• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of annrheumdAnnals of the Rheumatic DiseasesCurrent TOCInstructions for authors
Ann Rheum Dis. Jun 1999; 58(6): 357–365.
PMCID: PMC1752894

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


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.

Full Text

The Full Text of this article is available as a PDF (5.1M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Cawston TE, Billington C. Metalloproteinases in the rheumatic diseases. J Pathol. 1996 Oct;180(2):115–117. [PubMed]
  • Pelletier JP, Martel-Pelletier J, Malemud CJ. Proteoglycans from experimental osteoarthritic cartilage: degradation by neutral metalloproteases. J Rheumatol. 1987 May;14(Spec No):113–115. [PubMed]
  • McCachren SS. Expression of metalloproteinases and metalloproteinase inhibitor in human arthritic synovium. Arthritis Rheum. 1991 Sep;34(9):1085–1093. [PubMed]
  • Case JP, Lafyatis R, Remmers EF, Kumkumian GK, Wilder RL. Transin/stromelysin expression in rheumatoid synovium. A transformation-associated metalloproteinase secreted by phenotypically invasive synoviocytes. Am J Pathol. 1989 Dec;135(6):1055–1064. [PMC free article] [PubMed]
  • Dean DD, Martel-Pelletier J, Pelletier JP, Howell DS, Woessner JF., Jr Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage. J Clin Invest. 1989 Aug;84(2):678–685. [PMC free article] [PubMed]
  • Pelletier JP, Mineau F, Faure MP, Martel-Pelletier J. Imbalance between the mechanisms of activation and inhibition of metalloproteases in the early lesions of experimental osteoarthritis. Arthritis Rheum. 1990 Oct;33(10):1466–1476. [PubMed]
  • Martel-Pelletier J, McCollum R, Fujimoto N, Obata K, Cloutier JM, Pelletier JP. Excess of metalloproteases over tissue inhibitor of metalloprotease may contribute to cartilage degradation in osteoarthritis and rheumatoid arthritis. Lab Invest. 1994 Jun;70(6):807–815. [PubMed]
  • Martel-Pelletier J, Pelletier JP. Neutral proteases in human osteoarthritic synovium: quantification and characterization. J Rheumatol. 1987 May;14(Spec No):38–40. [PubMed]
  • Woessner JF, Jr, Gunja-Smith Z. Role of metalloproteinases in human osteoarthritis. J Rheumatol Suppl. 1991 Feb;27:99–101. [PubMed]
  • Wolfe GC, MacNaul KL, Buechel FF, McDonnell J, Hoerrner LA, Lark MW, Moore VL, Hutchinson NI. Differential in vivo expression of collagenase messenger RNA in synovium and cartilage. Quantitative comparison with stromelysin messenger RNA levels in human rheumatoid arthritis and osteoarthritis patients and in two animal models of acute inflammatory arthritis. Arthritis Rheum. 1993 Nov;36(11):1540–1547. [PubMed]
  • Sapolsky S, Malemud C, Sheff M. The proteoglycanase from human cartilage and cultured rabbit chondrocytes and its relation to osteoarthritis. J Rheumatol. 1987 May;14(Spec No):33–35. [PubMed]
  • Dean DD, Azzo W, Martel-Pelletier J, Pelletier JP, Woessner JF., Jr Levels of metalloproteases and tissue inhibitor of metalloproteases in human osteoarthritic cartilage. J Rheumatol. 1987 May;14(Spec No):43–44. [PubMed]
  • Martel-Pelletier J, Zafarullah M, Kodama S, Pelletier JP. In vitro effects of interleukin 1 on the synthesis of metalloproteases, TIMP, plasminogen activators and inhibitors in human articular cartilage. J Rheumatol Suppl. 1991 Feb;27:80–84. [PubMed]
  • Lefebvre V, Peeters-Joris C, Vaes G. Production of gelatin-degrading matrix metalloproteinases ('type IV collagenases') and inhibitors by articular chondrocytes during their dedifferentiation by serial subcultures and under stimulation by interleukin-1 and tumor necrosis factor alpha. Biochim Biophys Acta. 1991 Aug 13;1094(1):8–18. [PubMed]
  • Mehraban F, Kuo SY, Riera H, Chang C, Moskowitz RW. Prostromelysin and procollagenase genes are differentially up-regulated in chondrocytes from the knees of rabbits with experimental osteoarthritis. Arthritis Rheum. 1994 Aug;37(8):1189–1197. [PubMed]
  • Freemont AJ, Hampson V, Tilman R, Goupille P, Taiwo Y, Hoyland JA. Gene expression of matrix metalloproteinases 1, 3, and 9 by chondrocytes in osteoarthritic human knee articular cartilage is zone and grade specific. Ann Rheum Dis. 1997 Sep;56(9):542–549. [PMC free article] [PubMed]
  • Martel-Pelletier J, Pelletier JP. Wanted--the collagenase responsible for the destruction of the collagen network in human cartilage! Br J Rheumatol. 1996 Sep;35(9):818–820. [PubMed]
  • Freije JM, Díez-Itza I, Balbín M, Sánchez LM, Blasco R, Tolivia J, López-Otín C. Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J Biol Chem. 1994 Jun 17;269(24):16766–16773. [PubMed]
  • Knäuper V, López-Otin C, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem. 1996 Jan 19;271(3):1544–1550. [PubMed]
  • Reboul P, Pelletier JP, Tardif G, Cloutier JM, Martel-Pelletier J. The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis. J Clin Invest. 1996 May 1;97(9):2011–2019. [PMC free article] [PubMed]
  • Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PJ, Geoghegan KF, Hambor JE. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest. 1996 Feb 1;97(3):761–768. [PMC free article] [PubMed]
  • Shlopov BV, Lie WR, Mainardi CL, Cole AA, Chubinskaya S, Hasty KA. Osteoarthritic lesions: involvement of three different collagenases. Arthritis Rheum. 1997 Nov;40(11):2065–2074. [PubMed]
  • Marles PJ, Hoyland JA, Parkinson R, Freemont AJ. Demonstration of variation in chondrocyte activity in different zones of articular cartilage: an assessment of the value of in-situ hybridization. Int J Exp Pathol. 1991 Apr;72(2):171–182. [PMC free article] [PubMed]
  • Walsh L, Freemont AJ, Hoyland JA. The effect of tissue decalcification on mRNA retention within bone for in-situ hybridization studies. Int J Exp Pathol. 1993 Jun;74(3):237–241. [PMC free article] [PubMed]
  • Hoyland JA, Freemont AJ. Investigation of a quantitative post-hybridization signal amplification system for mRNA-oligodeoxyribonucleotide in situ hybridization. J Pathol. 1991 May;164(1):51–58. [PubMed]
  • Galis ZS, Sukhova GK, Libby P. Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue. FASEB J. 1995 Jul;9(10):974–980. [PubMed]
  • Pelletier JP, Roughley PJ, DiBattista JA, McCollum R, Martel-Pelletier J. Are cytokines involved in osteoarthritic pathophysiology? Semin Arthritis Rheum. 1991 Jun;20(6 Suppl 2):12–25. [PubMed]
  • Mort JS, Dodge GR, Roughley PJ, Liu J, Finch SJ, DiPasquale G, Poole AR. Direct evidence for active metalloproteinases mediating matrix degradation in interleukin 1-stimulated human articular cartilage. Matrix. 1993 Mar;13(2):95–102. [PubMed]
  • Bunning RA, Richardson HJ, Crawford A, Skjodt H, Hughes D, Evans DB, Gowen M, Dobson PR, Brown BL, Russell RG. The effect of interleukin-1 on connective tissue metabolism and its relevance to arthritis. Agents Actions Suppl. 1986;18:131–152. [PubMed]
  • Shingu M, Nagai Y, Isayama T, Naono T, Nobunaga M, Nagai Y. The effects of cytokines on metalloproteinase inhibitors (TIMP) and collagenase production by human chondrocytes and TIMP production by synovial cells and endothelial cells. Clin Exp Immunol. 1993 Oct;94(1):145–149. [PMC free article] [PubMed]
  • Buttle DJ, Bramwell H, Hollander AP. Proteolytic mechanisms of cartilage breakdown: a target for arthritis therapy? Clin Mol Pathol. 1995 Aug;48(4):M167–M177. [PMC free article] [PubMed]
  • Hollander AP, Pidoux I, Reiner A, Rorabeck C, Bourne R, Poole AR. Damage to type II collagen in aging and osteoarthritis starts at the articular surface, originates around chondrocytes, and extends into the cartilage with progressive degeneration. J Clin Invest. 1995 Dec;96(6):2859–2869. [PMC free article] [PubMed]
  • Vankemmelbeke M, Dekeyser PM, Hollander AP, Buttle DJ, Demeester J. Characterization of helical cleavages in type II collagen generated by matrixins. Biochem J. 1998 Mar 1;330(Pt 2):633–640. [PMC free article] [PubMed]
  • Panula HE, Hyttinen MM, Arokoski JP, Långsjö TK, Pelttari A, Kiviranta I, Helminen HJ. Articular cartilage superficial zone collagen birefringence reduced and cartilage thickness increased before surface fibrillation in experimental osteoarthritis. Ann Rheum Dis. 1998 Apr;57(4):237–245. [PMC free article] [PubMed]
  • Ståhle-Bäckdahl M, Sandstedt B, Bruce K, Lindahl A, Jiménez MG, Vega JA, López-Otín C. Collagenase-3 (MMP-13) is expressed during human fetal ossification and re-expressed in postnatal bone remodeling and in rheumatoid arthritis. Lab Invest. 1997 May;76(5):717–728. [PubMed]
  • Cazorla M, Hernández L, Nadal A, Balbín M, López JM, Vizoso F, Fernández PL, Iwata K, Cardesa A, López-Otín C, et al. Collagenase-3 expression is associated with advanced local invasion in human squamous cell carcinomas of the larynx. J Pathol. 1998 Oct;186(2):144–150. [PubMed]
  • Moldovan F, Pelletier JP, Hambor J, Cloutier JM, Martel-Pelletier J. Collagenase-3 (matrix metalloprotease 13) is preferentially localized in the deep layer of human arthritic cartilage in situ: in vitro mimicking effect by transforming growth factor beta. Arthritis Rheum. 1997 Sep;40(9):1653–1661. [PubMed]
  • Lindy O, Konttinen YT, Sorsa T, Ding Y, Santavirta S, Ceponis A, López-Otín C. Matrix metalloproteinase 13 (collagenase 3) in human rheumatoid synovium. Arthritis Rheum. 1997 Aug;40(8):1391–1399. [PubMed]
  • Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, Mitchell P, Hambor J, Diekmann O, Tschesche H, et al. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest. 1997 Apr 1;99(7):1534–1545. [PMC free article] [PubMed]
  • Wernicke D, Seyfert C, Hinzmann B, Gromnica-Ihle E. Cloning of collagenase 3 from the synovial membrane and its expression in rheumatoid arthritis and osteoarthritis. J Rheumatol. 1996 Apr;23(4):590–595. [PubMed]

Figures and Tables

Figure 1
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 ...
Figure 2
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 ...

Articles from Annals of the Rheumatic Diseases are provided here courtesy of BMJ Group


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...