摘要
目的探讨一种新型生物材料明胶微支架装载脂肪间充质干细胞(ADMSC)移植修复犬椎间盘退行性变的治疗效果。方法选用12只健康成年比格犬,采用穿刺抽吸髓核法建立椎间盘退行性变模型(L37),提取自体ADMSC并应用Luc-GFP慢病毒标记。4周后将犬椎间盘节段分为对照组(L7/S1)、模型组(L3/L4)、干细胞组(L4/L5,造模后注射ADMSC)、微支架组(L5/L6,造模后注射明胶微支架)、微支架装载干细胞组(L6/L7,造模后注射ADMSC和微支架共培养物)。分别于造模前及造模后4、8、12、24周行腰椎X线及MRI检查,观察椎间盘相对高度(术后椎间盘高度与术前椎间盘高度的比值)及相对灰度(MRI髓核组织灰度值与脑脊液灰度值的比值)的变化。24周后取椎间盘髓核组织,行冰冻切片荧光、HE染色及番红O染色观察,用ELISA法检测软骨相关蛋白SOX-9、PG和COL2的含量。结果影像学结果显示,造模后各组椎间盘相对高度及相对灰度与对照组相比均明显降低;造模后8、12、24周,微支架装载干细胞组椎间盘相对高度及相对灰度高于模型组、干细胞组和微支架组,差异均有统计学意义(P <0.05)。术后24周,微支架装载干细胞组和干细胞组髓核组织冰冻切片内能够检测到Luc-GFP+-ADMSC表达,且微支架装载干细胞组表达比干细胞组多。HE和番红O染色显示微支架装载干细胞组椎间盘退行性变比模型组、干细胞组和微支架组轻,蛋白定量检测结果显示微支架装载干细胞组髓核组织内SOX-9、PG和COL2表达高于模型组、微支架组和干细胞组,差异均有统计学意义(P <0.05)。结论明胶微支架装载ADMSC移植可延缓比格犬椎间盘退行性变,有望为发生退行性变的椎间盘组织的修复治疗提供新的策略。
Objective To investigate the therapeutic effect of new biomaterial gelatin micro-scaffolds loaded with adiposederived mesenchymal stem cells(ADMSCs) transplantation for the repair of dog intervertebral disc degeneration. Methods Degenerative models were established in L3/L4,L4/L5,L5/L6 and L6/L7 intervertebral discs by aspiration of the nucleus pulposus in 12 dogs. ADMSCs were isolated from canine and labeled with Luc-GFP. For each dog,L3/L4 was used as model group;L4/L5 was injected with ADMSCs as ADMSC group;L5/L6 was injected with micro-scaffolds as micro-scaffold group;L6/L7 was injected with micro-scaffolds+ADMSCs as micro-scaffold+ADMSC group. The intact disc L7/S1 was served as a normal control. Spinal image was evaluated at 0,4,8,12 and 24 weeks after operation respectively,and the changes of the disc relative height (the ratio of disc height between postoperative and preoperative) and relative gray value(the ratio of the gray value of nucleus pulposus tissue to the gray value of cerebrospinal fluid in MRI) were recorded. Fluorescence,HE staining and O staining were performed at 24 weeks. ELISA analysis for matrix-related genes(SOX-9,PG and COL2) was performed. Results The disc relative heights and relative gray values were lower in all the degenerative model groups than control group;in 8,12,and 24 weeks,the disc relative height and relative gray value of the micro-scaffold+ADMSC group were greater than those of model,micro-scaffold,and ADMSC groups;and the differences were statistically significant(P < 0.05). In 24 weeks,the Luc- GFP+-ADMSCs were detected in the nucleus pulposus of the canine in ADMSC group and micro-scaffold+ADMSC group,and microscaffold+ADMSC group expressed more Luc-GFP+-ADMSCs than ADMSC group. HE staining and O staining showed milder degeneration in micro-scaffold+ADMSC group as compared to the model,micro-scaffold,and ADMSC groups. The contents of SOX-9,PG and COL2 were significantly increased in micro-scaffold+ADMSC group than in model,micro-scaffold and ADMSC groups,and the differences were statistically significant(P < 0.05). Conclusion The micro-scaffold+ADMSC can delay intervertebral disc degeneration in beagle dogs,thus providing an experimental foundation for the clinical application of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.
作者
陈春
何凡
金广建
陈雷
CHEN Chun;HE Fan;JIN Guang-jian;CHEN Lei(Department of Orthopaedics,First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325000,Zhejiang,China)
出处
《脊柱外科杂志》
2019年第2期110-115,139,共7页
Journal of Spinal Surgery
基金
国家自然科学基金青年科学基金项目(81702132)
浙江省医药卫生科技计划项目(2017KY456)
关键词
腰椎
椎间盘退行性变
间质干细胞
组织支架
狗
Lumbar vertebrae
Intervertebral disc degeneration
Mesenchymal stem cells
Tissue scaffolds
Dogs
