Objective. To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations.Methods. The chondrogenic capacity of chondrocyte populations derived from different donors (n ؍ 21) or different clonal strains from the same cartilage biopsy specimen (n ؍ 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures.Results. Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells.Conclusion. We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.
Objective. Interleukin-1 (IL-1) is one potentially important cytokine during cartilage destruction. The aim of this study was to investigate whether there are different effects of low and high concentrations of IL-1 on the expression level of anabolic genes (type II collagen, aggrecan), catabolic genes (matrix metalloproteinase 1 [MMP-1], MMP-2, MMP-3, MMP-13, and ADAMTS-4), and cytokines (IL-1, IL-6, and leukemia inhibitory factor [LIF]) by articular chondrocytes (normal and osteoarthritic). Determination of whether there was a difference in reactivity between normal and osteoarthritic chondrocytes was also a goal of this study.Methods. Gene expression levels were detected by real-time polymerase chain reaction from isolated (nonpassaged) chondrocytes (normal [n ؍ 6]; osteoarthritic [n ؍ 7]) after stimulation with 0.01 ng, 0.1 ng, 1 ng, and 10 ng/ml IL-1.Results. In normal adult articular chondrocytes the expression of both aggrecan and type II collagen genes was significantly down-regulated, whereas matrixdegrading proteases (except MMP-2), as well as the investigated cytokines, were induced by IL-1 in a dose-dependent manner. The strongest regulation was found for IL-6 and LIF. Osteoarthritic chondrocytes showed strongly increased levels of catabolic enzymes and mediators, but were less responsive to further stimulation with IL-1. Conclusion.Our study confirms that IL-1 activity is critically dependent on both the applied concentration and the reactivity of the cells stimulated. The responsiveness appears to be significantly reduced in late-stage osteoarthritic chondrocytes. However, these cells show high basic expression levels of catabolic enzymes and mediators. Thus, it remains open whether our data indicate that osteoarthritic chondrocytes are per se not responsive to IL-1 or are already so strongly stimulated (e.g., by IL-1) during the disease process that they are refractory to further stimulation.Interleukin-1 (IL-1) is thought to be one of the most important cytokines relevant not only to rheumatoid arthritis, but also to osteoarthritic (OA) joint disease. Its level is increased in synovial fluids of rheumatoid arthritis and OA patients and its up-regulation in OA chondrocytes has been reported (1). IL-1 is thought to reduce the expression of anabolic genes such as aggrecan and type II collagen (2,3) as well as to upregulate various catabolic genes such as matrix metalloproteinases (MMP-1, MMP-3, and MMP-13) (4,5) and aggrecanase 1 (ADAMTS-4) (6). Both together might contribute to the anabolic-catabolic imbalance characteristic of OA cartilage degeneration. Also, intercellular mediators such as leukemia inhibitory factor (LIF) and IL-6 are strongly induced (7,8). In this study, we investigated whether there are different effects of low and high concentrations of IL-1 on human adult articular chondrocytes and whether there are differences between normal and OA chondrocytes with respect to the responsiveness to IL-1. MATERIALS AND METHODSCell isolation, culture, and stimulation with IL-1...
Intact human adult articular cartilage is central for the functioning of the articulating joints. This largely depends on the integrity of its extracellular matrix, given the high loading forces during movements in particular in the weight-bearing joints. Unlike the first impression of a more or less static tissue, articular cartilage shows -albeit in the adult organism -a slow tissue turnover. Thus, one of the most important questions in osteoarthritis research is to understand the balance of catabolic and anabolic factors in articular cartilage as this is the key to understand the biology of cartilage maintenance and degeneration.Anabolic and catabolic pathways are very much intermingled in articular cartilage. The balance between anabolism and catabolism is titrated on numerous levels, starting from the mediator-synthesizing cells which express either catabolic or anabolic factors. Also, on the level of the effector cells (i.e. chondrocytes) anabolic and catabolic gene expression compete for a balance of matrix homeostasis, namely the synthesis of matrix components and the expression and activation of matrix-degrading proteases. Also, there are multiple layers of intracellular cross-talks in between the anabolic and catabolic signalling pathways. Maybe the most important lesson from this overview is the notion that the anabolic-catabolic balance as such counts and not so much sufficient net anabolism or limited catabolism alone. Thus, it might be neither the aim of osteoarthritis therapy to foster anabolism nor to knock down catabolism, but the balance of anabolic-catabolic activities as a total needs proper titration and balancing.
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