基于软骨脱细胞外基质微载体体外构建软骨类器官  

In vitro construction of cartilage organoids based on extracellular matrix microcarriers of cartilage

作　　者：蒋洪宇 刘伟 陈嘉杰 管延军 贾志博 高宇阳 范伟 汪爱媛 彭江 阳运康 Jiang Hongyu;Liu Wei;Chen Jiajie;Guan Yanjun;Jia Zhibo;Gao Yuyang;Fan Wei;Wang Aiyuan;Peng Jiang;Yang Yunkang(Department of Joint Surgery,Affiliated Hospital of Southwest Medical University,Luzhou 646000,China;Institute of Orthopedics,Fourth Medical Center,Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]西南医科大学附属医院骨与关节外科,泸州646000 [2]中国人民解放军总医院第四医学中心骨科医学部研究所,北京100853

出　　处：《中华创伤杂志》2024年第1期29-39,共11页Chinese Journal of Trauma

基　　金：国家重点研发计划(2022YFB3804303)。

摘　　要：目的探讨基于软骨脱细胞外基质微载体体外构建具有功能化和自我更新能力的软骨类器官。方法取新鲜的猪关节软骨,将部分仅粉碎处理的软骨微粒设置为天然软骨组;利用物理离心化学萃取相结合的脱细胞方法,制备粒径合适的细胞外基质(ECM)微载体,设置为微载体组。通过旋转式生物反应器将人脐带干间充质干细胞(hUCMSCs)和人软骨细胞(hCho)按照3∶1的比例与微载体负载,体外构建软骨类器官,将诱导不同时间的类器官分为诱导0、7、14及21 d组。4′,6-二脒基-2-苯基吲哚(DAPI)荧光染色观察、DNA定量评估微载体组和天然软骨组细胞残留情况;番红O、甲苯胺蓝染色观察微载体ECM保留情况,二甲氨基苯甲醛比色法测定微载体组和天然软骨组胶原蛋白含量;二甲基亚甲蓝(DMMB)比色法测定微载体组和天然软骨组糖胺聚糖(GAGs)含量。扫描电镜及能谱分析对微载体进行进一步表征;将添加体积分数10%胎牛血清(FBS)的杜尔伯克改良伊戈尔低糖培养基(DMEM)培养的hUCMSCs作为对照组,将微载体浸提液培养的hUCMSCs作为实验组,两组分别设置培养1、3、5 d共3个时间点的亚组,细胞增殖及毒性检测试剂盒(CCK-8)检测两组生物相容性。活死染色检测诱导0、7、14及21 d组的软骨类器官细胞活性,Ki67荧光染色鉴定诱导14 d软骨类器官的自我更新能力。RT-PCR测定诱导7、14、21 d组的软骨类器官,包括软骨形成相关标记物聚蛋白聚糖(ACAN)、Ⅱ型胶原(COL2A1)、性别决定区Y框蛋白9(SOX9)及软骨肥大、矿化相关标记物Ⅰ型胶原(COL1A1)、Runt相关转录因子2(RUNX2)、骨钙素(OCN)的表达水平;比色法和DMMB法测定诱导0、7、14及21 d组的类器官分泌胶原蛋白和GAGs能力。结果DAPI荧光染色结果显示,天然软骨组具有大量细胞核,而微载体组则基本无细胞核。微载体组DNA含量为(7.8±1.8)ng/mg,较天然软骨组的(526.7±14.7)ng/mg显著降Objective To study the in vitro construction of functional and self-renewing cartilage organoids based on cartilage acellular extracellular matrix(ECM)microcarriers.Methods Fresh porcine articular cartilage was taken.The merely crushed cartilage particles were set as natural cartilage group and ECM microcarriers of appropriate particle size,which were prepared by the acellular method of combining physical centrifugation and chemical extraction,were set as microcarrier group.Cartilage organoids were constructed by loading human umbilical cord mesenchymal stem cells(hUCMSCs)and human chondrocytes(hCho)with a ratio of 3∶1 with microcarriers through a rotating bioreactor.The organoids with different induction times were divided into 0-,7-,14-,and 21-day induction groups.The cell residues of the microcarrier group and natural cartilage group were evaluated by 4′,6-diaminidine 2-phenylindole(DAPI)fluorescence staining and DNA quantitative analysis.The retention of microcarrier components was observed by Safranin O and toluidine blue stainnings,and the collagen and glycosaminoglycan(GAGs)levels in the microcarrier group and the natural cartilage group were determined by colorimetric method and dimethyl-methylene blue(DMMB)method.The microcarriers were further characterized by scanning electron microscopy and energy dispersive spectroscopy.The hUCMSCs cultured with Dulbecco′s Modified Eagle′s Medium(DMEM)supplemented with fetal bovine serum(FBS)in a volume fraction of 10%was used as the control group and the hUCMSCs cultured with the microcarrier extract was used as the experimental group.Subgroups of hUCMSCs cultured at 3 time points:1,3 and 5 days were set up in the two groups separately.Cell Counting Kit 8(CCK-8)was used to detect the biocompatibility of the two groups.The cellular activity of the organoids of the 0-,7-,14-,and 21-day induction groups was detected by live/dead staining and the self-renewal ability of the cartilage organoids of the 14-day induced group was identified by Ki67 fluorescence staini

关 键 词：类器官 软骨 细胞外基质 组织工程 微载体 

分 类 号：R-33[医药卫生]