仿生大孔骨支架的制备及生物相容性评估  

Fabrication and biocompatibility assessment of the scaffold with biomimetic interconnected macropore structure

作　　者：李智 谭春华 蔡贤华 王华松 丁晓明[2] 赵艳红[3] Li Zhi;Tan Chunhua;Cai Xianhua;Wang Huasong;Ding Xiaoming;Zhao Yanhong(Department of Orthopedics, General Hospital of Chinese PLA Central Theater Command & Hubei Provincial Center for Bone Trauma Treatment Clinical Medicine, Wuhan 430070, Hubei Province, China;Fifth Department of Orthopedics, Rizhao Hospital of Traditional Chinese Medicine, Rizhao 276800, Shandong Province, China;Department of Orthodontics, Stomatological Hospital of Tianjin Medical University, Tianjin 300070, China)

机构地区：[1]解放军中部战区总医院骨科/湖北省骨创伤救治临床医学研究中心,湖北省武汉市430070 [2]日照市中医医院骨伤五科,山东省日照市276800 [3]天津医科大学口腔医院正畸科,天津市300070

出　　处：《中国组织工程研究》2019年第14期2223-2227,共5页Chinese Journal of Tissue Engineering Research

基　　金：湖北省自然科学基金(2017CFB569);项目负责人:李智;国家自然科学基金(31470937);项目参与者:丁晓明;国家自然科学基金(31300798);项目负责人:赵艳红;天津市卫计委基金资助项目(16KG114);项目负责人:赵艳红;天津市卫计委基金资助项目(15KG125);项目参与者:赵艳红~~

摘　　要：背景:骨组织工程的发展,为解决骨缺损修复提供了新手段,而其中的支架载体选材和结构构建是目前的热点问题。目的:采用石蜡微球沥滤技术制备具有仿生大孔结构的丝素蛋白/纳米羟基磷灰石支架,分析其细胞相容性。方法:以丝素蛋白和纳米羟基磷灰石为原料,采用石蜡微球沥滤技术制成具有仿生大孔结构的丝素蛋白/纳米羟基磷灰石支架,利用体式显微镜和扫描电镜观察支架结构,测量支架的弹性模量。将第3代兔脂肪间充质干细胞接种于丝素蛋白/纳米羟基磷灰石支架上,培养48 h后,LIVE/DEAD染色观察细胞活性;培养1周,苏木精-伊红染色观察细胞黏附情况;培养3 d,扫描电镜观察支架-细胞复合物;培养1,3,5,7 d,CCK-8法检测细胞增殖,以单纯细胞培养为对照。结果与结论:①体式显微镜下可见丝素蛋白/纳米羟基磷灰石支架呈乳白色,扫描电镜显示无论在支架横截面还是纵切面上,均可见均匀排列的大孔结构,连通性好,支架孔径为(362.23±26.52)μm,弹性模量为(54.93±5.44) kPa;②扫描电镜显示,脂肪间充质干细胞能够很好地在支架孔壁及连通孔隙内黏附和伸展,分泌大量细胞外基质,填充在丝素蛋白/纳米羟基磷灰石支架孔隙内;③苏木精-伊红染色显示,脂肪间充质干细胞均匀黏附在丝素蛋白/纳米羟基磷灰石支架孔隙内壁上,并且大量增殖;④LIVE/DEAD染色显示脂肪间充质干细胞活性良好;⑤CCK-8检测结果显示,脂肪间充质干细胞在丝素蛋白/纳米羟基磷灰石支架可良好地生长与增殖;⑥结果表明,丝素蛋白/纳米羟基磷灰石支架具有良好的孔径与细胞相容性。BACKGROUND: Development of bone tissue engineering provides a new method to solve bone defect repair. Scaffold material and structure construction are issues of concern. OBJECTIVE: To fabricate a silk fibroin and hydroxyapatite scaffold with biomimetic interconnected macropores structure using the paraffin-sphere leaching technology and to evaluate its possibility of cytocompatibility. METHODS: The scaffold with biomimetic interconnected macropores structure was made by the paraffin-sphere leaching technology. The structure of scaffold was observed by the stereomicroscope and scanning electron microscope. The pore size and elasticity modulus were calculated. Passage 3 rabbit adipose-derived mesenchymal stem cells were seeded into the scaffold. The cell viability was detected by live/dead staining at 48 hours after culture. The cell adhesion was observed by hematoxylin-eosin staining at 1 week of culture. The scaffold-cell composite was observed under scanning electron microscope at 3 days of culture. The cell proliferation was detected by the cell counting-kit 8 assay at 1, 3, 5 and 7 days of culture. Those cells cultured alone were considered as control group. RESULTS AND CONCLUSION:(1) Stereomicroscope showed the ivory silk fibroin/hydroxyapatite scaffold. Scanning electron microscope revealed pore structures in cross-section and longitudinal-section with good connectivity. The scaffold pore size was (362.23±26.52)μm and the elasticity modulus was (54.93±5.44) kPa.(2) Scanning electron microscope showed that adipose-derived mesenchymal stem cells adhered and stretched well in the pore wall and connected pore, secreted abundant extracellular matrix, and filled in the pores of silk fibroin/hydroxyapatite scaffold.(3) Hematoxylin-eosin staining results found that adipose-derived mesenchymal stem cells evenly adhered onto the inner wall of silk fibroin/hydroxyapatite scaffold, and proliferated well.(4) Live/dead staining revealed a good viability of adipose-derived mesenchymal stem cells.(5) Cell counting-kit 8

关 键 词：骨缺损 丝素蛋白/纳米羟基磷灰石支架 生物材料 丝素蛋白 羟基磷灰石 脂肪干细胞 细胞黏附 羟基磷灰石类 细胞粘附 生物相容性材料 组织工程 

分 类 号：R459.9[医药卫生—治疗学] R918.08[医药卫生—临床医学]