猪脱细胞软骨基质生物相容性及细胞毒性评价系统的建立  

Porcine decellular endplate cartilage matrix:biocompatibility assessment and establishment of a cytotoxicity evaluation system

作　　者：王效 徐宏光[1] 肖良 刘晨[1] 金中行 沈阳 Wang Xiao;Xu Hong-guang;Xiao Liang;Liu Chen;Jin Zhong-xing;Shen Yang(Department of Spinal Surgery,the First Affiliated Hospital of Wannan Medical College,Wuhu 241001,Anhui Province,China)

机构地区：[1]皖南医学院弋矶山医院脊柱外科,安徽省芜湖市241001

出　　处：《中国组织工程研究》2018年第22期3539-3544,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81572185);安徽省自然科学基金面上项目(1708085MH185);安徽省自然科学基金青年项目(1708085QH205);安徽省自然科学基金青年项目(1808085QH275);安徽省科技厅对外科技合作项目(1704e1002229);皖南医学院中青年科研基金项目(WK2016F02)~~

摘　　要：背景:相对于其他用于支架材料的高分子化合物,脱软骨细胞基质能够模拟出更接近原生理状态下的细胞外环境,由其制备的椎间盘支架弹性模量也更加接近软骨组织。目的:制备脱细胞软骨基质,并从细胞和活体两方面评价其生物相容性。方法:将猪软骨粉碎后,经胰酶、核酶及TritonX-100处理得到脱细胞软骨基质。(1)细胞毒性实验:将脱细胞基质溶于醋酸,制备成脱细胞基质膜。将SD大鼠腰椎终板软骨干细胞接种于脱细胞基质膜上,培养1-5 d,采用CCK-8检测软骨干细胞在基质膜上的增殖情况,评价细胞毒性分级;(2)动物体内毒性实验:将含有脱细胞基质的醋酸(实验组)与醋酸(对照组)分别注射至SD大鼠背部皮下,形成皮丘,注射后36 h内观察大鼠体温与皮丘直径变化。结果与结论:(1)细胞毒性实验:倒置显微镜下可见,腰椎终板软骨干细胞接种于脱细胞基质膜2 d即贴壁,呈梭形生长,接种5 d内的细胞相对增殖率均在80%以上,细胞毒性分级为0至1级;(2)动物体内毒性实验:注射后即刻,两组大鼠背部均可见皮丘形成,注射36 h内皮丘内溶液被完全吸收,并且对照组吸收速度快于实验组,两组皮丘边缘均无红肿现象;注射36 h内,实验组与对照组大鼠体温均未超过37.5℃;(3)结果表明:脱细胞软骨基质具有很好的生物相容性,符合生物支架选材标准。BACKGROUND： Compared to other high-molecular compounds used for scaffold materials, the decellular endplate cartilage matrix（DEPM） can simulate the extracellular environment closer to the original physiological state, and the elastic modulus of the intervertebral disc scaffold made of DEPM therefore is closer to that of the cartilage tissue. OBJECTIVE： To prepare DEPM and explore its biocompatibility at cellular level and in vivo. METHODS： Porcine endplate cartilage samples were crushed and digested with Trypsin combined with ribozyme and Triton X-100 to obtain DEPM.（1） Cytotoxicity test： After dissolved in acetic acid, the DEPM was prepared into the acellular matrix membrane. Cartilage stem cells from the lumbar endplate of Sprague-Dawley rats were seeded onto the acellular matrix membrane, and cell proliferation was detected using cell counting kit-8 after 1-5 days of culture. Then cytotoxicity of the cells was graded.（2） Toxicity test in vivo： Acetic acid containing DEPM（experimental） or not（control） was subcutaneously injected into the back of Sprague-Dawley rats to form a bump. Rat body temperature and bump＇s size were observed within 36 hours after injection. RESULTS AND CONCLUSION：（1） Cytotoxicity test： Under the inverted microscope, cartilage stem cells from the lumbar endplate were adherent within 2 days after implantation, and grew in a fusiform shape. The relative cell growth rate was over 80% within 5 days after implantation, and the cell toxicity grade was level 0 to 1.（2） Toxicity test in vivo： A bump was formed on the rat back in the two groups immediately after injection, and completely disappeared within 36 hours after injection. Moreover, the absorption rate in the control group was faster than that in the experimental group. No red and swelling bump occurred in the two groups. the body temperature of the rats in the two groups did not exceed 37.5 ℃ within 36 hours after injection. To conclude, the DEPM has good biocompatibility, which is in line w

关 键 词：生物相容性材料 细胞外基质 椎间盘 细胞毒性试验 免疫 组织工程 脱细胞软骨基质 终板软骨干细胞 生物相容性评价 细胞毒性 国家自然科学基金 生物材料 

分 类 号：R318[医药卫生—生物医学工程]