Respiratory Activity of Isolated Chondrocytes with a Miniaturized Oxygen Electrade System

A technique for the isolation of chondrocytes from the articular cartilage of rabbits was modified and improved to yield 5 to 20 x 10 viable cells per preparation. A YSI Model 5331 O2 sensor was modified so that it could rapidly respond in as little as 1 ml of medium. Mean oxygen uptake of cell samples showed that chondrocytes obtained from mature rabbits (1.33 /J! O2/10 7 cells/hr) had a higher oxidative activity than chondrocytes from immature rabbits (0.8 (A O2/10 7 cells/hr). Elevation of the incubation temperature from 25 °C to 35 °C increased the chondrocyte oxygen uptake approximately 20% but incubation at 37 °C tended to decrease oxygen uptake. It is evident that articular chondrocyte cells have a real, but fairly low, temperature sensitive oxidative metabolism. OHIO J. SCI. 80(6): 262, 1980 It has long been assumed that the energy requirements of cartilage are met primarily through anaerobic pathways (Bywaters 1937, Rosenthal et al 1942a, b). During the past decade, interest in the study of articular cartilage has increased because of the development of better fractionation and biochemical methods. Mankin and Orlic (1964) indicated that there were well-developed anaerobic pathways in cartilage cells because of their tolerance to potassium cyanide and the minimal effects of short periods of oxygen deprivation. More recently, Fine and Person (1970) demonstrated the cytochrome activity of chondrocytes and several investigators have suggested the presence of aerobic metabolic activity by the cells. Part of the difficulty in assaying oxidative activity of chondrocytes was the difficulty of obtaining viable intact cells for investigation from the cartilagenous matrix. Kuroda (1964), Kawiak et al (1965) and Manning and Bonner (1967) pioneered the development of digestion procedures that yielded viable chondrocytes, and Green (1967) described a technique for the isolation of large numbers of chondrocytes from Manuscript received 1 March 1980 and in revised form 7 July 1980 (#80-11). Present address: Dept. of Orthopaedics, University Hospitals, Cleveland, Ohio 44106. cartilage sections. Green suggested that further improvements in isolation techniques would make it possible to isolate sufficient cells for metabolic studies in vitro. His suggestion led us to the present investigation of improvements in digestion techniques and miniaturization of the YSI oxygen sensor system to allow oxygen uptake studies of as little as 10 viable isolated chondrocytes. MATERIALS AND METHODS Male and non-pregnant female New Zealand rabbits, classed as mature (6-10 months old) or immature (6-10 weeks old), were used. Each rabbit was sacrificed by rapid injection of 100-150 cc of air into an ear vein. The leg area was shaved, washed, and prepared with povidone-iodine (Betadine) solution, and draped with sterile towels. The proximal tibia, distal and proximal femoral articular surface, proximal humerus, and glenoid fossa were removed from each animal with sterility maintained in this and all subsequent procedures. Cartilage shavings were removed from each articular surface and placed into a sterile Petri dish containing Gey's balanced salt solution (GBSS, Microbiological Associates #10-505). When all surfaces had been stripped, the GBSS was decanted and 15 ml of 0.05% testicular hyaluronidase in GBSS was added. The dish was then rocked gently by hand for 4 min at room temperature. The hyaluronidase solution was removed with a sterile syringe and the softened cartilage diced into fragments 1-2 mm square, washed with GBSS, and then placed into the inner compartment of the digestion chamber (figure IB). After addition of 4 ml