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中华神经创伤外科电子杂志 ›› 2021, Vol. 07 ›› Issue (06) : 331 -338. doi: 10.3877/cma.j.issn.2095-9141.2021.06.003

基础研究

吸附NT-3的3D打印胶原蛋白/壳聚糖支架改善脊髓损伤后的运动功能
隋曌1, 彭凤2, 余凯1, 严小虎3, 李英4, 钟琳5, 刘晓银6,†()   
  1. 1. 610500　成都，成都医学院第一附属医院神经外科
    2. 610500　成都，成都医学院第一附属医院麻醉科
    3. 610500　成都，成都医学院第一附属医院骨科
    4. 610500　成都，成都医学院第一附属医院胸心外科
    5. 610500　成都，成都医学院第一附属医院血液科
    6. 610041　成都，四川大学华西医院神经外科
  • 收稿日期:2021-03-30 出版日期:2021-12-15
  • 通信作者: 刘晓银

3D printing collagen/chitosan scaffold adsorbed NT-3 improved locomotor function after spinal cord injury

Zhao Sui1, Feng Peng2, Kai Yu1, Xiaohu Yan3, Ying Li4, Lin Zhong5, Xiaoyin Liu6,†()   

  1. 1. Department of Neurosurgery, First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China
    2. Department of Anesthesiology, First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China
    3. Department of Orthopaedics, First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China
    4. Department of Cardiothoracic Surgery, First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China
    5. Department of Hematology, First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China
    6. Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu 610065, China
  • Received:2021-03-30 Published:2021-12-15
  • Corresponding author: Xiaoyin Liu
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引用本文：

隋曌, 彭凤, 余凯, 严小虎, 李英, 钟琳, 刘晓银. 吸附NT-3的3D打印胶原蛋白/壳聚糖支架改善脊髓损伤后的运动功能[J]. 中华神经创伤外科电子杂志, 2021, 07(06): 331-338.

Zhao Sui, Feng Peng, Kai Yu, Xiaohu Yan, Ying Li, Lin Zhong, Xiaoyin Liu. 3D printing collagen/chitosan scaffold adsorbed NT-3 improved locomotor function after spinal cord injury[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2021, 07(06): 331-338.

目的

探究移植吸附神经营养素-3（NT-3）的3D打印胶原蛋白/壳聚糖支架对于脊髓损伤后运动功能的改善作用。

方法

制备吸附NT-3的3D打印胶原蛋白/壳聚糖支架。将80只成年雌性SD大鼠（220~250 g）随机分为4组：假手术组、模型组、3D打印支架组和3D打印支架-因子组，每组20只。假手术组：暴露脊髓后直接缝合硬脊膜无损伤；模型组：只进行T10脊髓全横断损伤；3D打印支架组：T10脊髓全横断损伤后植入3D打印胶原蛋白/壳聚糖支架；3D打印支架-因子组：T10脊髓全横断损伤后植入吸附NT-3的3D打印胶原蛋白/壳聚糖支架。脊髓损伤后1~8周采用BBB评分和斜坡爬壁实验。脊髓损伤后8周行电生理分析，评估大鼠的运动功能；行弥散张量纤维束成像观察脊髓缺损部位恢复情况。

结果

（1）脊髓损伤后1～8周，相对于假手术组、模型组和3D打印支架组，植入吸附NT-3的3D打印胶原蛋白/壳聚糖支架可以显著提高脊髓损伤大鼠的BBB评分和斜坡角度（P<0.05）；（2）脊髓损伤后8周，与3D打印支架组和模型组相比，3D打印支架-因子组展现出在左后肢和右后肢的运动诱发电位和体感诱发电位中振幅和潜伏期的改善（P<0.05）；（3）脊髓损伤后8周弥散张量纤维束成像的图像证明3D打印支架-因子组的脊髓损伤区域的再生神经纤维数量明显多于3D打印支架组和模型组。

结论

吸附NT-3的3D打印胶原蛋白/壳聚糖支架能改善脊髓损伤后的运动功能，有潜力成为脊髓损伤修复创新和安全的方法。

关键词: 脊髓损伤, 3D打印, 胶原蛋白, 壳聚糖, 神经营养素-3, 支架, 运动功能, 弥散张量纤维束成像
Objective

To explore the effect of transplantation of 3D printing collagen/chitosan scaffold adsorbed neurotrophin-3 (NT-3) on improving locomotor function after spinal cord injury (SCI).

