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. 1999 Jun;58(6):357–365. doi: 10.1136/ard.58.6.357

In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage

A Freemont 1, R Byers 1, Y Taiwo 1, J Hoyland 1
PMCID: PMC1752894  PMID: 10340960

Abstract

OBJECTIVES—Chondrocytic matrix metalloproteinases (MMPs) are believed to be important in osteoarthritic cartilage degradation. The cartilage lesion of osteoarthritis (OA) is focal and often progressive. During its development chondrocytes differentially up and down regulate production of mRNA for individual MMPs. This observation has potential implications for understanding the disease processes that lead to progressive cartilage loss in OA and designing appropriate targeted treatment. The complex regulation of MMP mediated effects means there is a pressing need to establish whether visualisation of MMP mRNA or protein equates to enzyme activity. The technique of in situ zymography (ISZ) offers a way of examining diseased human tissue for in vivo production of an excess of degrading enzyme over inhibitor. The primary objective of this study was to assess, and if positive follow, collagen II degrading activity in cartilage during development of the OA lesion. A secondary objective was to assess whether there was any correlation between sites of collagen II degrading activity and expression of the collagenase (MMP-13), recently implicated in type II collagen degredation in this lesion.
METHODS—Biopsied human normal and osteoarthritic cartilage, showing various degrees of damage, was examined by in situ zymography, with and without enzyme inhibitors, to establish sites of type II collagenase activity. Paired samples were probed for MMP-13 mRNA using 35S-labelled oligonucleotide probes. Comparative analyses were performed.
RESULTS—In situ zymography showed collagen II degrading activity over chondrocytes only in osteoarthritic cartilage. Distribution and amount varied with the extent of cartilage damage and position of chondrocytes, being greatest in deep cartilage and in cartilage lesions where fissuring was occurring. The enzyme causing the degradation behaved as a matrix metalloproteinase. MMP-13 mRNA expression codistributed with the type II collagenase activity.
CONCLUSION—In OA, chondrocytes can degrade type II collagen. The type II collagen degrading activity varies in site and amount as the cartilage lesion progresses and throughout codistributes with MMP-13 mRNA expression.



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Figure 1  .

Figure 1  

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Sections of articular surface reacted by radioactive ISH for MMP13 mRNA. (A)-(D) The cartilage surface is at the top. The darker staining area at the bottom is calcified cartilage (zone 4). The four sets of figures are grade 0, 1, 2, 3 lesions (A to D respectively). The figure on the left is probed with antisense oligonucleotides and that on the right with sense probes. Hybridisation has been demonstrated by autoradiography, and is seen as black grains over the cells. These slides have been exposed for 70 days so that the distribution could be demonstrated at low magnification (×10) to show spatial variation and relation to cartilage damage in each grade. (E) Both the photomicrographs in (E) are examples of the reaction product at 35 days viewed by dark field illumination. This is the length of exposure used for the measurements given in table 1. Magnification × 100.

Figure 2  .

Figure 2  

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Representative figures of the collagenase II ISZ to illustrate the appearances of the ISZ reaction. All the figures are from the same grade 1 cartilage specimen. (A) is time 0 and (B) is 48 hours with no inhibitors. (C) is a serial section reacted with MSI. It shows decreased reaction area. (D) is a similar area to (B) viewed in dark field Nomarski optics and shows the nuclei of the chondrocytes particularly well. All images taken at × 100 magnification. (E) is the positive control, rheumatoid synovium, also at 48 hours. Note the areas of gel digestion below regions of the subintima and a villous surface projection × 250.

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