兔软骨细胞与脱细胞软骨基质的生物相容性

Cytocompatibility of acellular cartilage matrix with chondrocyte in rabbits

目的:将体外培养的兔软骨细胞种植到脱细胞软骨基质上,进行细胞形态学观察、细胞黏附及增殖活性的测定,检测制备的脱细胞软骨基质与软骨细胞的相容性,分析脱细胞软骨基质作为软骨组织工程支架的可能性。方法:实验于2005-03/09在哈尔滨医科大学附属第二医院科研中心完成。选取兔龄6个月的清洁级健康雄性日本大耳白兔,麻醉后无菌条件下显露兔双侧膝关节,切取股骨远端和胫骨近端关节软骨,经脱细胞处理制备成脱细胞软骨基质微粒作为软骨支架,并分离培养软骨细胞。将培养至第2代的软骨细胞用于实验,随机分为脱细胞基质组和空白对照组,每组复孔为6孔。脱细胞基质组将脱细胞基质颗粒单层铺满培养板孔底,接种软骨细胞。空白对照组单纯接种软骨细胞。倒置显微镜及扫描电镜观察软骨细胞与脱细胞软骨基质的黏附情况,通过四甲基偶氮唑盐法检测细胞接种2,6,12,24h细胞黏附性,测定细胞接种后1,3,5,7d时细胞的增殖活性,测定细胞培养1,2,3d时上清液中的羟脯氨酸含量。结果:①软骨细胞在脱细胞基质上黏附的形态学观察:扫描电镜观察脱细胞基质颗粒呈不规则多角形,软骨巢内的软骨细胞消失,表面粗糙不平。倒置相差显微镜下观察软骨细胞刚开始为小圆形,折光性较强,接种2h后有少量细胞开始黏附于脱细胞基质上,随时间的延长细胞黏附数量增加。②接种后不同时间点两组软骨细胞黏附情况的测定结果:与空白对照组比较,接种2h时脱细胞基质组软骨细胞黏附性明显降低(P<0.05),而在接种6,12,24h时两组基本相似(P>0.05)。③接种后不同时间点两组软骨细胞增殖能力的比较:接种1,3,5,7d时,脱细胞基质组软骨细胞增殖活性分别高于空白对照组21.4%,32.7%,32.5%,25.4%,差异显著(P<0.05)。④接种后不同时间点两组软骨细胞羟脯氨酸含量的比较:接种后第1,2天,脱细胞基质组上清液中的羟脯氨酸含量与空白对照组接近(P>0.05)。接种第3天时,脱细胞基质组羟脯氨酸含量高于空白对照组20.28%,差异显著(P<0.05)。结论:实验制备的脱细胞软骨基质能够明显刺激增殖软骨细胞,且与软骨细胞具有良好的相容性,为组织工程支架的应用提供了新的材料。

AIM： To observe the cells morphology and detect the adherence and proliferation of cells after the chondrocytes cultured in vitro were implanted into the acellular cartilage matrix of rabbits, and evaluate the compatibility of prepared acellnlar cartilage matrix with chondrocytes and analyze the possibility of acellular cartilage matrix taken as cartilage tissue engineering scaffolds. METHODS： The experiment was conducted at the Science Research Center of Second Affiliated Hospital of Harbin Medical University between March and September 2005. The healthy male Japanese rabbits of 6-month old, cleaning grade were selected to expose bilateral knee joints in the sterile condition after anesthesia. The articular cartilages were cut down from far-end of femur and near-end of tibia, and acellular cartilage matrix particulates were prepared as cartilage scaffolds. After isolated and cultured, the second-generation chondrocytes were applied to the experiment and divided randomly into 2 groups： control group and acellular cartilage matrix group, with sextuple wells in each. The acellular cartilage matrix particulates were confluent cultured in the medium plate, and chondrocytes were implanted, which was performed in the control group only. The cell adhering of acellular cartilage matrix and chondrocytes were observed with inverted microscope and scanning electron microscope. The absorption coefficient of the ceil adhering at 2,6,12 and 24 hours and proliferation at 1, 3,5 and 7 days implantation were detected by methyl thiazolyl tetrazolium assay. The content of hydroxyproline in the serum was measured at 1,2 and 3 days culture. RESULTS：①The morphology of chondrocytes adhering to the acelluar matrix： In the scanning electron microscope, acellular cartilage matrix particulates exhibited abnormal polygon and cell disappeared in chondrocytes nest, with the rough and uneven surace. The result of inverted phase contrast microscope showed chondrocytes were initially micro-circle and strong in refraction, but some began to adhere to the acellular matrix after 2-hour implantation, and increased with time. ②The adhering of the chandrocytes at different times after implantation： The adhering of chondrocytes in acellular cartilage matrix group was markedly decreased compared with control group at 2-hour （P 〈 0.05）. There was no significant difference at 6,12 and 24 hours between two groups （P 〉 0.05）. ③The proliferation of chandro-cytes at different times after implantation： At 1,3,5 and 7 days, the proliferation in the acellular cartilage matrix group were 21.4%, 32.7%, 32.5% and 25.4% higher than those in the control group, with the significant difference （P 〈 0.05）.④The content of hydroxyproline at different times： Hydroxyproline content in the acellular cartilage matrix group was similar with that of control group at the 1^st and 2^nd days （P 〉 0.05）, but increased by 20.28% at the 3^rd day, compared with the control group （P 〈 0.05）. CONCLUSION： The prepared acellular cartilage matrix can strikingly stimulate the growth of ehondrocytes and has good compatibility with ehondrocytes, which provide a new material for application of tissue engineering scaffolds.