Methods

The 3D printing collagen/chitosan scaffold adsorbed NT-3 was prepared. Eighty adult female SD rats (220-250 g) were randomly divided into four groups: sham operation group, model group, 3D printing scaffold group and 3D printing scaffold-factor group, with 20 rats in each group. Sham operation group: the spinal cord was exposed to the spinal cord and directly sutured the endorachis without injury; Model group: only undergoes T10 spinal cord transection; 3D printing scaffold group T10 spinal cord transection is implanted with 3D printing collagen/chitosan; 3D printing scaffold-factor group: T10 spinal cord transection was implanted with 3D printing collagen/chitosan scaffold adsorbed by NT-3. 1-8 weeks after SCI, the BBB score and the inclined-grid climbing test were used to evaluate the locomotor function of rats 8 weeks after SCI. Diffusion tensor fiber tract imaging was performed to observe the recovery of spinal cord defect at 8 weeks after SCI.

Results

(1) 1-8 weeks after SCI, compared to sham operation group, model group and 3D printing scaffold group, implantation of 3D printing collagen/chitosan scaffold adsorbed by NT-3 could significantly improve the spinal cord BBB score and slope angle of injured rats (P<0.05). (2) 8 weeks after SCI, compared with the 3D printing scaffold and model groups, the 3D printing scaffold-factor group showed improvements in amplitude and latency in the motor and somatosensory evoked potentials of the left and right hind limbs (P<0.05). (3) The image of diffusion tensor fiber bundle imaging at 8 weeks after SCI proved that the number of regenerated nerve fibers in the SCI area in 3D printing scaffold-factor group was significantly more than that of the 3D printing scaffold and model groups.

Conclusion

3D printing collagen/chitosan scaffold adsorbed NT-3 can improve locomotor function after SCI, and has the potential to become an innovative and safe method for the repair of SCI.

Key words: Spinal cord injury, 3D printing, Collagen, Chitosan, Neurotrophin-3, Scaffold, Locomotor function, Diffusion tensor fiber bundle imaging
表1 3D打印胶原蛋白支架和3D打印胶原蛋白/壳聚糖支架的性能比较
组别 只数 孔隙率（%） 吸水率（%） 压缩模量（MPa） 压缩应力（kPa）
3D打印胶原蛋白支架 20 49.03±5.56 1298.45±39.34 1.77±0.14 1187.47±7.25
3D打印胶原蛋白/壳聚糖支架 20 64.88±4.50 1085.75±49.61 3.93±0.41 184.95±5.66
t值   9.910 15.023 22.208 45.236
P值   0.007 0.004 0.002 0.003
图1 支架的形态和力学性能A：扫描电镜下支架的表面观；B：扫描电镜下支架的截面观
图2 支架的生物相容性A：光学显微镜下的典型NSCs形态图像；B：荧光显微镜下的Nestin阳性细胞；C：吸附NT-3的3D打印胶原蛋白/壳聚糖支架与NSCs共培养的扫描电子显微镜图像；NSCs：神经干细胞
图4 4组大鼠脊髓损伤后的运动功能评价
图5 4组大鼠脊髓损伤后8周的电生理分析结果A：运动诱发电位的典型图像；B：体感诱发电位的典型图像比较；C：运动诱发电位和体感诱发电位的振幅比较；D：运动诱发电位和体感诱发电位的潜伏期比较；与模型组比较，aP<0.05；与3D打印支架组比较，bP<0.05
图6 4组大鼠脊髓损伤后8周的脊髓弥散张量纤维束成像图像
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