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. METHOD: 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-1ß (IL-1ß). 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-1ß reduced HOAC cell viability and increased GAG media levels. Acidosis and IL-1ß 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-1ß but reduced in hypoxia and anoxia. Mitochondrial membrane potential was reduced in low oxygen, acidosis and IL-1ß. Anoxia also resulted in intracellular acidosis. GSH/GSSG ratio was reduced in low oxygen conditions, acidosis and IL-1ß. 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.