明胶-海藻酸盐复合微球与凝胶修复软骨损伤  

Gelatin-alginate composite microspheres and gels for cartilage damage repair

作　　者：蒋浩然 高健明 林万程 李婷[2] 李获 王鹏[2] 冯靖 孟昊业 彭江 丁立祥 Jiang Haoran;Gao Jianming;Lin Wancheng;Li Ting;Li Huo;Wang Peng;Feng Jing;Meng Haoye;Peng Jiang;Ding Lixiang(Department of Spine Surgery,Beijing Shijitan Hospital Affiliated to Capital Medical University,the Ninth School of Clinical Medicine,Peking University,Beijing 100038,China;Institute of Orthopedics,Department of Orthopedics,Army Key Laboratory of Warfare Trauma,Beijing Key Laboratory of Orthopedic Regenerative Medicine PLA,Beijing 100853,China)

机构地区：[1]北京大学第九临床医学院,首都医科大学附属北京世纪坛医院脊柱外科,北京市100038 [2]中国人民解放军骨科研究所,全军骨科战创伤重点实验室,骨科再生医学北京市重点实验室,北京市100853

出　　处：《中国组织工程研究》2022年第28期4452-4457,共6页Chinese Journal of Tissue Engineering Research

基　　金：北京市医院管理中心重点医学专业发展计划(ZYLX202135),项目负责人:丁立祥。

摘　　要：背景:软骨损伤的微创治疗对于微载体的要求较高,需要其有较高的细胞相容性、较强的细胞黏附力、较好的力学性能与低免疫原性。同时,临床使用条件相比实验室更加苛刻,在微创或注射使用微载体时液态微载体要明显优于固态微载体。目的:制备一种全新的高分子有机微载体,以用于修复软骨缺损。方法:通过明胶与液体石蜡(W/O)混合搅拌的化学乳化法制备浓度为6%的明胶微球,冻干后用无水乙醇处理固定,再使用紫外交联法固定,电镜观察微球形态。配置浓度为7%的海藻酸钠凝胶,与明胶微球混合孵育2 h,制备明胶-海藻酸盐复合凝胶。将脂肪间充质干细胞悬液滴入明胶-海藻酸盐复合凝胶中孵育24 h,滴入5%CaCl_(2)溶液中充分交联,制备含细胞明胶-海藻酸盐复合微球。采用CCK-8法检测无细胞明胶-海藻酸盐复合微球浸提液的细胞毒性;利用死活染色观察含细胞明胶-海藻酸盐复合微球的细胞活性;将约1 mL明胶-海藻酸盐复合凝胶吸入10 mL针管进行注射,对未注射与注射1,3次的凝胶进行光镜观察。结果与结论:①扫描电镜下可见,明胶微球孔隙相对均一且表面有层次感,微球粒径大多分布在180-500μm之间;②死活染色显示,培养24 h含细胞明胶-海藻酸盐复合微球中的细胞生长良好,携带细胞数量多且分布均匀,培养1,3,7 d后的细胞活性均在90%以上;③CCK-8检测显示,无细胞明胶-海藻酸盐复合微球浸提液无明显的细胞毒性;④与未注射时相比,注射1,3次后明胶-海藻酸盐复合凝胶中的明胶微球形态无变化,显示明胶-海藻酸盐复合凝胶体力学性能良好;⑤结果表明,联合使用含细胞明胶-海藻酸盐复合微球及含细胞明胶-海藻酸盐复合凝胶可使凝胶发挥组织胶的作用,利于材料紧贴目标区域。BACKGROUND:Minimally invasive treatment of cartilage injury is highly demanding for microcarriers,which need to have high cytocompatibility,strong cell adhesion,better mechanical properties with low immunogenicity.Simultaneously,the clinical use conditions are more demanding compared to the laboratory,and liquid microcarriers are significantly better than solid microcarriers when they are used minimally invasively or for injection.OBJECTIVE:To prepare a brand-new polymer organic microcarrier for repairing cartilage.METHODS:Gelatin microspheres at a concentration of 6%were prepared by chemical emulsification of gelatin mixed and stirred with liquid paraffin(w/O),fixed by lyophilization followed by absolute ethanol treatment,then fixed using ultraviolet cross-linking.The microsphere morphology was observed by electron microscopy.The gelatin-alginate composite gel was prepared by dispensing the gel with sodium alginate at a concentration of 7%and incubating with gelatin microspheres for 2 hours.Cell-containing gelatin-alginate composite microspheres were prepared by dropping adipose mesenchymal stem cell suspension into gelatin-alginate composite gel and incubating for 24 hours,and fully crosslinking by dropping into 5%CaCl_(2) solution.The cytotoxicity of the extracts from cellfree gelatin-alginate composite microspheres was examined by CCK-8 assay.The cytocompatibility of cell-containing gelatin-alginate composite microspheres was observed by using live&dead staining.Approximately 1 mL of gelatin-alginate composite gel was aspirated into a 10 mL needle tube for injection,and the gel not injected versus injected 1 and 3 times was observed by light microscopy.RESULTS AND CONCLUSION:(1)As observed by scanning electron microscope,the pores of gelatin microspheres were relatively uniform and the surface was hierarchical,and most of the particles were distributed between 180 and 500μm in size.(2)As indicated by the live&dead staining,the cells in gelatinalginate composite microspheres containing cells grew well and car

关 键 词：明胶 海藻酸盐 复合微球 复合凝胶 高分子有机材料 化学乳化法 细胞相容性 光交联 软骨 

分 类 号：R459.9[医药卫生—治疗学] R322.72[医药卫生—临床医学]