The effect of low oxygen concentration on growth, glycolysis, and sulfate incorporation by articular chondrocytes in monolayer culture

Abstract

Radiosulfate incorporation, DNA synthesis, and glycoiysis by rabbit articular chondrocytes and skin fibrocytes in monolayer culture were compared under different concentrations of oxygen (19,6.8, 1.3, and 0.6%). Cell for cell, chondrocytes under each condition had higher levels of radiosulfate incorporation and giycoiysis than did fibrocytes. Growth of the fibrocytes was reduced approximately 20% when the oxygen concentration was reduced to 6.8%. but that of chondrocytes was unaffected. A progressive Pasteur effect was observed with both cell types. Radiosulfate incorporation was reduced at low oxygen concentrations. The findings in this cell culture system are thus at variance with other published evidence that hypoxia is a major histogenetic force favoring chondrogenic expression.Articular cartilage, being an avascular tissue, must somehow adapt itself to its anaerobic environment in order to survive, grow, and produce extracellular matrix in vim. T w o of these adaptations, a high rate of anaerobic glycolysis and a low rate of oxygen consumption, were described originally by investigators studying cartilage slices (1, 2). A variety of evidence, furthermore, suggests that low oxygen tension independently or in conjunction with other factors might play an important role in the differentiation of cartilage by enhancing its production of extracellular matrix (acid mucopolysaccharides) (3-This study was undertaken to investigate (a) whether low oxygen tensions are a measurable 10). Submitted for publication Nov. 8, 1972; accepted May 30. 1973. force in the differentiation of articular chondrocytes grown in monolayer culture, and (b) whether the cultured chondrocytes retain the high rate of glycolysis characteristic of whole cartilage. METHODS AND MATERIALS Technique of CultureThe articular chondrocytes and cutaneous fibrocytes were isolated from 6-to 8-week-old New Zealand white rabbits by a method described elsewhere (1 1). The cells (100,000 to 300,000) were inoculated into 75 sq cm plastir Falcon flasks (Falcon Plastic Co., Los Angeles, Calif.) containing 12 ml of Ham's F12 medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 10% fetal calf serum (Grand Island) and streptomycin-penicillin (LO &ml and 10 units/ml, respectively). The cells were fed three times a week and gassed with 90% air:lO% C02. When the cells became confluent (generally on the eighth day), Dulbecco's modified Eagle's medium (DMEM) (Grand Island) was substituted for the F12 medium. On the 1 Ith day the cells were trypsinized, and 100,000 to 200,000 cells were inoculated into 6 0 x 15 mm glass petri dishes containing 4 ml of supplemented DMEM. The dishes were placed in anaerobic culture jars which were gassed and incubated at 37O. They were refed and gassed on Day 2. On Day 4 the medium was replaced by supplemented DMEM in which MgSO, was replaced by MgCI,, 165 mg/liter. On

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage

“…In contrast, higher p O 2 and pH were associated with relatively more aerobic metabolism in groups 1, 2, and 4 (Y L/G ‫ס‬ 1.65-1.79, Table I). These findings are consistent with previous reports for monolayer cultures of rabbit articular chondrocytes that p O 2 values of 5-10 mmHg were associated with Y L/G ‫ס‬ 2 while a high p O 2 of 144 mmHg was associated with Y L/G ∼ 1.7 (Marcus, 1973). In contrast, periodic gas exchange in group 2 (3 times per week until equilibrated) was shown to be sufficient to maintain all culture parameters at levels comparable to the continuously gassed culture (group 1).…”

Computational Study of Culture Conditions and Nutrient Supply in Cartilage Tissue Engineering

“…Under conditions of nutrient limitations cellular regulation mechanisms become more important. Articular chondrocytes have been shown to display either a decrease in glycolysis as a function of decreasing oxygen (28,30,39) or by contrast, an increase (Pasteur effect) (27,29,32,59), modulated by differences in culture conditions and associated dedifferentiation. As a first order estimation, the model assumed that glucose and oxygen uptake were uncoupled, although such regulation mechanisms can be subsequently included.…”

“…Most modeling studies consider only one metabolite, most often oxygen. It has been shown, however, that GAG synthesis is affected by nutrient availability (25), pH (26), and oxygen (27)(28)(29)(30)(31)(32)(33)(34). Complicated interactions exist between chondrocyte energy balance and matrix metabolism, and the precise regulation mechanisms remain largely unknown (28,(35)(36)(37)(38)(39)(40).…”

HIF-1α controls extracellular matrix synthesis by epiphyseal chondrocytes

“…In previous studies, we and other groups have provided evidence that the embryonic growth plate of developing long bones is hypoxic (Rajpurohit et al, 1996;Schipani et al, 2001). Even chondrocytes in the deep zone of articular cartilage are in a hypoxic microenvironment, suggesting that chondrocytes require extensive adaptive mechanisms for low oxygen levels (Marcus, 1973;Silver, 1975).…”