title: In vitro model for the analysis of synovial fibroblast-mediated degradation of intact cartilage.
abstract: BACKGROUND
  Activated synovial fibroblasts are thought to play a major role in the destruction of cartilage in chronic, inflammatory rheumatoid arthritis (RA). However, profound insight into the pathogenic mechanisms and the impact of synovial fibroblasts in the initial early stages of cartilage destruction is limited. Hence, the present study sought to establish a standardised in vitro model for early cartilage destruction with native, intact cartilage in order to analyse the matrix-degrading capacity of synovial fibroblasts and their influence on cartilage metabolism.
  
  
  METHODS
  A standardised model was established by co-culturing bovine cartilage discs with early-passage human synovial fibroblasts for 14 days under continuous stimulation with TNF-alpha, IL-1beta or a combination of TNF-alpha/IL-1beta. To assess cartilage destruction, the co-cultures were analysed by histology, immunohistochemistry, electron microscopy and laser scanning microscopy. In addition, content and/or neosynthesis of the matrix molecules cartilage oligomeric matrix protein (COMP) and collagen II was quantified. Finally, gene and protein expression of matrix-degrading enzymes and pro-inflammatory cytokines were profiled in both synovial fibroblasts and cartilage.
  
  
  RESULTS
  Histological and immunohistological analyses revealed that non-stimulated synovial fibroblasts are capable of demasking/degrading cartilage matrix components (proteoglycans, COMP, collagen) and stimulated synovial fibroblasts clearly augment chondrocyte-mediated, cytokine-induced cartilage destruction. Cytokine stimulation led to an upregulation of tissue-degrading enzymes (aggrecanases I/II, matrix-metalloproteinase (MMP) 1, MMP-3) and pro-inflammatory cytokines (IL-6 and IL-8) in both cartilage and synovial fibroblasts. In general, the activity of tissue-degrading enzymes was consistently higher in co-cultures with synovial fibroblasts than in cartilage monocultures. In addition, stimulated synovial fibroblasts suppressed the synthesis of collagen type II mRNA in cartilage.
  
  
  CONCLUSIONS
  The results demonstrate for the first time the capacity of synovial fibroblasts to degrade intact cartilage matrix by disturbing the homeostasis of cartilage via the production of catabolic enzymes/pro-inflammatory cytokines and suppression of anabolic matrix synthesis (i.e., collagen type II). This new in vitro model may closely reflect the complex process of early stage in vivo destruction in RA and help to elucidate the role of synovial fibroblasts and other synovial cells in this process, and the molecular mechanisms involved in cartilage degradation.
authors.affiliation: Experimental Rheumatology Unit, Department of Orthopedics, University Hospital Jena, Friedrich Schiller University Jena, Klosterlausnitzer Strasse 81, Eisenberg D-07607, Germany. david.pretzel@med.uni-jena.de
authors.forename: David
authors.initials: D
authors.lastname: Pretzel
authors.forename: Dirk
authors.initials: D
authors.lastname: Pohlers
authors.forename: Sönke
authors.initials: S
authors.lastname: Weinert
authors.forename: Raimund W
authors.initials: RW
authors.lastname: Kinne
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002356:Cartilage / Q000378:metabolism* / Q000473:pathology; D002417:Cattle; D018920:Coculture Techniques / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D053583:Interleukin-1beta / Q000378:metabolism; D008297:Male; D020782:Matrix Metalloproteinases / Q000378:metabolism; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D013583:Synovial Membrane / Q000166:cytology* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; D020782:Matrix Metalloproteinases