脂肪血管基质成分复合骨软骨一体化支架的体外评价  

In vitro evaluation of adipose-derived stromal vascular fraction combined with osteochondral integrated scaffold

作　　者：陈磊[1] 郑蕊 杰永生[1] 綦惠[1] 孙磊[1] 舒雄[1] Chen Lei;Zheng Rui;Jie Yongsheng;Qi Hui;Sun Lei;Shu Xiong(Beijing Jishuitan Hospital,Beijing Institute of Traumatology and Orthopaedics,Beijing 100035,China)

机构地区：[1]北京积水潭医院/北京市创伤骨科研究所,北京市100035

出　　处：《中国组织工程研究》2021年第22期3487-3492,共6页Chinese Journal of Tissue Engineering Research

基　　金：北京市属医学科研院所科技发展项目(PXM2018_026275_000004),项目参与者:陈磊;北京市属医学科研院所公益发展改革试点项目(BMHC2018-4,BMHC2019-9),项目参与者:陈磊。

摘　　要：背景:大量的动物和临床实验已证实,脂肪来源的血管基质成分局部移植可促进关节软骨修复。目的:探讨脱细胞真皮基质/生物矿化胶原一体化骨软骨支架复合脂肪来源血管基质成分构建组织工程软骨的可行性。方法:无菌条件下取出雌兔双侧腹股沟脂肪垫,利用酶消法获得血管基质组分,采用流式细胞仪检测其表面特异性蛋白,观察其成脂、成骨与成软骨分化能力。将脂肪来源血管基质成分(实验组)、含转化生长因子β3的脂肪间充质干细胞(软骨诱导组)、含体积分数10%胎牛血清的脂肪间充质干细胞(对照组)分别接种于脱细胞真皮基质/生物矿化胶原一体化支架上共培养,CCK8法检测细胞增殖,荧光显微镜下观察细胞数量,实时定量PCR检测软骨相关基因表达,二甲基亚甲基蓝比色法测定胞外基质中糖胺多糖含量。结果与结论:①流式细胞仪检测结果显示,脂肪来源血管基质成分高表达CD44、CD105与CD90,低表达CD14、CD19与CD45,脂肪来源血管基质成分具有向脂肪细胞、成骨细胞与软骨细胞分化的能力;②CCK8检测显示,随着培养时间的延长,脱细胞真皮基质/生物矿化胶原一体化支架上的3种细胞数量增加,其中实验组细胞数量高于其他两组(P<0.05);培养7 d荧光显微镜下观察可见,实验组中的细胞数量多于其他两组;③培养21 d的实时定量PCR检测显示,实验组中的Sox9、蛋白聚糖和Ⅱ型胶原mRNA表达量高于其他两组(P<0.05);④培养7,14,21 d的二甲基亚甲基蓝比色检测显示,实验组中的糖胺多糖含量高于其他两组(P<0.05);⑤结果表明,脂肪来源血管基质成分复合脱细胞真皮基质/生物矿化胶原一体化支架可以在体外有效支持软骨形成。BACKGROUND:A large number of animal and clinical experiments have confirmed that local transplantation of adipose-derived stromal vascular fraction can promote articular cartilage repair.OBJECTIVE:To explore the feasibility of constructing tissue-engineered cartilage with acellular dermal matrix/biomineralized collagen integrated bone cartilage scaffold combined with autologous adipose-derived stromal vascular fraction.METHODS:The groin fat pads of both sides of the female rabbit were taken out under sterile conditions,and the stromal vascular fraction was obtained by enzyme digestion.Flow cytometry was used to detect the surface specific protein and observe the ability of adipogenic,osteogenic and chondrogenic differentiation.Adipose-derived stromal vascular fraction(experimental group),adipose-derived mesenchymal stem cells containing transforming growth factor-β3(cartilage induction group)and adipose-derived mesenchymal stem cells containing 10%fetal bovine serum(control group)were co-cultured on acellular dermal matrix/biomineralized collagen scaffold.CCK8 assay was used to detect cell proliferation.The number of cells was observed under fluorescence microscope.Real time-quantitative PCR detection was used to detect cartilage related gene expression.The content of glycosaminoglycan in extracellular matrix was determined by dimethylmethylene blue colorimetry.RESULTS AND CONCLUSION:(1)The results of flow cytometry showed that the adipose-derived stromal vascular fraction had high expression of CD44,CD105 and CD90,and low expression of CD14,CD19 and CD45.Adipose-derived stromal vascular fraction had the ability to differentiate into adipocytes,osteoblasts and chondrocytes.(2)CCK8 assay showed that with the extension of culture time,the number of three kinds of cells on the acellular dermal matrix/biomineralized collagen scaffold increased,and the number of cells in the experimental group was higher than that in the other two groups(P<0.05).Under the fluorescence microscope,the number of cells in the experimenta

关 键 词：骨 材料 脂肪来源血管基质成分 脱细胞真皮基质 生物矿化胶原 一体化支架 软骨缺损 骨软骨 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]