title: Oxygen and pH-sensitivity of human osteoarthritic chondrocytes in 3-D alginate bead culture system.
abstract: OBJECTIVE
  To identify the effect of alterations in physical parameters such as oxygen and pH on processes associated with cellular redox balance in osteoarthritic chondrocytes.
  
  
  METHODS
  Human osteoarthritic chondrocytes (HOAC) were isolated from total knee arthroplasty samples and cultured in 3-D alginate beads in four different oxygen tensions (<1%, 2%, 5% and 21% O2), at pH 7.2 and 6.2 and in the presence or absence of 10 ng/ml, interleukin-1β (IL-1β). Cell viability, media glycosaminoglycan (GAG) levels, media nitrate/nitrate levels, active matrix metalloproteinase (MMP)-13 and intracellular adenosine triphosphate (ATPi) were measured over a 96-h time course. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential, intracellular pH and reduced/oxidised glutathione (GSH/GSSG) were additionally measured after 48-h incubation under these experimental conditions.
  
  
  RESULTS
  Hypoxia (2% O2) and anoxia (<1% O2), acidosis (pH 6.2) and 10 ng/ml IL-1β reduced HOAC cell viability and increased GAG media levels. Acidosis and IL-1β increased nitrite/nitrate release, but increases were moderate at 2% O2 and significantly reduced at <1% O2. ATPi was significantly reduced following hypoxia and anoxia and acidosis. At 48 h cellular ROS levels were increased by acidosis and IL-1β but reduced in hypoxia and anoxia. Mitochondrial membrane potential was reduced in low oxygen, acidosis and IL-1β. Anoxia also resulted in intracellular acidosis. GSH/GSSG ratio was reduced in low oxygen conditions, acidosis and IL-1β.
  
  
  CONCLUSIONS
  This study shows that oxygen and pH affect elements of the redox system in HOAC including cellular anti-oxidants, mitochondrial membrane potential and ROS levels.
authors.affiliation: Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Leahurst Campus, Neston, Cheshire CH64 7TE, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Collins
authors.forename: R J
authors.initials: RJ
authors.lastname: Moots
authors.forename: R
authors.initials: R
authors.lastname: Winstanley
authors.forename: P D
authors.initials: PD
authors.lastname: Clegg
authors.forename: P I
authors.initials: PI
authors.lastname: Milner
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000368:Aged; D000464:Alginates; D002358:Cartilage, Articular / Q000378:metabolism / Q000473:pathology; D015687:Cell Hypoxia / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D003470:Culture Media; D020723:Glucuronic Acid; D006025:Glycosaminoglycans / Q000378:metabolism; D006603:Hexuronic Acids; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000378:metabolism / Q000473:pathology*; D010084:Oxidation-Reduction; D010641:Phenotype; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000464:Alginates; D003470:Culture Media; D006025:Glycosaminoglycans; D006603:Hexuronic Acids; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D020723:Glucuronic Acid; D000255:Adenosine Triphosphate; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13
keywords: Hypoxia; Mitochondria; Osteoarthritis; Redox imbalance; pH