin formation in the cross section of regeneration nerve under the light microscope; Loyez myelin staining and Bielschowsky silver stain were used for the nerve fiber regeneration of longitudinal section; Axonal dyeing of regeneration nerve was conducted by using the immunohistochemistry method of neurofilament proteins. While some normal sciatic nerves were taken as control group. （6） The ultramicro-structure of regeneration nerve was observed under transmission electron microscope. （7）Image analysis of regeneration nerve fiber included the section area of regeneration nerve, the density, quantity, diameter of opaque nerve fiber as well as neural sheath thickness. RESULTS： Totally 30 rats were involved in the result analysis.（1）Gross observation： 12 weeks after operation, there were many regeneration nerve fibers in experimental group and ECM group, the contour of which like normal nerve, but more slender. No regeneration nerve through gap was found in control group.（2）SFI： 8, 12 weeks after operation, the experimental group got higher score than ECM group, and the difference was significant [（-72.18±3.11）%, （-76.85± 2.76）%; （-62.91±9.87）%, （-69.63±9.52）%; F=7.85, P 〈 0.01].（3）Electrophysiological detection： 12 weeks after operation, the motor nerve conduction velocity and wave amplitude of experimental group were obviously higher than those of ECM group, with the significant difference [（41.29±3.83）, （32.64±3.52） m/s; （3.21±0.34）, （2.85±0.22） mV, F =6.39,P 〈 0.01]. The gastrocnemius electromyogram showed partly absence of neuropotential in experimental group and ECM group, but control group presented completely absence of neuropotential. （4）Gastrocnemius wet weight recovery rate was obviously higher in experimental group and ECM group than in control group, and the difference was extremely significant [（69.32±2.65）%, （66.72±1.75）%, （53.41±1.97）%, F =9.32, P 〈 0.01]. There were significant differences between experimental group and ECM group （F =6.25, P 〈 0.05）.（5）Histological examination： 12 weeks after operation, the regeneration nerve fiber of exPerimental group and ECM group lined up in order and intensively. There was no scar in nerve-tube juncture, and the regeneration nerve fiber of experimental group was thicker and more orderly. （6） Transmission electron microscope observation： There were medullated regenerated nerve fibers in both experimental group and ECM group.（7） Image analysis of regeneration nerve fiber： 12 weeks after operation, the density, quantity, diameter of opaque nerve fiber as well as neural sheath thickness of experiment group were all better than those of ECM group. CONCLUSION： Autologous mesenchymal stem cells compounded with chitosan nerve conduit can promote peripheral nerve regeneration and repair nerve defects as seed cells.