自体脂肪干细胞、壳聚糖/胶原蛋白支架材料及GDF-5复合物移植修复兔软骨缺损  

Repairing articular cartilage defects in rabbits by adipose-derived stem cells combined with chitosan/collagen scaffold and GDF-5

作　　者：王征[1] 于会 于尧[1] 吴柏霖 Wang Zheng;Yu Hui;Yu Yao;Wu Bo-lin(Department of Hand Surgery,Central Hospital Affiliated to Shenyang Medical College,Shenyang 110024,Liaoning Province,China;Shandong University,Jinan 250100,Shandong Province,China)

机构地区：[1]沈阳医学院附属中心医院手外科,辽宁省沈阳市110024 [2]山东大学,山东省济南市250100

出　　处：《中国组织工程研究》2018年第25期3969-3974,共6页Chinese Journal of Tissue Engineering Research

基　　金：沈阳医学院青年基金(20142050);沈阳市卫生和计划生育委员会科研基金~~

摘　　要：背景:GDF-5可诱导人脂肪干细胞分化为软骨样细胞,壳聚糖/胶原蛋白支架在软骨组织损伤修复中应用广泛。目的:利用兔的自体脂肪干细胞复合壳聚糖/胶原蛋白支架材料加入GDF-5修复兔关节软骨缺损,观察其修复效果。方法:取日本大耳兔皮下脂肪组织提取脂肪干细胞体外培养、纯化、扩增,并Brd U体外标记,应用紫外线交联法以1∶3比例构建壳聚糖/胶原蛋白复合支架载体,将标记后的兔脂肪干细胞种植到支架载体上共同培养形成复合物,以手术方式造成兔膝关节软骨直径为5 mm,深3 mm的圆柱形缺损,建立膝关节软骨缺损模型兔,随机分为4组,复合物组:脂肪干细胞+壳聚糖/胶原蛋白支架+GDF-5复合物植入干预;支架组:单纯植入壳聚糖/胶原蛋白支架;脂肪干细胞组:单纯植入脂肪干细胞;假手术组:单纯钻孔不作处理。各组干预4,8,12周时取材,行组织学检测、软骨Ⅱ型胶原免疫组织化学染色及Brd U免疫荧光染色。结果与结论:(1)术后12周,与其他3组相比,复合物组圆柱型缺损处完全被与周围正常软骨相同的乳白色透明样物质填充修复,软骨缺损区域由分化一致的透明软骨所替代修复,Wakitani评分最低(P<0.01);(2)Ⅱ型胶原免疫组化染色及Brd U免疫荧光染色:复合物组与术后4,8,12周免疫组化染色可见修复区组织Ⅱ型胶原染色胞浆内呈淡黄色阳性表达,修复区新生组织可见Brd U绿色荧光表达;(3)结果证实,自体脂肪干细胞+壳聚糖/胶原蛋白支架+GDF-5复合物移植修复兔膝关节软骨缺损效果较好。BACKGROUND:GDF-5 can induce the differentiation of human adipose-derived stem cells into chondrocyte-like cells,and chitosan/collagen scaffolds are widely used in cartilage tissue repair.OBJECTIVE:To repair rabbit articular cartilage defects by using rabbit autologous adipose-derived stem cells combined with chitosan/collagen scaffold and GDF-5 and to observe the repair effect.METHODS:Adipose-derived stem cells isolated from subcutaneous adipose tissues of Japanese rabbits were cultured,purified,amplified in vitro,and labeled with BrdU in vitro.The chitosan/collagen composite scaffold was prepared in a ratio of 1:3 using ultraviolet cross-linking method.Then labeled adipose-derived stem cells were seeded onto the scaffold and cultured to form complexes.A cylindrical defect(5 mm in diameter,3 mm in depth)in the knee articular cartilage was surgically made in each rabbit,and these rabbit models were thereafter randomly divided into four groups.Composite group:adipose-derived stem cells+chitosan/collagen scaffold+GDF-5 complex implantation;stent group:simple implantation of chitosan/collagen scaffold;cell group:simple implantation of adipose-derived stem cells;and sham operation group:simple drilling without treatment.All the rabbits were intervened for 4,8,and 12 weeks.Meanwhile,histological detection,type II collagen immunohistochemical staining and BrdU mmunofluorescence staining were performed.RESULTS AND CONCLUSION:(1)At 12 weeks after surgery,the cylindrical defect of the composite group was completely filled with the same milky white transparent substance as the surrounding normal cartilage,and the cartilage defect area was replaced by differentiated hyaline cartilage.Of all the groups,Wakitani score was the lowest in the composite group(P<0.01).(2)Type II collagen immunohistochemical staining and BrdU immunofluorescence staining:in the composite group,the type II collagen stained cytoplasm in the repaired area was detected by immunohistochemical staining at 4,8,and 12 weeks after surgery.The expression of Br

关 键 词：壳聚糖 胶原蛋白 支架 组织工程 脂肪干细胞 GDF-5 兔 软骨缺损 损伤修复 自体 干细胞 组织工程 壳聚糖 支架 

分 类 号：R394.2[医药卫生—医学遗传学]