title: Immunolocalization of matrix metalloproteinases in partial-thickness defects in pig articular cartilage. A preliminary report.
abstract: BACKGROUND
  Partial-thickness defects in mature articular cartilage do not heal spontaneously. Attempts at repair often result in limited integration between the repair tissue and the surrounding cartilage, with formation of chondrocyte clusters adjacent to a zone of cartilage necrosis. In wound repair, spatially and temporally controlled expression of matrix metalloproteinases and their inhibitors have been implicated in proteolytic degradation of damaged extracellular matrix components, but the sequence of events following damage to cartilage is unknown. To determine this sequence, we studied the distribution of matrix metalloproteinases and their inhibitors during early in vivo repair of partial-thickness defects in pig articular cartilage.
  
  
  METHODS
  With use of a model that elicits the ingrowth of mesenchymal cells into partial-thickness defects, partial-thickness defects were created in knee joint cartilage. The distributions of matrix metalloproteinase-1, 2, 3, 9, 13, and 14; tissue inhibitors of metalloproteinase-1 and 2; and the neoepitope DIPEN341 specifically generated following matrix metalloproteinase cleavage of aggrecan were determined by immunolocalization of repair tissue and surrounding cartilage excised from immature pigs during the first eight weeks of repair and from adult minipigs at eight days and three weeks.
  
  
  RESULTS
  Synthesis of matrix metalloproteinase-13 was usually confined to hypertrophic chondrocytes in immature cartilage and to the radial zone in adult cartilage. Following injury, strong induction of matrix metalloproteinase-13 synthesis was observed in chondrocyte clusters surrounding lesions in all of the animals. The migration of macrophages into defects was prominent at two and eight days, with synthesis and deposition of matrix metalloproteinase-9 onto damaged cartilage matrix and newly synthesized matrix in the defect. The DIPEN341 neoepitope was localized to damaged cartilage matrix at eight days and six weeks, indicating partial degradation of aggrecan. Focal synthesis of matrix metalloproteinase-1, 3, and 14 and of tissue inhibitor of metalloproteinase-1 occurred at later times, suggesting continuous remodeling of the increasingly compact repair tissue.
  
  
  CONCLUSIONS
  The expression of matrix metalloproteinase-13 by normal hypertrophic chondrocytes and the induction of synthesis in chondrocyte clusters adjacent to the zone of cartilage necrosis suggest that this enzyme participates in the pericellular proteolysis required for lacunar expansion. The localization of matrix metalloproteinase-9 to damaged cartilage matrix suggested matrix proteolysis, which was confirmed with DIPEN341 localization. Reduced matrix metachromasia persisted and was colocalized with DIPEN341 at six weeks. However, under the conditions investigated, there was only limited proteolytic degradation in the zone of cartilage necrosis. This may render the zone mechanically weakened, thereby contributing to subsequent instability of the region, and may form a barrier to integration of repair tissue with viable cartilage.
  
  
  CONCLUSIONS
  Osteoarthritis initially involves the superficial layers of cartilage. The development of procedures to promote the healing or repair of early defects will have major advantages in terms of disease alleviation as well as economic importance. Identification of the enzymes involved in the early repair of partial-thickness defects in articular cartilage is clinically relevant because proteolysis of damaged matrix has to take place in order for repair tissue to integrate with surrounding healthy cartilage.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. r.hembry@uea.ac.uk
authors.forename: R M
authors.initials: RM
authors.lastname: Hembry
authors.forename: J
authors.initials: J
authors.lastname: Dyce
authors.forename: I
authors.initials: I
authors.lastname: Driesang
authors.forename: E B
authors.initials: EB
authors.lastname: Hunziker
authors.forename: A J
authors.initials: AJ
authors.lastname: Fosang
authors.forename: J A
authors.initials: JA
authors.lastname: Tyler
authors.forename: G
authors.initials: G
authors.lastname: Murphy
mesh_terms: D053674:Aggrecans; D000375:Aging / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000737:chemistry* / Q000293:injuries / Q000473:pathology; D019902:Chondrocytes / Q000737:chemistry / Q000473:pathology; D016326:Extracellular Matrix Proteins; D005260:Female; D007150:Immunohistochemistry; D007719:Knee Joint; D037181:Lectins, C-Type; D008264:Macrophages / Q000473:pathology; D020781:Matrix Metalloproteinase 1 / Q000032:analysis; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D020780:Matrix Metalloproteinase 9 / Q000032:analysis; D020782:Matrix Metalloproteinases / Q000032:analysis*; D009336:Necrosis; D011509:Proteoglycans / Q000032:analysis; D013552:Swine; D013556:Swine, Miniature; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000032:analysis; D019714:Tissue Inhibitor of Metalloproteinases / Q000032:analysis
chemical_list: D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D019715:Tissue Inhibitor of Metalloproteinase-1; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1