激光微孔化脱细胞骨软骨支架的制备及表征  

Preparation and characterization of laser microporous acellular osteochondral scaffolds

作　　者：刘雪剑[1,2,3] 刘士臣 孙百川[1,2,3] 张凯红 孟昊业 王玉[2,3] 黄绍代 鲁长风[2,3] 王冲 余文 荆晓光 赵跃 杨建华[1,4] 彭江 Liu Xue-jian;Liu Shi-chen;Sun Bai-chuan;Zhang Kai-hong;Meng Hao-ye;Wang Yu;Huang Shao-dai;Lu Chang-feng;Wang Chong;Yu Wen;Jing Xiao-guang;Zhao Yue;Yang Jian-hua;Peng Jiang(First Affiliated Hospital of Jiamusi University,Jiamusi 154007,Heilongjiang Province,China;Institute of Orthopedics,General Hospital of Chinese PLA,Beijing 100853,China;Beijing Key Laboratory of Orthopaedic Regeneration Medicine,Beijing 100853,China;People＇s Hospital of Longgang District,Shenzhen 518172,Guangdong Province,China)

机构地区：[1]佳木斯大学,佳木斯大学附属第一医院,黑龙江省佳木斯市154007 [2]中国人民解放军总医院骨科研究所,北京市100853 [3]骨科再生医学北京市重点实验室,北京市100853 [4]深圳市龙岗区人民医院,广东省深圳市518172

出　　处：《中国组织工程研究》2018年第18期2836-2842,共7页Chinese Journal of Tissue Engineering Research

基　　金：北京市科技专项(Z161100005016059);国家自然科学基金(81572148);国家重点研发计划(2016YFC1102104);佳木斯大学科学技术重点项目(基础研究类)(12Z1201507);黑龙江省大学生创新创业训练计划项目(201610222058);黑龙江省教育厅基础研究项目(2016-KYYWF-0548)

摘　　要：背景:如何制备出既具有软骨源性成分又具有良好初始力学强度的支架,是组织工程软骨研究的方向。目的:制备微孔化脱细胞骨软骨支架,评价其力学性能及细胞相容性。方法:取猪膝关节骨软骨,采用激光微孔化技术制作微孔化骨软骨支架,随后利用化学方法制作微孔化脱细胞骨软骨支架,扫描电镜观察支架结构,检测支架的压缩模量。分别采用微孔化脱细胞骨软骨支架浸提液(实验组)、含体积分数10%胎牛血清的L-DMEM(对照组)培养骨髓间充质干细胞,培养5 d内CCK-8法检测细胞增殖。将骨髓间充质干细胞接种于微孔化脱细胞骨软骨支架上,共培养28 d内进行苏木精-伊红染色与甲苯胺蓝染色,观察细胞生长情况。结果与结论:(1)扫描电镜:微孔化脱细胞骨软骨支架表面平整,孔与孔之间分布均匀,纵切面孔深达软骨下骨;(2)力学性能:微孔化脱细胞骨软骨支架的平均压缩模量为0.77 MPa,接近于正常骨软骨压缩模量(1.15 MPa);(3)CCK-8实验:实验组培养1,2,3,4,5 d的细胞增殖与对照组无差异;(4)共培养实验:苏木精-伊红染色和甲苯胺蓝染色显示,培养1 d后,大量骨髓干细胞在微孔化脱细胞骨软骨支架孔隙中生长,培养7 d后有少量细胞贴孔壁生长,孔隙中有少量细胞生长;培养21 d后,孔壁上有少量细胞贴壁生长,孔隙中有少量细胞生长;培养28 d后,孔壁上有少量细胞贴壁增殖,孔隙中有大量细胞成团增殖;(5)结果表明:微孔化脱细胞骨软骨支架具有良好的力学性能及细胞相容性。BACKGROUND： Preparing a scaffold with cartilage derived components and good initial mechanical strength is the direction of tissue engineering cartilage research. OBJECTIVE： To prepare porous acellular osteochondral scaffolds, and to explore their mechanical properties and cell compatibility. METHODS： Osteochondral bone from the porcine knee joint was taken, and then porous osteochondral scaffolds were made by laser microporation technology. Subsequently, the scaffolds were decellularized chemical methods. Scaffold structure was observed by scanning electron microscopy, and the compression modulus of the scaffolds was determined. Bone marrow mesenchymal stem cells were cultured in L-DMEM containing 10% fetal bovine serum （control group） and cultured in the medium extract of porous acellular osteochondral scaffolds （experimental group）, respectively. Cell proliferation was detected by cell counting kit-8 method within 5 days of culture. Bone marrow mesenchymal stem cells were seeded on the porous acellular osteochondral scaffolds, and within 28 days of co-culture, cell growth was observed by hematoxylin-eosin staining and toluidine blue staining. RESULTS AND CONCLUSION： （1） Observation under scanning electron microscopy： The porous acellular osteochondral scaffolds had the smooth surface with evenly distributed pores. The pores of the scaffold extended longitudinally into the subchondral bone. （2） Mechanical properties： The average compressive modulus of porous acellular osteochondral scaffolds was 0.77 MPa, which was close to the compression modulus of the normal cartilage （1.15 MPa）. （3） Cell counting kit-8 test： There were no differences in cell proliferation between the control and experimental groups at 1, 2, 3, 4 and 5 days of culture. （4） Cell-scaffold co-culture： A large amount of bone marrow mesenchymal stem cells were observed to be adherent to the scaffold after 1 day of culture through hematoxylin-eosin and toluidine blue staining. However, as time went on, a

关 键 词：软骨组织工程 脱细胞 骨软骨支架 共培养 骨髓间充质干细胞 激光微孔化 α-Gal抗原检测 生物相容性 生物材料 

分 类 号：R318[医药卫生—生物医学工程]