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本研究的目的是构建一种新型的组织诱导性神经导管并评价其生物学性能。壳聚糖包被中药川芎嗪制备微球,利用体外缓释方法检测壳聚糖/川芎嗪微球的缓释效果;壳聚糖/川芎嗪微球与胶原蛋白复合构建组织诱导性神经导管,2%京尼平交联导管;万能材料试验机评价交联前后神经导管的力学特征;体外降解试验分析交联前后神经导管的降解性能;应用织物手感评价仪检测神经导管的柔韧性;扫描电镜(SEM)观察神经导管交联前后空间结构及微球的分布;光学显微镜(LMS)和免疫荧光方法观察并评价壳聚糖微球/胶原蛋白神经导管与大鼠间充质干细胞(MSCs)共培养对MSCs向神经细胞定向分化的影响;SEM和细胞免疫荧光方法分别评价神经导管与MSCs的复合情况及对细胞定向分化的影响。结果显示:壳聚糖微球具有良好的缓释效果;交联前后神经导管的最大载荷和断裂载荷分别为(0.23±0.09)N、(0.76±0.15)N和(0.20±0.12)N、(0.69±0.17)N,两者相比具有统计学意义(P<0.05);体外降解实验表明,交联前后导管的平均失重率分别为(58.62±7.59)mg和(9.23±2.47)mg,两者相比具有统计学意义(P<0.01);干湿态神经导管的平均线性度分别为(0.597±0.012)LC和(0.333±0.015)LC,两者相比差异具有统计学意义(P<0.01),湿态神经导管的柔韧性较干态好;交联后神经导管中胶原蛋白排列紧密,微球均匀分布于胶原支架材料;神经导管与MSCs共培养后,微球缓释的川芎嗪能促进MSCs表达神经细胞相关标志分子NSE和MAP2;神经导管与MSCs复合培养,通过缓释的川芎嗪促进MSCs增殖和NSE的表达。构建的组织工程化神经导管具有良好的组织相容性和组织诱导性功能。
The purpose of this study was to construct a new tissue-inducible nerve catheter and evaluate its biological properties. Chitosan was coated with ligustrazine to prepare microspheres. The in vitro sustained release method was used to detect the sustained release of chitosan / ligustrazine microspheres. Chitosan / ligustrazine microspheres were combined with collagen to construct tissue-inducible nerve conduits, 2% Jing Niuping cross-linked catheters; universal testing machine before and after mechanical evaluation of mechanical characteristics of nerve conduits; in vitro degradation test before and after cross-linked degradation of nerve conduits; using fabric feel evaluation instrument to detect nerve conduit flexibility; scanning electron microscopy (SEM) were used to observe the spatial structure and distribution of microspheres before and after cross-linking of nerve conduits. The effects of chitosan microsphere / collagen neural tube and rat mesenchymal stem cells (MSCs) were observed and evaluated by light microscopy (LMS) and immunofluorescence Culture on the directional differentiation of MSCs to nerve cells; SEM and cell immunofluorescence method to evaluate the neural tube and MSCs composite situation and the impact of cell differentiation. The results showed that the chitosan microspheres had a good sustained-release effect. The maximum and the fracture load of the nerve conduit before and after crosslinking were (0.23 ± 0.09) N, (0.76 ± 0.15) N and (0.20 ± 0.12) N, 0.69 ± 0.17) N, respectively (P <0.05). The in vitro degradation experiments showed that the average weight loss of the catheter before and after cross-linking were (58.62 ± 7.59) mg and (9.23 ± 2.47) mg, respectively (0.597 ± 0.012) LC and (0.333 ± 0.015) LC, respectively. The difference between the two groups was statistically significant (P <0.01). The average linearity of the wet and dry nerve conduits was (0.597 ± 0.012) 0.01). The wet nerve conduits were more flexible than the dry ones. Collagen in the nerve conduit was tightly packed and the microspheres were evenly distributed in the collagen scaffolds after the cross-linking. After the co-culture of the nerve conduits and MSCs, the ligustrazine Can promote MSCs to express neuron-related marker molecules NSE and MAP2; nerve conduit and MSCs compound culture, through sustained release of tetramethylpyrazine promote MSCs proliferation and NSE expression. The constructed tissue engineered nerve conduit has good histocompatibility and tissue-induced function.