软骨细胞与异体软骨微粒脱细胞基质体外相容性的研究  

Experimental study of compatibility between chondrocytes and allogenic cartilage microparticle acellular tissue matris in vitro

作　　者：韩雪峰[1] 杨大平[1] 郭铁芳[1] 方冬云[1] 徐学武[1] 张颖[2] 

机构地区：[1]哈尔滨医科大学附属第二临床医学院整形外科,150086 [2]哈尔滨医科大学附属第二临床医学院病理科,150086

出　　处：《中华医学杂志》2005年第27期1895-1898,共4页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30170970);国家杰出青年基金资助项目(30325042);黑龙江省自然基金重点项目(ZJY031);黑龙江省杰出青年基金资助项目(JC0207)

摘　　要：目的探索以异体软骨微粒脱细胞基质为支架构建组织工程化软骨。方法多步骤酶法将绵羊关节软骨制成微粒脱细胞基质,行大体、光镜(苏木素伊红、胶原、甲苯胺蓝染色)、扫描电镜观察,同时测定微粒的胶原、氨基葡聚糖、DNA含量。异体绵羊关节软骨细胞分离、体外扩增,并与软骨微粒脱细胞基质混合体外培养0～35d,倒置显微镜及扫描、透射电镜观察,同时测定羟脯氨酸,氨基葡聚糖,DNA含量及细胞黏附率。结果软骨微粒脱细胞基质只含有基质成分,直径0.100～0.154mm,胶原,氨基葡聚糖及DNA含量分别为204.4±3.1μg/mg,18.3±2.0μg/mg和0.042±0.013μg/mg。异体软骨细胞紧紧围绕于异体软骨微粒脱细胞基质四周,二者形成的复合物中胶原、氨基葡聚糖及DNA含量于第7d与0d相比具有统计学意义(P<0.05),分别与14d(胶原、DNA)和21d(氨基葡聚糖)达高峰,随后维持在高水平。细胞黏附率为92%。结论软骨细胞与异体软骨微粒脱细胞基质有良好的生物相容性,为组织工程方法再造软骨提供又一支架材料。Objective To construct tissue engineered cartilage using cartilage microparticle acellular tissue matrix(CMACTM)as scaffold. Methods To determine the content of hydroxyproline, glycosaminoglycan and DNA of CMACTM prepared from sheep′s articular cartilage with multistep enzymic method, and to analyze CMACTM with gross observation, histology and scanning electron microscopy. Allogenic chondrocytes were mixed with CMACTM and cultured in vitro from 0 to 35 days. Observations through inverted microscope, scanning and transmission electron microscope, quantifications of hydroxyproline, glycosaminoglycan and DNA in the composite, cells adhesion rate were applied to analyze the results. Results The diameter of CMACTM was 0.100-0.154 mm, which contain extracellular matrix only. Hydroxyproline, glycosaminoglycan and DNA quantifications in CMACTM were 204.374±3.120 μg/mg, 18.302±2.037 μg/mg and 0.042 ±0.013 μg/mg respectively. Allogenic chondrocytes enclosed CMACTM tightly, hydroxyproline, glycosaminoglycan and DNA quantifications in the composite of the two formers increased with difference on 7th day compared with that on 0 day, reached to the peaks on 14th day(hydroxyproline , DNA)and on 21st day(glycosaminoglycan), and retained at a high level on the following days. Cells adhesion rate was 92%. Conclusion Allogenic CMACTM possessed satisfactory biocompatibility for chondrocytes and provided a new scaffold for cartilage tissue engineering.

关 键 词：脱细胞基质 体外相容性 微粒 氨基葡聚糖 DNA含量 组织工程化软骨 苏木素-伊红 甲苯胺蓝染色 扫描电镜观察 软骨细胞分离 透射电镜观察 异体软骨细胞 组织工程方法 同时测定 倒置显微镜 生物相容性 关节软骨 体外扩增 体外培养 

分 类 号：R318.0[医药卫生—生物医学工程]