骨髓干细胞在羟基磷灰石涂层钛片上向软骨的分化  

Chondrogenic differentiation of bone marrow stem cells on a hydroxyapatite coated titanium plate

作　　者：覃李玲 严妍 余卓 覃巧玲 黎伟 刘莉 Qin Liling;Yan Yan;Yu Zhuo;Qin Qiaoling;Li Wei;Liu Li(Department of Stomatology,Third Clinical College of China Three Gorges University,Central Hospital of China Gezhouba(Group)Corporation,Yichang 443002,Hubei Province,China)

机构地区：[1]三峡大学第三临床医学院.葛洲坝集团中心医院口腔科,湖北省宜昌市443002

出　　处：《中国组织工程研究》2019年第1期35-40,共6页Chinese Journal of Tissue Engineering Research

基　　金：宜昌市医疗卫生科研项目(A18-301-27);项目负责人:刘莉;三峡大学青年科学基金项目(KJ2017A009);项目负责人:黎伟~~

摘　　要：背景:羟基磷灰石具有良好的生物相容性和生物活性,具有促进干细胞黏附、增殖和骨诱导分化的作用。目的:观察羟基磷灰石涂层钛片对大鼠骨髓间充质干细胞增殖、软骨性分化的影响。方法:采用电沉积法在钛金属基底表面沉积羟基磷灰石涂层(电沉积时间分别为30 min,1.5 h),用场发射扫描电子显微镜观察涂层形态,X射线衍射分析其化学组成。取4只3周龄SD雄性大鼠(购于湖北省实验动物研究中心)双侧股骨的骨髓,采用全骨髓贴壁法培养骨髓间充质干细胞,将第3代细胞接种于有涂层钛片及没有涂层纯钛片上,并进行4周的成软骨诱导分化。通过CCK-8试剂盒分析羟基磷灰石涂层对大鼠骨髓间充质干细胞增殖的影响,二甲基亚甲基蓝比色法检测各组细胞糖胺多糖含量,RT-PCR法检测各组细胞软骨相关基因的表达,评价各组骨髓间充质干细胞的成软骨能力。结果与结论:(1)羟基磷灰石涂层均匀分布在钛基底上,大小结构较一致,X射线衍射图谱与羟基磷灰石标准峰基本吻合;(2)羟基磷灰石涂层促进骨髓间充质干细胞增殖,羟基磷灰石涂层亦表现了比空白钛片组具有更好的生物活性;(3)羟基磷灰石涂层组较空白钛片组产生了更多的糖胺多糖,差异有显著性意义(P <0.01);(4)羟基磷灰石涂层组软骨相关基因(Sox9、多聚蛋白聚糖、Ⅱ型胶原、Ⅹ型胶原)的相对表达量显著高于空白钛片组(P <0.05),电沉积时间为1.5 h的羟基磷灰石涂层组的Ⅱ型胶原表达量明显高于电沉积时间30 min羟基磷灰石涂层组和空白钛片组(P <0.01);(5)结果表明,应用电化学沉积技术可以在钛片上制备均匀的羟基磷灰石涂层,且该涂层可以促进大鼠骨髓间充质干细胞增殖,具有良好的生物相容性,有利于骨髓间充质干细胞向软骨方向分化。BACKGROUND:Hydroxyapatite has good biocompatibility and bioactivity and can promote adhesion,proliferation and chondrogenic differentiation of stem cells.OBJECTIVE:To investigate the effect of hydroxyapatite coated titanium plate on chondrogenic differentiation of bone marrow mesenchymal stem cells.METHODS:Hydroxyapatite coating was fabricated on the titanium surface by electrochemical deposition technique(deposition time:3 minutes and 1.5 hours)and characterized by field emission scanning electron microscopy for coating morphology and by X-ray diffraction for the chemical composition of the coating.Bone marrow samples from four Sprague-Dawley rats of 3 weeks old(purchased from Hubei Provincial Laboratory Animal Center,Wuhan,China)were taken to culture bone marrow mesenchymal stem cells using whole bone marrow adherent method.Passage 3 cells were inoculated onto the titanium plate with or without coating followed by 4 weeks chondrogenic induction.For assessing the ability of chondrogenesis,cell counting kit-8 assay was applied to test the cell proliferation;dimethylmethylene blue colorimetric assay was used to detect the content of glycosaminoglycans;and RT-PCR was used to test the expression of chondrocyte specific genes.RESULTS AND CONCLUSION:(1)The hydroxyapatite coating was evenly distributed on the titanium plate and its structure was uniform.The X-ray diffraction peaks of hydroxyapatite powder generally matched the characteristic X-ray diffraction pattern of hydroxyapatite.(2)The hydroxyapatite-coated titanium plate improved the proliferation of bone marrow mesenchymal stem cells and showed better bioactivity compared to the non-coated titanium plate.(3)The glycosaminoglycan content was significantly higher in the hydroxyapatite coating group than the non-coated group(P<0.01).(4)The RT-PCR results showed higher expression levels of chondrocyte specific genes(SOX9,type II collagen,type X collagen,aggrecan)in the hydroxyapatite coating group than the non-coated group(P<0.05).The expression of type II collagen

关 键 词：钛片 羟基磷灰石 骨髓间充质干细胞 糖胺多糖 Ⅱ型胶原 软骨分化 电化学沉积 骨髓 间质干细胞 钛 羟基磷灰石类 电化学技术 组织工程 

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