title: Low oxygen tension increased fibronectin fragment induced catabolic activities--response prevented with biomechanical signals.
abstract: BACKGROUND
  The inherent low oxygen tension in normal cartilage has implications on inflammatory conditions associated with osteoarthritis (OA). Biomechanical signals will additionally contribute to changes in tissue remodelling and influence the inflammatory response. In this study, we investigated the combined effects of oxygen tension and fibronectin fragment (FN-f) on the inflammatory response of chondrocytes subjected to biomechanical signals.
  
  
  METHODS
  Chondrocytes were cultured under free-swelling conditions at 1%, 5% and 21% oxygen tension or subjected to dynamic compression in an ex vivo 3D/bioreactor model with 29 kDa FN-f, interleukin-1beta (IL-1β) and/or the nitric oxide synthase (NOS) inhibitor for 6 and 48 hours. Markers for catabolic activity (NO, PGE2), tissue remodelling (GAG, MMPs) and cytokines (IL-1β, IL-6 and TNFα) were quantified by biochemical assay. Aggrecan, collagen type II, iNOS and COX-2 gene expression were examined by real-time quantitative PCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse data.
  
  
  RESULTS
  Both FN-fs and IL-1β increased NO, PGE2 and MMP production (all P< 0.001). FN-f was more active than IL-1β with greater levels of NO observed at 5% than 1% or 21% oxygen tension (P < 0.001). Whilst FN-f reduced GAG synthesis at all oxygen tension, the effect of IL-1β was significant at 1% oxygen tension. In unstrained constructs, treatment with FN-f or IL-1β increased iNOS and COX-2 expression and reduced aggrecan and collagen type II (all P < 0.001). In unstrained constructs, FN-f was more effective than IL-1β at 5% oxygen tension and increased production of NO, PGE2, MMP, IL-1β, IL-6 and TNFα. At 5% and 21% oxygen tension, co-stimulation with compression and the NOS inhibitor abolished fragment or cytokine-induced catabolic activities and restored anabolic response.
  
  
  CONCLUSIONS
  The present findings revealed that FN-fs are more potent than IL-1β in exerting catabolic effects dependent on oxygen tension via iNOS and COX-2 upregulation. Stimulation with biomechanical signals abolished catabolic activities in an oxygen-independent manner and NOS inhibitors supported loading-induced recovery resulting in reparative activities. Future investigations will utilize the ex vivo model as a tool to identify key targets and therapeutics for OA treatments.
authors.forename: Eleanor
authors.initials: E
authors.lastname: Parker
authors.forename: Sandrine
authors.initials: S
authors.lastname: Vessillier
authors.forename: Belinda
authors.initials: B
authors.lastname: Pingguan-Murphy
authors.forename: Wan
authors.initials: W
authors.lastname: Abas
authors.forename: Dan L
authors.initials: DL
authors.lastname: Bader
authors.forename: Tina T
authors.initials: TT
authors.lastname: Chowdhury
mesh_terms: D053674:Aggrecans / Q000235:genetics; D000704:Analysis of Variance; D000818:Animals; D001696:Biomechanical Phenomena; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics; D051546:Cyclooxygenase 2 / Q000235:genetics; D016207:Cytokines / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005353:Fibronectins / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D053583:Interleukin-1beta / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics; D010100:Oxygen / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D053674:Aggrecans; D024043:Collagen Type II; D016207:Cytokines; D004791:Enzyme Inhibitors; D005353:Fibronectins; D053583:Interleukin-1beta; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D020782:Matrix Metalloproteinases; D015232:Dinoprostone; D010100:Oxygen