Semiquantitative analysis of ECM molecules in the different cartilage layers in early and advanced osteoarthritis of the knee joint
Andreas Lahm1,2, Richard Kasch1, Eike Mrosek3,4, Heiko Spank1, Christoph Erggelet5, Jan Esser1 and Harry Merk1
1Section Research and Cell Biology of Department of Orthopaedics and Orthopaedic Surgery, Ernst-Moritz-Arndt University Hospital, Greifswald, Germany, 2Municipal Clinic Munich Bogenhausen University Hospital, Munich, Germany, 3Cartilage and Connective Tissue Research Laboratory, Department of Orthopaedic Surgery, Mayo Clinic College of Medicine, USA, 4Department of Trauma and Reconstructive Surgery, Schwarzwald-Baar-Klinikum Villingen-Schwenningen, VS-Schwenningen and 5Arthrose Clinic Zürich, Zürich, Switzerland.
Offprint requests to: Prof. Dr. med Andreas Lahm, Section of Orthopaedic Research and Cell Biology, Department of Orthopaedics and Orthopaedic Surgery, University Hospital of the Ernst-Moritz-Arndt University Greifswald, F.-v.-Sauerbruch Strasse, 17475 Greifswald, Germany. e-mail: alahm@uni-greifswald.de
Summary. The study was conducted to examine the expression of collagen type I and II in the different cartilage layers in relation to other ECM molecules during the progression of early osteoarthritic degeneration in human articular cartilage (AC).
Quantitative real-time (RT)-PCR and colorimetrical techniques were used for calibration of Photoshop-based image analysis in detecting such lesions.
Immunohistochemistry and histology were performed with 40 cartilage tissue samples showing mild (ICRS grade 1b) respectively moderate/advanced (ICRS grade 3a or 3b) (20 each) osteoarthritis compared with 15 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. The digitized images of histology and immunohisto-chemistry stains were analyzed with Photoshop software. T-test and Spearman correlation analysis were used for statistical analysis.
In the earliest stages of AC deterioration the loss of collagen type II was associated with the appearance of collagen type I, shown by increasing amounts of collagen type I mRNA. During subsequent stages, a progressive loss of structural integrity was associated with increasing deposition of collagen type I as part of a natural healing response. A decrease of collagen type II is visible especially in the upper fibrillated area of the advanced osteoarthritic samples, which then leads to an overall decrease. Analysis of proteoglycan showed losses of the overall content and a loss of the classical zonal formation. Correlation analysis of the proteoglycan Photoshop measurements with the RT-PCR revealed strong correlation for Safranin O and collagen type I, medium for collagen type II, alcian blue and glycoprotein but weak correlation with PCR aggrecan results.
Photoshop based image analysis might become a valuable supplement for well known histopathological grading systems of lesioned articular cartilage.
The evidence of collagen type I production early in the OA disease process coupled with the ability of chondrocytes to up-regulate collagen type II production suggests that therapeutic agents that suppress collagen type I production and increase collagen type II production may enable chondrocytes to generate a more effective repair response. Histol Histopathol 27, 609-615 (2012)
Key words: Cartilage, Osteoarthritis, RT-PCR, Photoshop image analysis
DOI: 10.14670/HH-27.609