Section of Orthopaedic Research and Cell Biology, Department of Orthopaedics and Orthopaedic Surgery, University Hospital of the Ernst-Moritz-Arndt University Greifswald, Greifswald, Germany.
Arch Orthop Trauma Surg. 2010 Apr;130(4):557-64. doi: 10.1007/s00402-009-0981-y. Epub 2009 Oct 17.
The different cartilage layers vary in synthesis of proteoglycan and of the distinct types of collagen with the predominant collagen Type II with its associated collagens, e.g. types IX and XI, produced by normal chondrocytes. It was demonstrated that proteoglycan decreases in degenerative tissue and a switch from collagen type II to type I occurs. The aim of this study was to evaluate the correlation of real-time (RT)-PCR and Photoshop-based image analysis in detecting such lesions and find new aspects about their distribution.
We performed immunohistochemistry and histology with cartilage tissue samples from 20 patients suffering from osteoarthritis compared with 20 healthy biopsies. Furthermore, we quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorimetrically. Using Adobe Photoshop the digitized images of histology and immunohistochemistry stains of collagen type I and II were stored on an external data storage device. The area occupied by any specific colour range can be specified and compared in a relative manner directly from the histogram using the "magic wand tool" in the select similar menu. In the image grow menu gray levels or luminosity (colour) of all pixels within the selected area, including mean, median and standard deviation, etc. are depicted. Statistical Analysis was performed using the t test.
With the help of immunohistochemistry, RT-PCR and quantitative RT- PCR we found that not only collagen type II, but also collagen type I is synthesized by the cells of the diseased cartilage tissue, shown by increasing amounts of collagen type I mRNA especially in the later stages of osteoarthritis.
A decrease of collagen type II is visible especially in the upper fibrillated area of the advanced osteoarthritic samples, which leads to an overall decrease. Analysis of proteoglycan showed a loss of the overall content and a quite uniform staining in the different zones compared to the healthy cartilage with a classical zonal formation. Correlation analysis of the proteoglycan Photoshop measurements with the RT-PCR using Spearman correlation analysis revealed strong correlation for Safranin O and collagen type I, medium for collagen type II and glycoprotein but weak correlation between PCR aggrecan results.
Photoshop-based image analysis might become a valuable supplement for well known histopathological grading systems of lesioned articular cartilage.
不同的软骨层在蛋白聚糖和不同类型的胶原合成方面存在差异，正常软骨细胞主要合成胶原 II 及其相关胶原，如胶原 IX 和 XI。研究表明，退变组织中蛋白聚糖减少，胶原 II 向 I 型转换。本研究旨在通过实时（RT）-PCR 和基于 Photoshop 的图像分析来评估这些病变，并发现其分布的新方面。
我们对 20 例骨关节炎患者和 20 例健康活检患者的软骨组织样本进行了免疫组织化学和组织学检查。此外，我们利用实时（RT）-PCR 对胶原 I 和 II 以及聚集蛋白聚糖的基因表达进行了定量。通过比色法测量蛋白聚糖含量。使用 Adobe Photoshop，将组织学和免疫组织化学染色的胶原 I 和 II 的数字化图像存储在外部数据存储设备上。可以使用“魔术棒工具”在“选择相似”菜单中指定并直接从直方图中以相对方式指定任何特定颜色范围的面积。在“图像生长”菜单中，显示所选区域内所有像素的灰度级或亮度（颜色），包括平均值、中位数和标准差等。使用 t 检验进行统计分析。
通过免疫组织化学、RT-PCR 和定量 RT-PCR，我们发现不仅胶原 II，而且胶原 I 也由病变软骨组织的细胞合成，特别是在骨关节炎后期，胶原 I mRNA 的量增加。
在晚期骨关节炎样本的上层纤维状区域，胶原 II 的减少尤为明显，导致整体减少。蛋白聚糖分析显示，与具有典型层状结构的健康软骨相比，整体含量减少，不同区域的染色均匀。通过 Spearman 相关分析对 Photoshop 测量的蛋白聚糖与 RT-PCR 进行相关性分析，发现 Safranin O 与胶原 I 之间存在强相关性，与胶原 II 和糖蛋白之间存在中等相关性，而与 PCR 聚集蛋白聚糖之间存在弱相关性。
基于 Photoshop 的图像分析可能成为有价值的补充，用于现有的关节软骨病变的组织病理学分级系统。
