BMSCs-Pluronic F-127水凝胶复合体移植治疗椎间盘退变的实验研究

BMSCs-pluronic F-127 hydrogel complex transplantation for treating intervertebral disc degeneration:an experimental study

目的:探讨兔BMSCs-Pluronic F-127凝胶复合体移植治疗椎间盘髓核缺损退变的效果,为临床应用提供实验依据。方法:6只1月龄健康新西兰白兔,雌雄不限,各取骨髓2 ml,分离培养BMSCs。取第3代骨髓间充质干细胞(BMSCs),与Pluronic F-127水凝胶混匀,制备成BMSCs-Pluronic F-127水凝胶复合体。将18只新西兰大白免建立椎间盘髓核缺损退变动物模型,并随机分为3组(n=6)。正常对照组仅分离暴露椎间盘,不作任何处理;移植治疗组将25μl BMSCs-Pluronic F-127水凝胶复合体注入缺损椎间盘中心;缺损退变组仅注射入0.01 mol.L-1PBS液25μl。8周后处死动物,取出脊柱,应用MRI测量各椎间盘的信号强度和邻近椎旁肌肉的信号强度,计算信噪比;取出相应椎间盘行阿尔辛蓝过碘雪夫(AB-PAS)染色及Ⅱ型胶原免疫组织化学染色,切片行灰度值测定和分光光度计测定蛋白多糖含量。结果:3组椎间盘信噪比差异有统计学意义(P<0.05)。AB-PAS染色显示,正常对照组及移植治疗组椎间盘染色明显,椎间盘结构清晰;缺损退变组椎间盘结构紊乱,髓核组织和外周纤维化分界不清。移植治疗组Ⅱ型胶原和蛋白多糖的含量明显高于缺损退变组(P<0.05),但与正常对照组比较差异无统计学意义(P>0.05)。结论:兔BMSCss-PluronicF-127水凝胶复合体可修复椎间盘缺损退变,本研究为临床应用可注射式组织工程髓核移植治疗椎间盘退变奠定了实验基础。

Objective： To investigate the therapeutic effect of BMSCs-Pluronic F-127 hydrogel complex transplantation on intervertebral disc degeneration and to provide experimental basis for its clinical application.Methods： Two milliliter of bone marrow from 6 healthy one-month-old New Zealand rabbits were selected to isolate and culture BMSCs.Then,BMSCs at passage 3 were mixed with Pluronic F-127 hydrogel to prepare BMSCs-Pluronic F-127 hydrogel complex.Eighteen rabbits were selected to establish the model of intervertebral disc degeneration and divided into 3 groups randomly（n=6 per group）： control group in which intervertebral disc was separated and exposed but without further processing;transplantation group in which 25 μl of autogenous BMSCs-Pluronic F-127 hydrogel complex was injected into the center of defected intervertebral disc;degeneration group in which only 25 μl of 0.01 mol·L^-1 PBS solution was injected.Animals were killed 8 weeks later and the repaired discs were obtained.The changes of the intervertebral disc signal-noise ratio were showed by MRI and the proteoglycan was quantitated by spectrophotometer to observe the repair effect.The collagenaseⅡ expression was detected by immunohistochemistry.Results： The signal-noise ratio had a significant difference among the three groups（P〈0.05）.AB-PAS staining showed that intervertebral disc in the control and transplantation groups were stained obviously,with a clear structure;while the intervertebral disc in the degeneration group demonstrated a deranged structure with an indistinct division between the nucleus pulposus and the anulus fibrosus.The content of collagenaseⅡand proteoglycan of transplantation group had significant difference comparing with that of degeneration group（P〈0.05）,but there was no significant difference between the control group（P〉0.05）.Conclusion： The rabbit BMSCs-Pluronic F-127 hydrogel complex can repair intervertebral disc degeneration and it provide an experimental foundation for the clinical application of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.