Objective. To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OAj cartilage, using type X collagen as a specific marker.Methods. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with 3H-proline, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and I1 collagen was analyzed immunohistochemically . Chondrocytes of normal adult articular cartilage are phenotypically similar to fetal chondrocytes of the resting zone, in that they synthesize the same pattern of collagens: types 11, IX, XI, and VI ( 1 4 ) . There are conflicting reports concerning the modulation of the chondrocyte phenotype and associated alterations of collagen synthesis during osteoarthritic (OA) degeneration of articular cartilage. In early stages of human and experimentally induced OA, articular chondrocytes respond to the degenerative events by increasing the synthesis of proteoglycan and type I1 collagen several times higher than normal (5,6); this suggests stability of the chondrocyte phenotype. Other investigators have observed that chondrocytes in the upper zone of OA cartilage stain pericellularly with antibodies to types 1 and I11 collagen (7). Biochemical analyses of articular cartilage, however, have clearly shown that more than 95% of the collagen in both normal and OA cartilage is type I1 collagen, while significant amounts of type I collagen are found only in fibrocartilage (3,8). ResultsThe frequent occurrence of clusters of proliferating, hypertrophic chondrocytes in OA cartilage (9) prompted us to search for type X collagen during various stages of osteoarthritic degeneration of cartilage. Type X collagen is a short, non-fibril-forming collagen which has thus far been found only in hyper-

Normal and osteoarthritic human articular cartilage was investigated by in situ hybridization for expression patterns of the fibrillar collagens type I, II, and III to evaluate phenotypic changes of articular chondrocytes related to the disease. In 11 out of 20 samples, a defined subset of chondrocytes in the superficial and upper middle zone of osteoarthritic cartilage showed significant levels of cytoplasmic al (III) mRNA, whereas strong signals of at (II) mRNA were found in the upper and lower middle zone, partially overlapping with the zone of al (III) mRNA-expressing cells. The extent of type II and III collagen expression depended on the integrity of the extracellular matrix surrounding the chondrocytes, and the location within the articular cartilage. No atl (I) mRNA was detectable in osteoarthritic original articular cartilage. The at (I) probe did, however, reveal signals in pannuslike tissue, osteophytes, and bone cells. In normal articular cartilage, no detectable levels of cytoplasmic mRNA for Al (I), a2(I), or al (III) were seen. Using specific mono-and polyclonal antibodies, we found deposition of type III collagen but hardly any of type I collagen in the superficial zone of osteoarthritic cartilage that is consistent with the in situ hybridization results.These results indicate a phenotypic alteration in a defined subset of chondrocytes in conditions of diseased cartilage, expressing and synthesizing collagen type III independently from type I collagen, but in part simultaneously with type II collagen. (J. Clin. Invest. 1993. 91:829-837.)

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Osteocalcin (OC), which is a marker of the mature osteoblasts, can also be found in posthypertrophic chondrocytes of the epiphyseal growth plate, but not in chondrocytes of the resting zone or in adult cartilage. In human osteoarthritis (OA), chondrocytes can differentiate to a hypertrophic phenotype characterized by type X collagen. The protein- and mRNA-expression pattern of OC was systematically analyzed in decalcified cartilage and bone sections and nondecalcified cartilage sections of human osteoarthritic knee joints with different stages of OA to investigate the differentiation of chondrocytes in OA. In severe OA, we found an enhanced expression of the OC mRNA in the subchondral bone plate, demonstrating an increased osteoblast activity. Interestingly, the OC protein and OC mRNA were also detected in osteoarthritic chondrocytes, whereas in chondrocytes of normal adult cartilage, both the protein staining and the specific mRNA signal were negative. The OC mRNA signal increased with the severity of OA and chondrocytes from the deep cartilage layer, and proliferating chondrocytes from clusters showed the strongest signal for OC mRNA. In this late stage of OA, chondrocytes also stained for alkaline phosphatase and type X collagen. Our results clearly show that the expression of OC in chondrocytes correlates with chondrocyte hypertrophy in OA. Although the factors including this phenotypic shift in OA are still unknown, it can be assumed that the altered microenvironment around osteoarthritic chondrocytes and systemic mediators could be potential inducers of this differentiation.

We conclude that matrilin-3 is an integral component of human articular cartilage matrix and that the enhanced expression of matrilin-3 in OA may be a cellular response to the modified microenvironment in the disease.

Objective-Thrombospondin-1 (TSP-1), a trimeric glycoprotein, is involved in cellmatrix interactions of various tissues, particularly in cartilage. Biochemical analyses show expression of TSP-1 in human cartilage, but its cellular source as well as the presence of its main surface receptors CD36 and CD51 in normal and osteoarthritic cartilage remain unknown. Therefore, to localise TSP-1 and its receptors immunohistochemistry and in situ hybridisation were used. Methods-Radioactive in situ hybridisations with an RNA probe that encodes TSP-1 combined with immunostaining were carried out to investigate the expression patterns of TSP-1, CD36, and CD51 in seven normal and 23 osteoarthritic human cartilage samples. Results-In normal cartilage TSP-1 was present mainly in the middle and upper deep zone. RNA expression was predominantly seen over chondrocytes of the middle zone. CD36 was found in chondrocytes of the superficial and upper middle zone. In mild and moderate osteoarthritic cartilage an increased number of TSP-1 expressing chondrocytes were seen and an increased pericellular staining close to the surface. In severe osteoarthritic cartilage a decrease in the number of TSP-1 synthesising chondrocytes and a strong reduction in matrix staining were observed. Most of these severe osteoarthritic samples showed a strongly enhanced number of CD36 positive chondrocytes. Conclusion-The cellular source of TSP-1 in normal cartilage is mainly mid-zone chondrocytes, which also express CD36. In early osteoarthritic cartilage lesions an increase of TSP-1 was seen, whereas reduced TSP-1 synthesis is paralleled by a strong decrease in TSP-1 protein staining in severe osteoarthritis. Furthermore, in severe osteoarthritic cartilage the number of CD36 immunostained chondrocytes is significantly increased. (Ann Rheum Dis 2000;59:448-454) Articular cartilage is a complex tissue, composed of a highly organised extracellular matrix and only a small number of embedded chondrocytes.1 The extracellular matrix itself consists of large type II collagen fibrils cross linked by type IX collagen, large proteoglycans (mainly aggrecan) and small proteoglycans, hyaluronic acid, cations, and water.2 3 Furthermore, diVerent minor components, such as collagen types VI, XI, X, and non-collagenous proteins like COMP, CMP, fibronectin, 58 kDa protein, tenascin, 32 kDa protein, and others, are present. [4][5][6][7][8] The chondrocytes are responsible for the integrity of this highly organised matrix, by maintaining a balance between the synthesis and degradation of the cartilage matrix. TSP-1 is a member of the thrombospondin family. TSP-1 and TSP-2 have many structural similarities and should be distinguished from TSP-3, TSP-4, and TSP-5 (COMP), which share homologies with TSP-1 and TSP-2 only in the C terminal domain. 9TSP-1 is a trimeric glycoprotein with a molecular weight of 420 kDa. It consists of six diVerent domains which are the N terminal domain, the procollagen homology domain, three type I (properdin-like) repeats, three type II ...

These results demonstrate that in normal and osteoarthritic cartilage, type VI collagen is expressed in a zone specific pattern. The observed increase of type VI collagen expression in osteoarthritis suggests a potential role in the disease process.