We demonstrate the direct involvement of increased collagenase activity in the cleavage of type II collagen in osteoarthritic human femoral condylar cartilage by developing and using antibodies reactive to carboxy-terminal (COL2-3/ 4C short ) and amino-terminal (COL2-1/4N1) neoepitopes generated by cleavage of native human type II collagen by collagenase matrix metalloproteinase (MMP)-1 (collagenase-1), MMP-8 (collagenase-2), and MMP-13 (collagenase-3). A secondary cleavage followed the initial cleavage produced by these recombinant collagenases. This generated neoepitope COL2-1/4N2. There was significantly more COL2-3/ 4C short neoepitope in osteoarthritis (OA) compared to adult nonarthritic cartilages as determined by immunoassay of cartilage extracts. A synthetic preferential inhibitor of MMP-13 significantly reduced the unstimulated release in culture of neoepitope COL2-3/4C short from human osteoarthritic cartilage explants. These data suggest that collagenase(s) produced by chondrocytes is (are) involved in the cleavage and denaturation of type II collagen in articular cartilage, that this is increased in OA, and that MMP-13 may play a significant role in this process. ( J. Clin. Invest. 1997. 99:1534-1545.)

The degeneration of articular cartilage is a key feature of osteoarthritis (OA)'. It is characterized by a loss of the tensile strength (1-3) of this tissue. The incidence of OA increases progressively with age (4). This is in association with a progressive reduction in tensile properties during aging (5). Whereas the large aggregating proteoglycan (now called aggrecan) is responsible for the compressive stiffness of cartilage (6, 7), it is the collagen fibrillar network that determines the tensile properties of this tissue ( 1, 2). These collagen fibrils are primarily composed of type H collagen (-90-95%) but also contain type IX collagen, covalently bound to type II, and type XI collagen which together comprise 2-4% of the total collagen (8).These biomechanical changes are indicative of damage to the collagen fibrils in aging and OA. To investigate these changes at the molecular level first polyclonal (9) and then monoclonal (10) antibodies were developed that only react with epitopes on the collagen a, (II) chains when the triple helix has been denatured, such as occurs following cleavage by interstitial collagenase (MMP1) which results in unwinding of the triple helix.Using a monoclonal antibody in an immunoassay, quantitative evidence was obtained to indicate that there is indeed increased denaturation of type II collagen in osteoarthritic cartilage (10). In the study presented here we describe the use of this antibody to determine where, in both aging and osteoarthritis, this damage to type II collagen occurs. We show that it starts at the articular surface and spreads into the mid and deep zones with increasing degeneration, denaturation being first observed around chondrocytes implicating these cells as primary mediators of collagen degradation in aging and OA. Tissue. Full depth human articular femoral condylar cartilages (0.5-1.0 cm2 surface area) were removed within 15 h of death with a sharp scalpel from the anterior loaded regions of the adult knee joint from a total of 11 individuals, of various ages and sexes, with no observable arthritic joint abnormalities nor recent (2-3 mo) chemotherapy (see Table I). A total of 51 patients underwent total knee arthroplasty for osteoarthritis diagnosed using the criteria of the American College of Rheumatology. Site matched femoral condylar cartilages that remained were immediately removed to the laboratory. By definition, osteophytic cartilages were excluded from this study. Single samples from 30 patients (see Table I) and 12 multiple samples from three other patients 1. Abbreviation used in this paper: OA, osteoarthritis. MethodsType II Collagen Denaturation 2859 J. Clin. Invest.

There is evidence to suggest that the synthesis of type II collagen is increased in osteoarthritis (OA). Using an immunoassay, we show that the content of the C-propeptide of type II procollagen (CPII), released extracellularly from the newly synthesized molecule, is directly related to the synthesis of this molecule in healthy and osteoarthritic articular cartilages. In OA cartilage, CPII content is often markedly elevated (mean 7.6-fold), particularly in the mid and deep zones, reaching 29.6% of the content in newborn. Synthesis is also directly related to total collagen II content in OA, suggesting its importance in maintaining collagen content and cartilage structure. The release of CPII from cartilage is correlated directly with cartilage content. However, the increase in CPII in OA cartilage is not reflected in serum, where a significant reduction is observed. Together these studies provide evidence for alterations in procollagen II synthesis in vivo in patients with OA. ( J. Clin. Invest. 1998. 102:2115-2125 . )

These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.