成骨微环境仿生支架用于骨组织工程的可行性分析  

Preparation and Cytocompatibility of Osteogenic Microenvironmental Biomimetic Scaffold

作　　者：慈政 张起新 王雅慧 张沛灵 张伟[1,3,4] 周广东[1,3,4] CI Zheng;ZHANG Qixin;WANG Yahui;ZHANG Peiting;ZHANG Wei;ZHOU Guangdong(Plastic Surgery Research Institute,Weifang Medical College,Weifang 261042,China;Department of Health Care,Weifang People's Hospital,Weifang 261000,China;Department of Plastic and Reconstructive Surgery,Shanghai Key Laboratory of Tissue Engineering,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China;National Tissue Engineering Center of China,Shanghai 200241,China)

机构地区：[1]潍坊医学院整形外科研究所,山东省潍坊市261042 [2]潍坊市人民医院,山东省潍坊市261000 [3]上海交通大学医学院附属第九人民医院整复外科,上海市组织工程研究重点实验室,上海市200011 [4]组织工程国家工程研究中心,上海市200241

出　　处：《组织工程与重建外科杂志》2020年第6期437-441,共5页Journal of Tissue Engineering and Reconstructive Surgery

基　　金：国家重点研发计划(2017YFC1103900);国家自然科学基金项目(81871502,81701843)。

摘　　要：目的探索使用羟基磷灰石(HA)、脱钙骨基质(DBM)粉末及明胶(GT)制备成骨微环境仿生支架的可行性及其细胞相容性和成骨诱导活性。方法将骨组织粉碎后经脱脂、脱钙、脱细胞处理,并使用DNA定量检测DBM脱细胞效果,再将DBM粉末与一定比例羟基磷灰石及明胶一同制备成为成骨微环境仿生支架。比较不同浓度比例的HA-DBM-GT支架的孔径、孔隙率。取羊骨髓基质干细胞,接种于合适浓度比例的HA-DBM-GT支架,体外培养,于第1、3、7天进行大体、电镜观察,DNA定量及活死细胞染色;于第1、7、14天进行成骨分化相关基因检测。结果DNA定量显示脱钙骨基质粉末的制备过程可有效去除骨组织中的细胞;随着脱钙骨及羟基磷灰石比例的提高,支架孔径明显增大;各组HA-DBM-GT支架孔隙率均可达到84%以上;活死细胞染色及DNA定量证实细胞可在支架上黏附、增殖;qt-PCR检测证实支架材料可诱导干细胞特异性表达成骨基因。结论成功制备了一种成骨微环境仿生支架,并证实其具有良好的材料表征、细胞相容性及成骨诱导活性,可用于组织工程骨构建。Objective To explore the feasibility,cytocompatibility and osteogenic induction activity of osteogenic microenvironmental biomimetic scaffold made with hydroxyapatite(HA),demineralised bone matrix(DBM)and gelatin(GT).Methods The bone tissue was defatted,decalcified and decellularized after completely pulverization,and the effect of DBM decellularization was detected by DNA quantification.And then combined with a certain proportion of hydroxyapatite(HA)and gelatin(GT)to prepare osteogenic microenvironmental biomimetic scaffold.The pore size and porosity of scaffold with different concentration ratios were compared,and the cytocompatibility analysis and osteogenic gene detection were performed.Goat bone marrow stromal stem cells were cultured in suitable HA-DBM-GT scaffold.Gross observation,SEM observation,DNA quantification and Living&Dead cell staining were carried out at day 1,3 and 7.Osteogenic differentiation related genes were tested at day 1,7 and 14.Results DNA quantification showed that the preparation process of decalculated bone matrix powder could effectively remove the cells in bone tissue.With the increase of the proportion of decalcified bone and hydroxyapatite,the diameter of the scaffold increased obviously.The porosity of HA-DBM-GT stent in each group was above 84%.Live&Dead staining and DNA quantification confirmed that the cells could adhere and proliferate on the scaffold.qt-PCR assay confirmed that scaffold materials could induce specific expression of osteogenic genes in stem cells.Conclusion A biomimetic osteogenicmicroenvironment scaffold has been successfully prepared and proved to have good material characterization,cytocompatibility and osteogenic induction activity,which can be used in the construction of tissue-engineered bone.

关 键 词：脱钙骨基质 明胶 支架材料 骨再生 组织工程骨 

分 类 号：R318.08[医药卫生—生物医学工程]