Viability of equine articular chondrocytes in alginate beads exposed to different oxygen tensions

Abstract

Ischaemia and reperfusion are suspected to alter chondrocyte metabolism. Here, we studied the effects of three oxygen (O 2 ) tensions on the viability of equine articular chondrocytes isolated from the cartilage of the distal interphalangeal joint of horses. Chondrocytes were cultured in alginate beads under 1%, 5% or 21% gas phase O 2 concentration for 14 days, cellular growth kinetics were measured (n = 6), and the cells were observed by light microscopy after staining for necrotic and apoptotic cell detection. For information about the metabolic status, the intracellular adenosine triphosphate (ATP) content was measured.The number of chondrocytes remained stable for the first eight days, then decreased especially at 1% and 21% O 2 . At 21% O 2 , normal cells decreased and necrotic cells increased at the end of the 14 day-period. No significant variations were found at 5% O 2 except for a decrease in necrotic cells at day 14. Most apoptotic cells were found at 1% O 2 from days 5 to 11, and normal cells decreased during the same period. But an unexpected increase in normal cells and decrease in apoptotic cells were observed at day 14. The intracellular ATP content remained stable. It was concluded that, in a three-dimensional culture model of equine articular chondrocytes, O 2 tension affected the viability of the cells after an 11-day period, with the most important effects observed at 21% and 1% O 2 conditions.

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

“…At 0 g/L glucose, the number of cells decreased whatever the O 2 tension was, but the decrease reached statistical significance only for 21% O 2 tension. These observations confirmed our previous results demonstrating the capacity of the chondrocytes cultured in alginate beads to survive at different O 2 tensions until at least eight days of culture (Schneider et al, 2004) and the results of the new study of the mature equine articular chondrocytes in alginate beads during 11 days with different O 2 and glucose concentrations (Fig. 6).…”

“…In another study, we demonstrated that the intracellular ATP levels were not significantly different for equine chondrocytes cultured in alginate beads for eight days with 4.5 g glucose/L, at 1%, 5% or 21% O 2 as regulated in the gas phase, suggesting that the rate of the mitochondrial respiration did not intensively change whatever the applied O 2 tension was (Schneider et al, 2004). Furthermore, we observed a cell survival of 40-60% at 1% O 2 tension, even in the complete absence of glucose.…”

“…An area of about 20 × 20 cm at the stifle was clipped, and the skin was removed without preliminary opening of the joint cavity. The joint capsule was dissected, and the isolation of equine articular cartilage was performed aseptically as previously described (Schneider et al, 2004). The cartilage, cut in small pieces, was submitted to a triple enzymatic digestion (sequentially: hyaluronidase, pronase, collagenase) in DMEM without glutamine, added with 10 mM HEPES, 2% Ultroser G, 1 g/L of glucose and a mixture of antibiotics and amphotericin B (Sanchez et al, 2002;Schneider et al, 2004).…”

“…The joint capsule was dissected, and the isolation of equine articular cartilage was performed aseptically as previously described (Schneider et al, 2004). The cartilage, cut in small pieces, was submitted to a triple enzymatic digestion (sequentially: hyaluronidase, pronase, collagenase) in DMEM without glutamine, added with 10 mM HEPES, 2% Ultroser G, 1 g/L of glucose and a mixture of antibiotics and amphotericin B (Sanchez et al, 2002;Schneider et al, 2004). After digestion, the cell suspension was filtered (filter porosity 70 µm), centrifuged (5 min at 2500 g) and washed three times in culture medium.…”

Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation

“…Articular chondrocytes in vivo live under poor oxygen (O 2 ) tension [3]. We already observed that equine articular chondrocytes in culture could survive for more than 10 days to near anoxia conditions (<1% O 2 in the surrounding atmosphere) [4], and we recently observed that the O 2 uptake of equine articular chondrocytes was very low (20.5 pmol O 2 /min/10 6 cells), even if they were cultured at 21% O 2 during 48 h before the assays. Their respiratory rate did not increase after an anoxia period of 30 min (data submitted for publication).…”