酶消化结合组织块培养法用于颞下颌关节盘组织工程细胞扩增的初步研究

Preliminary Research on Obtaining Seeded Cells for Temporomandibular Joint Disc Tissue Engineering Using Modified Explant Culture

目的采用酶消化结合组织块培养法对山羊颞下颌关节(temporomandibular joint,TMJ)盘细胞进行体外培养和扩增,探索TMJ关节盘细胞体外培养及扩增的新方法。方法在无菌条件下,切取一月龄山羊TMJ关节盘,剪至1.0 mm^3的碎块,用0.25%胰酶、0.01%Ⅰ型胶原酶消化关节盘组织块,将消化好的组织块置入6孔板中培养。在倒置显微镜下连续观察细胞的形态变化及贴壁率,甲苯胺蓝染色、Ⅰ型胶原免疫组化染色进行细胞鉴定,测定其生长曲线。结果原代培养的关节盘纤维软骨细胞4天可观察到贴壁细胞,7天贴壁细胞逐渐增多,第10天时细胞彼此相连,铺满平底,细胞以梭形为主,部分多角形。传代后12小时贴壁率达90%,大部分为多角形,4～5天即可长满瓶底。甲苯胺蓝染色可见异染颗粒,胶原免疫组化染色胞浆内可见棕黄色颗粒。结论酶消化结合组织块培养法培养的山羊TMJ关节盘细胞具有较强的增殖能力,可作为TMJ关节盘组织工程中获取大量原代细胞的实用方法。

Objective To search a novel method suitable for proliferation of TMJ disc cells, and to observe the biological characteristics of cultured TMJ disc cells （fibroehondrocytes）. Methods The small pieces （ 1.0 mm^3 in size） of isolated and chopped TMJ disc tissues, which were dissected from two one-month-goats under sterile conditions,were digested with 0. 25% trypsin and 0. 01% type I collagenase,then put into 6-well plate to be cultured with DMEM. The phase contrast microscope was used to observe the morphological changes and attachment efficiency of TMJ cells. Immunohistochemical staining for type I collagen and toluidine blue staining for proteoglyeans were used for extracellular matrix characterization, and the cell growth curve was plotted. Results At the fourth day of culture, primary TMJ disc cells （fibroehondro- cytes） could be seen in adherence to the dishes. After the 7th days, the number of adhesive fibroehondrocytes was increased. On the day 10th,the cells were spindle-shaped and some were polygon-shaped, started to contact with each other to form a monolayer ceils covering the bottom of the 6-well plate. At the 12th hour after passage,the seeding efficiency of the fibroehondrocytes was 90%. From the 5th to 6th day after passage, the bottom of the culture bottle was filled with these cells. Immunohistoehemical staining of type I collagen and toluidine blue staining exhibited positive resultd. Conclusion The goat TMJ disc cells presented proliferous ability in vitro culture using enzyme digestion with explant culture method and .can be a practical way to obtain numberous primary cells for the TMJ disc tissue engineering.