Banking of osteochondral allografts. Part I. Viability assays adapted for osteochondrol and cartilage studies
Banking of osteochondral allografts. Part I. Viability assays adapted for osteochondrol and cartilage studies
复制标题
骨软骨同种异体移植物的储存。
作者：
L. Csönge;Daniel Bravo;H. Newman;T. Rigley;E. Conrad;A. Bakay;D. Strong;S. Pellet
本研究的目的是采用一种可靠的，可重复的和简单的可行性测定软骨和骨软骨研究。先前的测定（放射性同位素摄取、基质成分评估、组织学方法、耗氧量等）复杂、费力、耗时或难以解释。选择MTT测定法是因为其已广泛且成功地用于不同的细胞和组织研究，但尚未在人固体关节软骨上发表。新鲜完整的软骨样品的人距骨进行了测试，以研究测定。还通过荧光染料组合测试了MTT测定的可靠性。MTT测定法基于活软骨细胞的线粒体酶从甲基四唑盐产生紫色甲瓒色素。还研究了反应的酶动力学，因为在软骨的情况下它是未知的。在用甲基溶纤剂提取后，可以通过分光光度法测量形成的色素的量。颜色密度与线粒体酶活性成比例，反映了活软骨细胞的数量。确定了最佳试剂浓度、活检大小和孵育时间。软骨重量与色素生成活性之间存在线性关系。在阴性对照中观察到9.8%的非特异性反应。并对反应的酶动力学进行了研究。最高达0.1%（w/v）的MTT曲线符合米氏动力学。计算了反应的米氏常数（2835 ± 130 μM）、最大速度（36 ± 3.2 × 10−5μMsec−1）和速度常数（1.27 ± 0.2 × 10−7sec−1）。后者是每种组织类型的重要标志物。还通过包含1 μg/ml碘化丙啶（PI）和4 μM/ml SYTO-16染色剂的荧光染料组合来评估和计算软骨的活力。PI染色死细胞（红色荧光），SYTO-16染色活细胞（绿色荧光）。染色可以同时可视化，并且可以通过图像分析软件从保存的图像文件计算活/死比率。MTT法是一种简单、廉价、有效、可靠、可重复、灵敏的软骨活性检测方法。MTT还原法和染色法具有较好的确证性。
The aim of this study was to adapt a reliable, reproducible and simple viability assay for cartilage and osteochondral studies. The previous assays (radioisotope uptake, assessment of matrix components, histological methods, oxygen consumption etc.) were complex, laborious, time consuming or suffer from difficulty of interpretation. MTT assay was chosen because it has been widely and successfully used in different cell and tissue studies, but has not been published on human solid articular cartilage. Fresh intact cartilage samples of human tali were tested to investigate the assay. The reliability of the MTT assay was also tested by an fluorescent dye combination. The MTT assay is based on the production of purple formazan pigment from methyltetrazolium salt by the mitochondrial enzymes of viable chondrocytes. The enzyme kinetics of the reaction was also investigated because it was unknown in the case of cartilage. The amount of pigment formed can be measured by spectrophotometry after extraction by methyl cellosolve. The color density is proportional to mitochondrial enzyme activity, reflecting the number of viable chondrocytes. The optimal reagent concentration, biopsy size, and incubation period were established. There is a linear relationship between the cartilage weight and the pigment production activity. A 9.8% nonspecific raction was observed in the negative controls. The enzyme kinetics of the reaction was also investigated. The MTT clevage up to 0.1% (w/v) follows the Michaelis kinetics. We calculated the Michaelis constant (2835 ± 130 μM), the maximal velocity (36 ± 3.2 × 10−5μMsec−1) and the velocity constant (1.27 ± 0.2 × 10−7sec−1) of the reaction. The latter is a significant marker for each tissue type. The viability of cartilage was also assessed and calculated by a fluorescent dye combination comprising 1 μg/ml propidium iodide (PI) and 4 μM/ml SYTO-16 stains. The PI stains dead cells (red fluorescence), the SYTO-16 stains live cells (green fluorescence). The staining can be visualised simultaneously, and the live/dead ratio can be calculated by image analysis software from saved image files. The MTT assay is a simple, non-expensive, efficient, reliable, reproducible, sensitive viability test for cartilage studies. The MTT reduction assay and the staining method were corrobative.
影响因子：
2.2
作者：
GREEN, LM;READE, JL;WARE, CF
通讯作者：
WARE, CF
影响因子：
6.4
作者：
LEVITZ, SM;DIAMOND, RD
通讯作者：
DIAMOND, RD