负载外泌体的可注射葡聚糖/明胶复合水凝胶修复大鼠椎间盘退变  

An injectable dextran/gelatin composite hydrogel loaded with exosomes for repair of rat intervertebral disc degeneration

作　　者：潘玉军 时长江 曹胜 穆怀昭[1] 张义龙[1] 王凯华[1] Pan Yujun;Shi Changjiang;Cao Sheng;Mu Huaizhao;Zhang Yilong;Wang Kaihua(Affiliated Hospital of Chengde Medical College,Chengde 067000,Hebei Province,China)

机构地区：[1]承德医学院附属医院,河北省承德市067000

出　　处：《中国组织工程研究》2023年第34期5477-5482,共6页Chinese Journal of Tissue Engineering Research

基　　金：承德市科学技术研究与发展计划项目(201801A050);项目负责人:潘玉军。

摘　　要：背景:外泌体可改善退变椎间盘的炎症微环境,但将单纯外泌体直接注射于退变椎间盘内的局部浓度与一过性地释放不能保证持续的作用,因此单纯的外泌体体内治疗效果有限。目的:观察负载外泌体的葡聚糖/明胶复合水凝胶修复大鼠退变椎间盘的效果。方法:提取大鼠骨髓间充质干细胞外泌体。利用Schiff交联反应制备负载外泌体的葡聚糖/明胶复合水凝胶,检测水凝胶的微观结构、弹性模量与体外缓释性能。取60只SD大鼠,采用随机数字表法分为5组,每组12只:A组为正常对照,B、C、D、E组采用经皮穿刺法建立椎间盘退变模型,依次向退变椎间盘内注射PBS、外泌体、葡聚糖/明胶复合水凝胶与负载外泌体的葡聚糖/明胶复合水凝胶,术后8周,分别进行影像学、病理组织学与Western blot检测。结果与结论:(1)负载外泌体的葡聚糖/明胶复合水凝胶呈现三维多孔结构,孔隙之间相互连通,孔径为50-200μm,弹性模量(4.41±0.23)kPa,在体外可持续稳定释放外泌体达20 d以上。(2)术后8周X射线片显示,与A组比较,B-E组椎间盘高度指数百分比降低(P<0.05);与B组比较,C-E组椎间盘高度指数百分比升高(P<0.05),其中E组升高最明显。术后8周MRI检查显示,与A组比较,B-E组髓核组织结构均遭到不同程度破坏;与B组比较,C-E组髓核组织结构均得到不同程度改善,其中E组改善最明显。(3)术后8周的苏木精-伊红染色与番红O染色显示,B组髓核结构遭到严重破坏,仅含有少量的胶原组织;C-E组髓核结构相较于B组有所改善,其中E组髓核组织结构更细清晰、规则且胶原含量最多。(4)Western blot检测显示,与A组比较,B-E组Ⅱ型胶原、蛋白聚糖蛋白表达减少(P<0.05);与B组比较,C-E组Ⅱ型胶原、蛋白聚糖蛋白表达增加(P<0.05),其中E组增加最明显。(5)结果表明,将负载外泌体的葡聚糖/明胶复合水凝胶注射至退变椎间盘内,可在一定程度�BACKGROUND:Exosomes can improve the inflammatory microenvironment of the degenerated intervertebral disc.However,direct injection of exosomes into the degenerated intervertebral disc at local concentrations and transient release cannot guarantee sustained effects,so the in vivo therapeutic effect of exosomes alone is limited.OBJECTIVE:To observe the effect of exosome-loaded glucan/gelatin composite hydrogel on repairing rat degenerative intervertebral disc.METHODS:Exosomes were extracted from bone marrow mesenchymal stem cells of rats.Exosome-loaded dextran/gelatin composite hydrogels were prepared by Schiff crosslinking reaction,and the microstructure and elastic modulus of the hydrogels were detected.Sixty Sprague-Dawley rats were randomly divided into five groups,with 12 rats in each group.Group A was the normal control.In groups B,C,D,and E,the intervertebral disc degeneration model was established by percutaneous puncture.PBS,exosomes,glucan/gelatin composite hydrogel and exosome-loaded glucan/gelatin composite hydrogel were injected into the intervertebral disc in turn.At postoperative 8 weeks,imaging,histopathology and western blot assay were performed.RESULTS AND CONCLUSION:(1)The exosome-loaded glucan/gelatin composite hydrogels presented a three-dimensional porous structure with interconnected pores.The pore size was 50-200μm,and the elastic modulus was(4.41±0.23)kPa,respectively.The exosomes could be released continuously and stably in vitro for more than 20 days.(2)At 8 weeks after operation,X-ray films exhibited that the intervertebral disc height index percentages of rats in groups B-E were lower than those in group A(P<0.05).The intervertebral disc height index percentages of rats in groups C,D and E were higher than those in group B(P<0.05),of which group E was the most obvious.At 8 weeks after operation,MRI examination showed compared with group A,the tissue structure of nucleus pulposus in groups B-E was damaged to different degrees.Compared with group B,the tissue structure of nucleus pulposu

关 键 词：椎间盘退变 水凝胶 外泌体 复合水凝胶 动物实验 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学] R-332