第1代骺板软骨细胞离心管内培养生成软骨组织的观察  

Cartilage tissue regeneration from passage-one epiphyseal plate chondrocytes pellet cultured in centrifuge tube

作　　者：杨国勇[1] 李箭[2] 

机构地区：[1]四川省骨科医院五病区运动创伤科,四川省成都市610041 [2]四川大学华西医院骨科,川省成都市610041

出　　处：《中国组织工程研究与临床康复》2008年第42期8239-8242,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:骺板软骨细胞聚集体离心管内培养能生成具有良好组织结构和较高含量的软骨基质特异性成分,然而应用这一技术一般均以原代软骨细胞为种子细胞。观察第1代骺板软骨细胞高数量聚集离心管培养生成软骨组织的可行性及其生物学特点,并对此技术进行初步评价。方法:实验于2006-07/2007-04在四川大学华西医院科研楼组织工程实验室完成。自4周龄新西兰大白兔骺板按三步酶消化法分离软骨细胞,行单层传代培养,取第1代细胞,以5×105个细胞/管接种于15mL离心管内,离心沉降聚集,连续培养2周。大体、倒置相差显微镜观察软骨细胞形态;光学显微镜观察软骨的组织学结构;透射电镜观察了解细胞及细胞外基质的超微结构。结果:①第1代骺板软骨细胞经离心管培养能形成软骨,随时间的延长,生成的软骨组织体积逐渐增大,到第2周时直径为1.5～2.0mm,高为0.8～1.4mm。②外周组织结构类似于骺软骨的生发层或软骨膜,由多层细胞构成,随时间的延长而减少。其中心为肥大软骨细胞,且随培养时间的延长而增多。2周时,软骨组织甲苯胺蓝染色呈强的红色异染反应,Ⅱ型胶原免疫组织化学染色呈强阳性,番红"O"染色呈强阳性。③细胞核内以常染色质为主,且分布均匀,细胞周围及细胞间质结构稀疏、排列紊乱,缺乏紧密的胶原网络结构。结论:高数量第1代骺板软骨细胞聚集离心管培养能形成软骨组织,有独特的超微结构,富含软骨特异性蛋白基质。AIM： Epiphyseal plate chondrocytes, almost primary cells, cultured in centrifuge tube can generate cartilage matrix specific components, which possess good structure and high content. This study observes the feasibility of cartilaginous tissue formation and the biological characteristics of the regenerated cartilaginous tissue from the pellets of large number of passage one epiphyseal plate chondrocytes cultured in centrifuge tube, and evaluate this technique of cartilaginous tissue regeneration. METHODS： The experiment was carried out in the Laboratory of Tissue Engineering, West China Hospital, Sichuan University between July 2006 to April 2007. Chondrocytes were isolated with three-step enzyme digestion method from the epiphyseal plate of four-week old New Zealand rabbits, and were passaged in monolayer. Passage one chondrocytes were collected and the pellet mass of 5 × 10^5 chondrocytes by centrifugation were cultured in a plastic centrifuge tube （15 mL） for 2 weeks continuously. The pellet morphology and histological structure were investigated grossly and with light and inverted microscopy, respectively. The ultrastructures of the cell and the extracellular matrix were investigated by transmission electron microscope. RESULTS： （1）There was cartilaginous tissue regenerated from passage one epiphyseal plate chondrocytes cultured in centrifuge tube, which gradually became mature in structure along with the time elapsing, and with the diameter of 1.5 2.0 rnm, and height of 0.8-1.4 mm at the time of 2 weeks.（2）The periphery of the cartilaginous tissue resembled the germinal layer of epiphyseal plate or the cartilage membrane, consisting of several cellular layers that decreased with time. The central part was composed of hypertrophic chondrocytes, for which the number increased with time. The cartilaginous tissue at the time of 2 weeks＇ cultivation appeared strongly positive staining of collagen type II immunohistochemistry and safranine ＂O＂, as well as strongly metachromatic to toluidin

关 键 词：骺板 组织工程 离心管培养 组织工程化软骨 

分 类 号：R318[医药卫生—生物医学工程]