B. Swoboda

Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies

For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.

Expression of Early and Late Differentiation Markers (Proliferating Cell Nuclear Antigen, Syndecan-3, Annexin VI, and Alkaline Phosphatase) by Human Osteoarthritic Chondrocytes

Although osteoarthritis is characterized by a progressive loss of the extracellular cartilage matrix, very little is known about the fate of articular chondrocytes during the progression of the disease. In this study we examined the expression of syndecan-3, a marker of early chondrocyte differentiation, and annexin VI, a marker of late chondrocyte differentiation, in mammalian embryonic growth plate cartilage and normal and osteoarthritic human articular cartilage. Whereas syndecan-3 was expressed in the proliferative and hypertrophic zones of growth platecartilage, immunostaining for annexin VI waspredominately found in the hypertrophic and mineralizing zones of fetal bovine growth plate cartilage. Approximately 20% of chondrocytes were immunopositive for syndecan-3 in normal human articular cartilage, the number of syndecan-3-expressing chondrocytes significantly increased during the progression of osteoarthritis with more than 80% syndecan-3-positive cells in the upper zone of severely affected osteoarthritic cartilage. Similarly, the number of annexin VI-expressing cells significantly increased in the upper cartilage zones during the progression of osteoarthritis. Furthermore, immunostaining for proliferating cell nuclear antigen, a marker for cell proliferation, was detected in chondrocytes in the upper zone of osteoarthritic cartilage. Double-labeling experiments with antibodies against syndecan-3 and annexin VI revealed chondrocytes that expressed only syndecan-3, and cells that expressed both syndecan-3 and annexin VI. These results suggest that the expression of early (proliferating cell nuclear antigen, syndecan-3) and late differentiation markers (annexin VI, alkaline phosphatase) is activated in chondrocytes of osteoarthritic cartilage.

Vascular endothelial growth factor in articular cartilage of healthy and osteoarthritic human knee joints

OBJECTIVE To determine the levels of vascular endothelial growth factor (VEGF) mRNA and protein expression in normal and osteoarthritic (OA) human articular cartilage, and whether VEGF expression alters during the progression of OA. METHODS Sections from normal and OA human knee cartilage were immunotained with a polyclonal antibody recognising VEGF. In addition, total RNA was isolated from normal and osteoarthritic human knee cartilage and analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) for VEGF mRNA expression. RESULTS VEGF was found to be present in normal and OA human knee cartilage in all cartilage layers. A significant increase of VEGF immunopositive chondrocytes to up to ∼82% was detected in severe OA cartilage compared with normal articular cartilage (∼56% of immunopositive chondrocytes). RT-PCR analysis showed the expression of VEGF also on the mRNA level. CONCLUSIONS VEGF is expressed by articular chondrocytes in normal and OA human knee cartilage. The percentage of VEGF immunopositive chondrocytes significantly increases in late stages of the disease. The VEGF transcript levels encoding all four isoforms shows a big variability in samples from different donors, suggesting a distinct regulation of the expression of the four VEGF isoforms in normal and OA cartilage.

Osteopontin is expressed by adult human osteoarthritic chondrocytes: protein and mRNA analysis of normal and osteoarthritic cartilage

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.

Signature of circulating microRNAs in osteoarthritis

BACKGROUND Osteoarthritis is the most common form of arthritis and a major socioeconomic burden. Our study is the first to explore the association between serum microRNA levels and the development of severe osteoarthritis of the knee and hip joint in the general population. METHODS We followed 816 Caucasian individuals from 1995 to 2010 and assessed joint arthroplasty as a definitive outcome of severe osteoarthritis of the knee and hip. After a microarray screen, we validated 12 microRNAs by real-time PCR in the entire cohort at baseline. RESULTS In Cox regression analysis, three microRNAs were associated with severe knee and hip osteoarthritis. let-7e was a negative predictor for total joint arthroplasty with an adjusted HR of 0.75 (95% CI 0.58 to 0.96; p=0.021) when normalised to U6, and 0.76 (95% CI 0.6 to 0.97; p=0.026) after normalisation to the Ct average. miRNA-454 was inversely correlated with severe knee or hip osteoarthritis with an adjusted HR of 0.77 (95% CI 0.61 to 0.97; p=0.028) when normalised to U6. This correlation was lost when data were normalised to Ct average (p=0.118). Finally, miRNA-885-5p showed a trend towards a positive relationship with arthroplasty when normalised to U6 (HR 1.24; 95% CI 0.95 to 1.62; p=0.107) or to Ct average (HR 1.30; 95% CI 0.99 to 1.70; p=0.056). CONCLUSIONS Our study is the first to identify differentially expressed circulating microRNAs in osteoarthritis patients necessitating arthroplasty in a large, population-based cohort. Among these microRNAs, let-7e emerged as potential predictor for severe knee or hip osteoarthritis.

