摘要
目的：研究采用PLGA构建的新型三维神经导管修复大鼠周围神经缺损。观察神经导管内不同直径的微丝支架对神经再生的影响,为研制理想的人工神经提供理论和实验依据。
方法：新型三维神经导管材料采用聚乳酸聚羟基乙酸共聚物(PLGA,85:15)。先将这种可吸收材料制备成管壁有微孔,内含有不同直径微丝支架的三维管状结构；再随机将60只成年SD大鼠分为六组,每组10只,在左侧制造12mm坐骨神经缺损,用PLGA导管桥接大鼠坐骨神经缺损。A组：PLGA神经导管内纵形放入直径为60um的20根PLGA微丝；B组：PLGA神经导管内纵形放入直径为80um的20根PLGA微丝;C组：PLGA神经导管内纵形放入直径为100um的20根PLGA微丝;D组：PLGA神经导管内纵形放入直径为120um的20根PLGA微丝；E组：PLGA神经导管内纵形放入直径为140um的20根PLGA微丝；以上各组神经导管内均注入层粘蛋白与神经生长因子混合液0.3ml；F组：自体神经移植组。所有大鼠左后肢为实验侧,右后肢为对照侧。术后动态观察大鼠肌肉萎缩及跛行等情况,术后12周测量含有不同直径微丝支架的神经导管内再生神经的运动神经传导速度、小腿三头肌湿重恢复率。对再生神经中1/3段行组织学观察及图像分析以评价神经修复的效果。
结果：术后12周,各组大鼠术侧足底溃疡愈合并已有刺痛反应,但A组和E组大鼠反应较为迟钝；B组、C组、D组和F组大鼠肌肉萎缩有所恢复,跛行减轻,而A组和E组则未见明显恢复；各组再生神经均已通过神经导管长入远端,B、C、D、F组再生神经较A、E组粗大,PLGA神经导管与微丝均基本降解；再生神经的运动神经传导速度B组、C组、D组和F组明显快于A组和E组(P<0.01);A组、E组肌肉萎缩最明显,而B组、C组、D组和F组肌肉萎缩较轻且肌肉萎缩程度基本相当,其差异无统计学意义(P>0.05)；再生神经中1/3段光镜和电镜观察,A组、E组与B组、C组、D组、F组比较神经纤维较稀疏、轴突较细小、髓鞘较薄、发育不成熟,且E组水肿明显,结构松散,髓鞘内微丝和微管较模糊；而B组、C组、D组和F组轴突较粗大,髓鞘厚,髓鞘内微丝和微管清晰且排列整齐。病理图像分析神经纤维计数以F组最多,C组次之,神经纤维直径、轴突直径和髓鞘厚度B组、C组、D组和F组比较差异无统计学意义(P>0.05),而与A组、E组比较差异均有统计学意义(P<0.01),B组、C组、D组和F组的再生神经纤维数量及成熟程度均要明显优于A组和E组。
结论：1.研制的PLGA三维导管能有效引导SD大鼠坐骨神经再生并长过12mm的神经缺损,是一种较理想的修复材料。
2、三维神经导管内微丝直径影响周围神经再生,直径为80-120um微丝为最利于神经生长。
Objective:To study the feasibility of using a new type of PLGA three-dimensional nerve conduit containing various of microfilaments in repairing rat's peripheral nerve defect, and investigate the effect of the diameter of microfilaments on nerve regeneration, in order to provide experimental basis for further study of ideal artificial nerves.
Methods:60 adult SD rats (300-350g) were randomly divided into 6 groups (n=10), whose sciatic nerves were resected 12mm. In all groups except F group nerve defects were repaired using the PLGA nerve conduit containing 20 microfilaments with different diameter (60 um、80 um、100 um、120 um、140um). The nerve conduits of all groups were filled with o.3ml laminin and never growth factor(NGF). group F:nerve autograft (control group).The left-behind legs were done as experiment sides, the right ones as control sides. To evaluate the repairing result, special assessments were performed including EMG testing, immunohistochemisty to test the number of regeneration nerves at 12 weeks after operation.
Results:12 weeks after operation, all the ulcers in claw's palm were healed. The sense of pain and atrophy of triceps muscle of calf recovered better in group B,C,D and F than in group A and E; All the PLGA nerve conduits and microfilaments were degraded on the whole, the regeneration nerves succeeded passing through the gap, which were thicker in group B,C,D and F than in group A and E. Compared with group A and E, the regeneration nerve conduction velocity and the weighing of triceps muscle of calf were better in group B,C,D and F(P<0.01). However, there was no significant difference of atrophy of triceps muscle between group B,C,D and F(P>0.05). Under light microscope and electron microscope, we also discovered that there were significant difference of nerve fiber diameter, thickness of myelin sheath, and fiber density in group B,C,D and F from those in group A and E (P<0.01). Furthermore, image analysis suggested the group B,C,D and F had the nerve regeneration better than group A and E in quantity of nerve fiber, thickness of myelin sheath, and nerve fiber diameter (P<0.01).And the nerve regeneration of PLGA nerve conduit containing 20 microfilaments group was as good as that of nervous autograft group (P>0.05).
Conclusion:1. The new PLGA three-dimensional nerve conduit containing 20 microfilaments can help repair 12mm nerve defect effectively, which can be considered as an ideal nerve conduit;
2. The diameter of microfilaments can influence nerve regeneration, and the best diameter is 80-120um.
引文
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