负载脑源性神经营养因子胶原/肝素硫酸支架促进颅脑创伤大鼠神经运动功能的恢复

Collagen/heparin sulfate scaffolds loaded with brain-derived neurotrophic factor promote neurological and locomotor function recovery in rats after traumatic brain injury

背景:研究表明胶原和硫酸肝素交联后可有效固定生长因子,显著改善脊髓损伤大鼠的神经运动功能恢复。目的:观察负载脑源性生长因子胶原/硫酸肝素支架移植修复颅脑创伤的效果。方法:制作胶原支架、胶原/硫酸肝素支架与负载脑源性神经营养因子的胶原/硫酸肝素支架,检测胶原支架、胶原/硫酸肝素支架的孔隙率、吸水率、压缩模量和压缩应力;检测载脑源性生长因子胶原/硫酸肝素支架的体外缓释性能;检测胶原/硫酸肝素支架与负载脑源性神经营养因子的胶原/硫酸肝素支架的细胞毒性与细胞相容性。将48只SD大鼠随机分4组干预:对照组开骨窗后缝合,空腔组仅制作颅脑创伤空腔模型,支架组、生长因子支架组颅脑创伤后空腔处分别植入胶原/硫酸肝素支架、负载脑源性神经营养因子的胶原/硫酸肝素支架,术后分别进行改良神经功能缺损评分、水迷宫测试与脑损伤形态学观察。动物实验获得武警特色医学中心伦理委员会批准。结果与结论:①胶原/硫酸肝素支架的孔隙率、压缩模量和压缩应力高于胶原支架(P<0.05),吸水率低于胶原支架(P<0.05);②胶原/硫酸肝素支架无细胞毒性,负载脑源性神经营养因子后更加有利于脑微血管内皮细胞的增殖;脑微血管内皮细胞在负载脑源性神经营养因子支架微孔内生长良好,分布均匀;③形态学观察显示,两支架组损伤灶处可见大量新生神经细胞及神经纤维,其中以生长因子支架组的新生神经细胞数量及神经纤维密度更高;④生长因子支架组大鼠的逃逸潜伏期短于空腔组、支架组(P<0.01,P<0.05),象限停留时间和平台穿越次数高于支架组、空腔组(P<0.01,P<0.05);⑤生长因子支架组术后3-7周的改良神经功能缺损评分低于支架组、空腔组(P<0.05,P<0.01);⑥结果表明,负载脑源性神经营养因子胶原/硫酸肝素支架移植可以促进大鼠颅脑创伤后神经运动功能的恢复。

BACKGROUND:The porous scaffold fabricated with collagen and heparin sulfate can effectively immobilize neurotrophic factor and greatly improve neurological and locomotor function recovery.OBJECTIVE:To investigate the effect of collagen/heparan sulfate scaffold loaded with brain-derived neurotrophic factor in the treatment of traumatic brain injury(TBI).METHODS:Collagen scaffolds,collagen/heparan sulfate scaffolds and collagen/heparan sulfate scaffolds loaded with brain-derived neurotrophic factor were prepared.The porosity,water absorption,compression modulus and compression stress of collagen scaffolds and collagen/heparan sulfate scaffolds were measured.The in vitro sustained-release performance of collagen/heparan sulfate scaffolds loaded with brain-derived neurotrophic factor was measured.The cytotoxicity and cytocompatibility of collagen/heparin sulfate scaffolds and collagen/heparin sulfate scaffolds loaded with brain-derived neurotrophic factor were detected.Forty-eight Sprague-Dawley rats were randomly assigned to undergo corresponding interventions:opening bone window followed by suture(control group),induction of craniocerebral trauma(model group),induction of craniocerebral trauma+implantation of collagen/heparin sulfate scaffolds(scaffold group),and induction of craniocerebral trauma+implantation of collagen/heparin sulfate scaffolds loaded with brain-derived neurotrophic factor(brain-derived neurotrophic factor group).After surgery,modified neurological severity scoring and Morris water maze test were performed to evaluate the recovery of neurological and locomotor function.Rat cerebral morphology was performed.This study was approved by the Animal Ethics Committee of Characteristic Medical Center of Chinese People’s Armed Police Force,China.RESULTS AND CONCLUSION:(1)The porosity,compression modulus and compression stress of the collagen/heparin sulfate scaffold were significantly higher than those of the collagen scaffold(P<0.05),and the water absorption rate of the collagen/heparin sulfate scaffold was lower than that of the collagen scaffold(P<0.05).(2)Collagen/heparin sulfate scaffolds had no cytotoxicity,and collagen/heparin sulfate scaffolds loaded with brain-derived neurotrophic factor were more conducive to the proliferation of cerebral microvascular endothelial cells;cerebral microvascular endothelial cells grew well and distributed evenly in the micropores of the collagen/heparin sulfate scaffolds loaded with brain-derived neurotrophic factor.(3)There were a large number of newly formed nerve cells and nerve fibers in the lesions in the scaffold and brain-derived neurotrophic factor groups.The number of nerve cells and the density of nerve fibers in the scaffold group were higher compared with the brain-derived neurotrophic factor group.(4)In the brain-derived neurotrophic factor group,escape latency was shorter(P<0.01,P<0.05),quadrant stay time and the number of crossing the platform were higher(P<0.01,P<0.05)compared with the scaffold and model groups.(5)The modified neurological severity score in the brain-derived neurotrophic factor group was significantly lower than that in the scaffold and model groups,respectively at postoperative 3-7 weeks(P<0.05,P<0.01).(6)These results suggest that collagen/heparin sulfate scaffolds loaded with brain-derived neurotrophic factor can promote neurological and locomotor function recovery in rats after traumatic brain injury.