Chondromodulin 1 stabilizes the chondrocyte phenotype and inhibits endochondral ossification of porcine cartilage repair tissue

OBJECTIVE To investigate the effect of chondromodulin 1 on the phenotype of osteochondral progenitor cells in cartilage repair tissue. METHODS Self-complementary adeno-associated virus (AAV) vectors carrying chondromodulin 1 complementary DNA (AAV-Chm-1) were applied to cartilage lesions in the knee joints of miniature pigs that were treated by the microfracture technique. Alternatively, isolated porcine osteochondral progenitor cells were infected with AAV-Chm-1 or with AAV-GFP control vectors ex vivo prior to being transplanted into cartilage lesions in which the subchondral bone plate was left intact. The quality of the repair tissue and the degree of endochondral ossification were assessed by histochemical and immunohistochemical methods. The effects of chondromodulin 1 overexpression were also analyzed by angiogenesis assays and quantitative reverse transcriptase-polymerase chain reaction. RESULTS AAV-Chm-1-infected cells efficiently produced chondromodulin 1, which had strong antiangiogenic effects, as verified by the inhibition of tube formation of endothelial cells. Gene expression analyses in vitro revealed the cell cycle inhibitor p21WAF1/Cip1 as one target up-regulated by AAV-Chm-1. Direct application of AAV-Chm-1 vectors into microfractured porcine cartilage lesions stimulated chondrogenic differentiation of ingrowing progenitor cells, but significantly inhibited terminal chondrocyte hypertrophy, the invasion of vessel structures, and excessive endochondral ossification, which were otherwise observed in untreated lesions. Indirect gene transfer, with infection of porcine osteochondral progenitor cells by AAV-Chm-1 ex vivo, also supported chondrogenic differentiation of these transplanted cells. AAV-Chm-1-infected cells maintained a chondrocyte-like phenotype and formed a hyaline-like matrix that was superior to that formed by uninfected or AAV-GFP-infected cells. CONCLUSION Our findings indicate that the antiangiogenic factor chondromodulin 1 stabilizes the chondrocyte phenotype by supporting chondrogenesis but inhibiting chondrocyte hypertrophy and endochondral ossification.

Molecular differentiation between osteophytic and articular cartilage – clues for a transient and permanent chondrocyte phenotype

OBJECTIVE To identify the molecular differences between the transient and permanent chondrocyte phenotype in osteophytic and articular cartilage. METHODS Total RNA was isolated from the cartilaginous layer of osteophytes and from intact articular cartilage from knee joints of 15 adult human donors and subjected to cDNA microarray analysis. The differential expression of relevant genes between these two cartilaginous tissues was additionally validated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. RESULTS Among 47,000 screened transcripts, 600 transcripts were differentially expressed between osteophytic and articular chondrocytes. Osteophytic chondrocytes were characterized by increased expression of genes involved in the endochondral ossification process [bone gamma-carboxyglutamate protein/osteocalcin (BGLAP), bone morphogenetic protein-8B (BMP8B), collagen type I, alpha 2 (COL1A2), sclerostin (SOST), growth arrest and DNA damage-induced gene 45ß (GADD45ß), runt-related transcription factor 2 (RUNX2)], and genes encoding tissue remodeling enzymes [matrix metallopeptidase (MMP)9, 13, hyaluronan synthase 1 (HAS1)]. Articular chondrocytes expressed increased transcript levels of antagonists and inhibitors of the BMP- and Wnt-signaling pathways [Gremlin-1 (GREM1), frizzled-related protein (FRZB), WNT1 inducible signaling pathway protein-3 (WISP3)], as well as factors that inhibit terminal chondrocyte differentiation and endochondral bone formation [parathyroid hormone-like hormone (PTHLH), sex-determining region Y-box 9 (SOX9), stanniocalcin-2 (STC2), S100 calcium binding protein A1 (S100A1), S100 calcium binding protein B (S100B)]. Immunohistochemistry of tissue sections for GREM1 and BGLAP, the two most prominent differentially expressed genes, confirmed selective detection of GREM1 in articular chondrocytes and that of BGLAP in osteophytic chondrocytes and bone. CONCLUSIONS Osteophytic and articular chondrocytes significantly differ in their gene expression pattern. In articular cartilage, a prominent expression of antagonists inhibiting the BMP- and Wnt-pathway may serve to lock and stabilize the permanent chondrocyte phenotype and thus prevent their terminal differentiation. In contrast, osteophytic chondrocytes express genes with roles in the endochondral ossification process, which may account for their transient phenotype.

