Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16 h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5 h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

背景胶原酶经常用于从关节软骨中分离软骨细胞。然而，这种酶在建立原代人软骨细胞培养物中的充分性仍然未知。方法将全关节置换术患者（16髋、8膝）股骨头或胫骨平台刮取的软骨切片用0.02%胶原酶IA消化16 h，加（N=19）或不加（N= 5  ）预处理 。 -0.4%链霉蛋白酶E处理1.5小时。比较两组之间的软骨细胞产量和活力。软骨细胞表型通过 II 型胶原蛋白与 I 型胶原蛋白的表达比来确定。用光学显微镜监测培养的软骨细胞的形态。结果经过链霉蛋白酶E预处理的软骨比未经预处理的软骨产生显着更高的软骨细胞（3,399 ± 1,637 个细胞/mg 湿软骨 vs. 1,895 ± 688 个细胞/mg 湿软骨；P = 0.0067 ）  。前一组的细胞活力也显着高于后一组（94%±2% vs. 86%±6%；P  = 0.03）。当单层培养时，经过链霉蛋白酶E预处理的软骨细胞在单个平面上生长，呈圆形，而另一组细胞在多平面上生长，呈不规则形状。在用链霉蛋白酶E预处理的软骨中分离的细胞中，II型胶原蛋白与I型胶原蛋白的mRNA表达比为13.2±7.5，表明典型的软骨细胞表型。结论胶原酶 IA 不足以建立原代人软骨细胞培养物。在应用胶原酶 IA 之前，必须先用链霉蛋白酶E处理软骨。