壳聚糖导管复合辛伐他汀/泊洛沙姆407水凝胶修复大鼠外周神经缺损的研究

Bridging sciatic nerve defect with simvastatin delivered by injectable thermosensitive pluronic F-127 hydrogel repairs peripheral nerve defect in rats

目的探讨壳聚糖导管复合辛伐他汀/泊洛沙姆407水凝胶作为无细胞人工神经支架修复坐骨神经缺损的可行性。方法制备壳聚糖导管和辛伐他汀/泊洛沙姆407水凝胶,通过体视显微镜、扫描电镜、体外降解实验和流变学检测来观察复合材料的性能。选取SPF级SD大鼠40只,分为单纯导管组、导管复合辛伐他汀0mg组、导管复合辛伐他汀0.5 mg组,和导管复合辛伐他汀1 mg组共4组其中前2组为对照组,后2组为辛伐他汀治疗组,每组10只,造成左侧坐骨神经10 mm缺损模型,用壳聚糖导管桥接缺损,其内填充不同浓度的辛伐他汀水凝胶。移植后10周,取再生神经的中间段进行HE染色、透射电镜观察再生神经的形态学改变,并对再生神经的轴突数量、髓鞘厚度、G-ratio等进行统计学分析;免疫组化观察再生神经中NF200和S100蛋白的表达和神经营养因子PTN、HGF、GDNF和VEGF的表达。结果壳聚糖导管和辛伐他汀/泊洛沙姆407水凝胶是适合神经缺损的无细胞修复材料。植入10周后四组均可见再生神经,但HE染色显示辛伐他汀治疗组的神经干明显较对照组粗;透射电镜显示辛伐他汀治疗组的再生轴突数量显著增多,髓鞘显著增厚,G-ratio显示髓鞘化程度亦明显好于对照组;免疫组化显示辛伐他汀治疗组再生神经中标记轴突的NF200和标记雪旺细胞的S100的阳性表达明显增强,且内源性神经营养因子PTN、HGF、VEGF和GDNF呈现高表达。结论壳聚糖导管复合辛伐他汀/泊洛沙姆407水凝胶明显促进神经缺损组织学的重建,可用于修复坐骨神经缺损。

Objective To explore the feasibility of combining chitosan conduit filled with simvastatin/pluronic F-127 thermosensitive hydrogel to repair peripheral nerve defects in rats. Methods The chitosan conduits and simvastatin/ pluronic F-127 hydrogel were prepared and tested by stereomicroscope, SEM, vitro degradation testing and rheometer property. A total of 40 Sprague-Dawley rats were randomly divided into 4 groups （ n=10 per group） ： the chitosan conduitalone （conduit group） or the conduit filled with pluronic F-127 hydrogel containing 0 （c ＋ sim 0mg）, 0. 5 （c＋sim 0.5mg） , and 1 mg （c＋sim 1mg） simvastatin. A 10mm nerve defect was created in the rat left sciatic nerve and bridged with the chitosan conduit filled with pluronic F-127 hydrogel containing different doses of simvastatin. At 10 weeks after surgery, the regenerated nerves were performed the H＆E and TEM to observe the morphological change. The diameters of the myelinated axons, the thickness of the myelin sheath, and the G-ratios （ the ratio of the axon diameter to the total fiber diameter） were calculated to make statistical analyses. And the immuuohistochemical method was used to measure the expression of NF200 and SIO0 protein in the regenerated nerves and to detect the expression of neurotrophins including pleiotrophin （PTN）, hepatocyte growth factor （HGF）, vascular endothelial growth factor （VEGF） and glial cell line- derived neurotrophic factor （GDNF）. Results The chitosan conduits filled with simvastatin/ pluronic F-127 promote nerve regeneration in rats. All groups showed the regenerated nerves, but H＆E staining showed that the regenerated nerves in the simvastatin treated groups were much thicker than those without simvastatin; And the same trend was also found for the diameter of the myelinated axons, the thickness of the myelin sheath and G-ratio; In addition, the numbers of NF- positive cells indicating regenerated axons and S100-positive cells indicating Schwann cells were significantly larger in simvastatin treated groups than those without simvastatin. And immunohistochemical staining indicated that simvastatin induced an increased expression of PTN, HGF, VEGF and GDNF compared to those found in the control group. Conclusions Taken together, the simvastatin/ pluronic F-127 hydrogel filling in the chitosan conduits promoted the reconstruction of histological in peripheral nerve defects, which can be used for peripheral nerve regeneration.