Ruthenium hexaammine trichloride chemography for aggrecan mapping in cartilage is a sensitive indicator of matrix degradation
Ruthenium hexaammine trichloride chemography for aggrecan mapping in cartilage is a sensitive indicator of matrix degradation
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DOI：
10.1177/002215540004800108
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发表时间：
2000-01-01
影响因子：
3.2
通讯作者：
Hunziker, EB
中科院分区：
文献类型：
--
作者：
Buschmann, MD;Maurer, AM;Hunziker, EB
我们开发了一种新的定量组织化学方法来绘制关节软骨中聚集蛋白的含量，并将其应用于软骨降解模型。三氯化六胺钌（RHT）与软骨的主要蛋白聚糖组分聚集蛋白形成共沉淀，以前发现它是一种很好的固定剂，有助于维持软骨细胞的形态。我们发现，在组织切片中没有任何放射性的情况下，通过类似于以前用于组织中痕量金属离子定位的自动金相方法，这些rht聚集蛋白沉淀物在自放射成像乳剂上产生阳性化学信号。通过利用成人关节软骨中聚集蛋白浓度固有的深度依赖性，我们证明了rht衍生化学成像在放射自显影乳剂上产生的银颗粒密度与局部测量的聚集蛋白浓度相关，这是由来自这些不同软骨深度的微解剖组织的二甲基亚甲基蓝测定法确定的。为了探索这种新方法在监测组织降解方面的益处，软骨外植体在培养过程中使用白细胞介素-1 （IL-1）降解，或在培养后使用软骨素酶和角化酶消化。与对照组相比，来自这些样品的RHT化学信号显示出对聚集蛋白损失的敏感性，并区分了由于添加外源酶而导致的细胞介导的损失（IL-1）。rht衍生的化学颗粒密度代表了一种有趣的新的定量工具，用于软骨生理学和关节炎的组织学分析。
We developed a new quantitative histochemical method for mapping aggrecan content in articular cartilage and applied it to models of cartilage degradation. Ruthenium hexaammine trichloride (RHT) forms co-precipitates with aggrecan, the main proteoglycan component of cartilage, and was previously found to be a good fixative in aiding the maintenance of chondrocyte morphology. We show that these RHT-aggrecan precipitates generate a positive chemographic signal on autoradiographic emulsions, in the absence of any radioactivity in the tissue section, via a process similar to the autometallographic process used previously for localization of trace metals ions in tissues. By exploiting the inherent depth-dependence of aggrecan concentration in adult articular cartilage, we demonstrated that the density of silver grains produced by RHT-derived chemography on autoradiographic emulsions correlated with locally measured aggrecan concentration as determined by the dimethylmethylene blue assay of microdissected tissue from these different depths of cartilage. To explore the benefits of this new method in monitoring tissue degradation, cartilage explants were degraded during culture using interleukin-1 (IL-1) or digested after culture using chondroitinase and keratinase. The RHT chemographic signal derived from these samples, compared to controls, showed sensitivity to loss of aggrecan and distinguished cell-mediated loss (IL-1) from degradation due to addition of exogenous enzymes. The RHT-derived chemographic grain density represents an interesting new quantitative tool for histological analysis of cartilage in physiology and in arthritis.