负载抗结核药物与骨形态发生蛋白2缓释微球的3D打印人工骨能促进骨髓间充质干细胞成骨  

Three-dimensional printed artificial bone loaded with anti-tuberculosis drugs and bone morphogenetic protein 2 sustained-release microspheres can promote osteogenesis of bone marrow mesenchymal stem cells

作　　者：刘昌昊 郑建平[2] 施建党[3] 朱禧[3] 周占文 张旭 Liu Changhao;Zheng Jianping;Shi Jiandang;Zhu Xi;Zhou Zhanwen;Zhang Xu(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750000,Ningxia Hui Autonomous Region,China;Department of Orthopedic Trauma,General Hospital of Ningxia Medical University,Yinchuan 750000,Ningxia Hui Autonomous Region,China;Department of Spinal Orthopedics,General Hospital of Ningxia Medical University,Yinchuan 750000,Ningxia Hui Autonomous Region,China)

机构地区：[1]宁夏医科大学临床医学院,宁夏回族自治区银川市750000 [2]宁夏医科大学总医院创伤骨科,宁夏回族自治区银川市750000 [3]宁夏医科大学总医院脊柱骨科,宁夏回族自治区银川市750000

出　　处：《中国组织工程研究》2021年第28期4447-4453,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(8176090169),项目负责人:施建党。

摘　　要：背景:脊柱结核病灶清除术后局部有效药物浓度不足及残留骨缺损的修复是目前脊柱结核治疗的难题,新型抗结核药物联合骨形态发生蛋白2成为解决该问题的新思路,可有效促进骨缺损修复。目的:观察PA-824、莫西沙星和吡嗪酰胺/骨形态发生蛋白2/纳米羟基磷灰石抗结核人工骨对兔骨髓间充质干细胞成骨分化和黏附能力的影响。方法:提取并纯化兔骨髓间充质干细胞;以纳米羟基磷灰石为材料,制备含抗结核药物PA-824、莫西沙星和吡嗪酰胺与骨形态发生蛋白缓释微球的3D打印人工骨,并与第3代骨髓间充质干细胞共培养,作为实验组;将骨形态发生蛋白2与骨髓间充质干细胞共培养,作为阳性对照组;将骨髓间充质干细胞单独培养,作为空白对照组。培养第1,3,7,14,21天检测兔骨髓间充质干细胞增殖、成骨分化及细胞黏附效果,以评估该人工骨对兔骨髓间充质干细胞成骨分化的影响,以CCK-8法检测细胞增殖和细胞毒性,以钙结节沉积程度及碱性磷酸酶表达活性评价成骨分化效果,荧光定量PCR和Western blot法检测成骨相关基因和蛋白表达水平,扫描电镜观察细胞黏附特性。结果与结论:①新合成的抗结核人工骨微观结构呈现良好的空间性,利于细胞附着;②CCK-8法检测结果表明各组细胞均增殖良好,其中实验组、阳性对照组持续14 d呈现增殖状态,空白对照组7 d后趋于平稳状态,说明此人工骨具有良好的生物相容性;③碱性磷酸酶活性呈先上升后降低的变化趋势,至14 d达到高峰,在第7,14天时阳性对照组碱性磷酸酶活性高于实验组(P<0.05),但第21天时实验组高于阳性对照组,而空白对照组在各时间点变化不大;④茜素红染色实验显示实验组、阳性对照组均有钙结节形成,两组钙结节数量和大小差别不明显;两组成骨相关基因和蛋白(Runx2和Ⅰ型胶原蛋白)表达均高于空白对照组(P<0.05);⑤扫�BACKGROUND:Insufficient local effective drug concentration after debridement of spinal tuberculosis lesions and repair of residual bone defects are the current problems in the treatment of spinal tuberculosis.New anti-tuberculosis drugs combined with bone morphogenetic protein 2(BMP-2)have become a new idea to solve this problem,which can effectively promote bone defect repair.OBJECTIVE:To observe the effect of PA-824,moxifloxacin and pyrazinamide(PaMZ)/BMP-2/nano-hydroxyapatite(nHA)anti-tuberculosis artificial bone on osteogenic differentiation and adhesion of rabbit bone marrow mesenchymal stem cells.METHODS:Rabbit bone marrow mesenchymal stem cells were extracted and purified.The nHA was used to fabricate the three-dimensional printed artificial bone containing anti-tuberculosis drugs PA-824,moxifloxacin,pyrazinamide and BMP sustained-release microspheres(PaMZ/BMP-2/nHA),and co-cultured with the third generation of bone marrow mesenchymal stem cells as the experimental group.BMP-2 was co-cultured with bone marrow mesenchymal stem cells as a positive control group.Bone marrow mesenchymal stem cells were cultured separately as blank control group.The proliferation,osteogenesis and cell adhesion effects of rabbit bone marrow mesenchymal stem cells were respectively tested at 1,3,7,14,and 21 days of culture to evaluate the influence of the artificial bone on the osteogenic differentiation of rabbit bone marrow mesenchymal stem cells.The cell proliferation and cytotoxicity were detected by CCK-8 assay.The degree of calcium nodule deposition and alkaline phosphatase expression activity were measured to evaluate the influence of osteogenic differentiation.Fluorescence quantitative PCR and western blot assay were used to detect the expression levels of osteogenicgenes and proteins,respectively.The adhesion characteristics of cells were observed by scanning electron microscopy.RESULTS AND CONCLUSION:(1)The newly synthesized anti-tuberculosis artificial bone showed good spatial microstructure,which was conducive to cell

关 键 词：PA-824 莫西沙星 吡嗪酰胺 骨形态发生蛋白2 纳米羟基磷灰石 抗结核人工骨 间充质干细胞 成骨分化 

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