Hypoxia and HIF-1α in osteoarthritis

We have previously shown that functional inactivation of hypoxia-inducible factor-1α (HIF-1α) in growth-plate chondrocytes will dramatically inhibit anaerobic energy generation and matrix synthesis. Using immunohistochemistry, we have now analyzed the spatial distribution of HIF-1α and its target genes in normal cartilage and in cartilage from knee joints with osteoarthritis. We detected HIF-1α and its target genes in both types of cartilage. In cartilage from joints with osteoarthritis, the number of HIF-1α-, Glut-1-, and PGK-1-stained chondrocytes increased with the severity of osteoarthritis. Activated matrix synthesis and strongly decreased oxygen levels are hallmarks of osteoarthritic cartilage. Thus, we assume that chondrocytes are depending on the adaptive functions of HIF-1α in order to maintain ATP levels and thereby matrix synthesis during the course of osteoarthritis.RésuméNous avons montré précédemment que l’inactivation du facteur de l’hypoxie 1-α (HIF-1α) dans les chondrocytes du cartilage de conjugaison inhibe très nettement la génération d’énergie anaérobie et la synthèse de la matrice. Utilisant l’immuno-histochimie nous avons analysé la distribution spatiale de HIF-1, et ses gène- cibles dans le cartilage normal et dans le cartilage d’articulations avec arthrose. Nous avons détecté des HIF-1α et ses gène-cibles dans les deux types de cartilage. Dans le cartilage arthrosique le nombre de chondrocytes marqués HIF-1α, Glut-1 et PGK-1 a augmenté avec la sévérité de l’arthrose. La synthèse de la matrice activée et le niveau d’oxygène fortement diminué sont des caractéristiques du cartilage arthrosique. Donc nous supposons que les chondrocytes dépendent de la fonction adaptative de HIF-1 pour maintenir le niveau d’ATP et de cette façon la synthèse de la matrice pendant l’évolution de l’arthrose.

Expression of thrombospondin-1 and its receptor CD36 in human osteoarthritic cartilage.

OBJECTIVE Thrombospondin-1 (TSP-1), a trimeric glycoprotein, is involved in cell-matrix 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.

Comparison of low-field (0.2 Tesla) and high-field (1.5 Tesla) magnetic resonance imaging of the knee joint.

In order to evaluate the reliability of low-field magnetic resonance imaging (MRI), we examined 22 patients using a 0.2-Tesla magnet unit in comparison with a 1.5-Tesla system. The MRI findings were compared with the intraoperative findings. Concerning the diagnosis of meniscal tears, the gradings of both systems differed only in three cases. The specificity was 97% (both systems), the sensitivity 83% (1.5 T) versus 75% (0.2 T). The sensitivity and specificity for detection of tears of the anterior cruciate ligament were 100% and 75%, respectively, for both systems. The gradings differed only in two cases. In our series we found 6 full-thickness cartilage defects that were all detected with the high-field imaging system. They were missed by the low-field imaging system in 5 cases. The results suggest that both systems are reliable in diagnosing meniscal tears and ruptures of the anterior cruciate ligament.