Lee G M, Poole C A, Kelley S S, Chang J, Caterson B
Thurston Arthritis Research Center, University of North Carolina at Chapel Hill 27599-7280, USA.
Osteoarthritis Cartilage. 1997 Jul;5(4):261-74. doi: 10.1016/s1063-4584(97)80022-2.
To develop and test a simple enzymatic procedure for isolating chondrons, which consist of the chondrocytes and their surrounding pericellular microenvironment.
Chondrons were obtained by digesting adult human articular cartilage with a mixture of dispase and collagenase. Chondrons and chondrocytes were cultured in alginate beads, immunofluorescence labeled and examined by confocal microscopy.
Comparison of freshly isolated chondrons with cryostat sections of cartilage revealed that type VI collagen, type II collagen and aggrecan were retained, but fibronectin and a unique chondroitin sulfate epitope recognized by the antibody, 7D4, were lost. Comparison of enzymatic and mechanical homogenization methods revealed subtle changes in chondron morphology and retention of fibronectin in mechanically isolated chondrons. Average yield of enzyme-isolated chondrons was slightly lower than that of chondrocytes isolated by pronase and collagenase digestion, but was much greater than that reported for mechanically isolated chondrons. Enzyme-isolated chondron viability was greater than 80% 1 day after isolation, and continued to be above 80% through 7 weeks of alginate bead culture. Viability of isolated chondrocytes was initially greater than 80% but fell to 60-80% with time in culture. Chondrons and isolated chondrocytes had a similar division rate except osteoarthritic chondrons were significantly slower after 2 weeks in culture. Cell division was more rapid for nonosteoarthritic chondrons than for osteoarthritic ones.
Enzymatic isolation of chondrons is relatively simple, gives better yield and viability than mechanical isolation, but comparable yield and viability of traditional chondrocyte isolation. Enzymatic chondron isolation allows the effect of the in vivo-formed pericellular matrix on chondrocyte metabolism to be studied in vitro.
开发并测试一种用于分离软骨粒的简单酶法，软骨粒由软骨细胞及其周围的细胞周微环境组成。
用 dispase 和胶原酶混合物消化成人关节软骨获取软骨粒。将软骨粒和软骨细胞培养于藻酸盐珠中，进行免疫荧光标记并通过共聚焦显微镜检查。
将新鲜分离的软骨粒与软骨的冷冻切片比较发现，VI 型胶原、II 型胶原和聚集蛋白聚糖得以保留，但纤连蛋白和抗体 7D4 识别的一种独特硫酸软骨素表位丢失。酶解和机械匀浆方法的比较显示，软骨粒形态有细微变化，且机械分离的软骨粒中纤连蛋白得以保留。酶法分离软骨粒的平均产量略低于用链霉蛋白酶和胶原酶消化分离的软骨细胞，但远高于机械分离软骨粒的报道产量。酶法分离的软骨粒在分离后 1 天活力大于 80%，在藻酸盐珠培养 7 周期间持续高于 80%。分离的软骨细胞活力最初大于 80%，但随培养时间下降至 60 - 80%。软骨粒和分离的软骨细胞有相似的分裂率，除骨关节炎软骨粒在培养 2 周后明显较慢。非骨关节炎软骨粒的细胞分裂比骨关节炎软骨粒更快。
酶法分离软骨粒相对简单，产量和活力比机械分离更好，但与传统软骨细胞分离的产量和活力相当。酶法分离软骨粒可在体外研究体内形成的细胞周基质对软骨细胞代谢的影响。
