构建兔软骨脱落细胞三维支架及与干细胞相容性评价的实验研究  

Experimental study to construct the three-dimensional scaffold of rabbit cartilage-shedding cells and evaluate their compatibility with stem cells

作　　者：徐菊菊 谢燕燕 郭志义 马雨凯 王霖虹 杨美荣 邓昭玲 华宝来 闫振宇 XU Juju;XIE Yanyan;GUO Zhiyi;MA Yukai;WANG Linhong;YANG Meirong;DENG Zhaoling;HUA Baolai;YAN Zhenyu(North China University of Science and Technology,Tangshan 063000,China;Affiliated Hospital ofNorth China University of Science and Technology,Tangshan 063000,China;Department of ExperimentalMedical Technology,School of Public Health,North China University of Science and Technology,Tangshan063000,China;Beijing Shijitan Hospital,Capital Medical University,Beijing 100015,China)

机构地区：[1]华北理工大学,河北唐山063000 [2]华北理工大学附属医院,河北唐山063000 [3]华北理工大学公共卫生学院实验医学技术系,河北唐山063000 [4]首都医科大学附属北京世纪坛医院,北京100015

出　　处：《中国实验动物学报》2024年第8期1012-1022,共11页Acta Laboratorium Animalis Scientia Sinica

基　　金：2019年河北省政府资助临床医学优秀人才培养项目。

摘　　要：目的制备不同浓度的兔软骨脱落细胞支架,并评估其理化性能和干细胞的相容性,为软骨修复提供实验依据。方法Percoll密度分离法培养骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),并进行流式细胞学鉴定和成骨、成软骨分化能力检测。从兔膝、股关节中切取软骨片,进行物理粉碎、反复冻融以及各种酶混合消化脱细胞。为比较并观察不同浓度脱细胞支架的理化性能差异,设计3组支架,浓度分别为100%(A组)、50%(B组)、30%(C组),每组3只。将第3代PKH26示踪的BMSCs接种于最适浓度支架上培养1周,观察细胞生长情况。结果流式技术检测BMSCs表面抗原,CD44、CD90呈阳性表达,CD45阴性表达;成骨诱导茜素红染色可见红色钙结节;软骨诱导阿利新蓝染色见软骨结节呈蓝色;3组支架经苏木素-伊红(HE)、甲苯胺蓝染色后未观察到明显细胞形态。样本脱细胞后的DNA浓度与未脱支架的指标均值差异具有显著性(P<0.05)。糖胺聚糖含量较正常值稍偏低。3组支架两两比较孔径、吸水膨胀率、孔隙率、支架断裂强度、杨氏模量差异具有显著性(P<0.05);干细胞与支架共培养后细胞附着良好。结论Percoll密度分离法可获取高纯度的兔BMSCs;应用混合脱细胞方法脱细胞较彻底。B组支架可作为构建组织工程软骨修复的最适方案。体外培养的BMSCs在B组支架生长良好。Objective To prepare decellularized scaffolds from rabbit cartilage at various concentrations and assess their physicochemical properties and compatibility with stem cells to provide an experimental basis for cartilage repair.Methods Bone marrow mesenchymal stem cells(BMSCs)were cultured using the Percoll density gradient separation method,and this was followed by flow cytometric analysis and testing of their osteogenic and chondrogenic differentiation capabilities.Cartilage pieces were excised from rabbit knees and hip joints and subjected to physical crushing,repeated freeze-thaw cycles,and mixed enzymatic digestion for decellularization.To compare and observe the physicochemical properties of the decellularized scaffolds at different concentrations,three groups of scaffolds(labelwd A,B,and C)were designed with concentrations of 100%,50%and 30%,with three replicates each.Third-generation PKH26-labeled BMSCs were seeded onto optimally concentrated scaffolds and cultured for 1 week to observe cell growth.Results Flow cytometry detected BMSC surface antigens with positive expression of CD44 and CD90 and negative expression of CD45.Osteogenic induction stained with alizarin red showed red calcific nodules,and chondrogenic induction stained with alcian blue showed blue cartilaginous nodules.No apparent cell morphology was observed in the three groups of scaffolds stained with hematoxylin-eosin,and toluidine blue.There was a significant difference in DNA concentration between decellularized samples and non-decellularized scaffolds(P<0.05).The content of glycosaminoglycans was slightly lower than the normal values.Significant differences were observed between the three groups of scaffolds in terms of pore size,water absorption,porosity,tensile strength,and Young’s modulus(P<0.05).After co-cultivation of stem cells with the scaffolds,cell adhesion was found to be good.Conclusions Percoll density gradient separation can obtain high-purity rabbit BMSCs,and the mixed decellularization method is superior.Group B scaffold

关 键 词：骨髓间充质干细胞 组织工程 软骨脱细胞支架 共培养 

分 类 号：Q95-33[生物学—动物学]