Stimulation of DNA Synthesis in Isolated Chondrocytes by Sulfation Factor11

Growth hormone dependent serum factors (e.g. sulfation factor or SF) regulate both proliferation of chondrocytes and formation of matrix. Previous work in this field has generally employed explants of rat cartilage. We have investigated the action of sulfation factor on isolated chick chondrocytes. Pelvic rudiments from 15-day chick embryos were treated with collagenase and trypsin to liberate chondrocytes. Chondrocytes were then isolated by differential centrifugation. Aliquots containing about 300,000 cells were pipetted for subsequent incubations. Normal human serum (NHS) added to the medium stimulated incorporation of [3H]thymidine into the cold TCA ppt, as compared to serum from hypopituitary subjects. When a patient with low serum SF was treated with growth hormone the serum SF activity increased. In contrast, bovine growth hormone added in vitro did not have any significant effect, either alone or in the presence of 0.5% NHS. Pulse labeling after various periods of incubation demonstrated maximal stimulation of uptake at 31 hr. A graded response of thymidine incorporation was obtained with most NHS at concentrations of 0.6 to 6% of the final vol. Stimulation with 5% NHS was as great as 100- fold at 24 hr. In some NHS and most rat sera there appeared to be inhibitory factors. This inhibitory effect was decreased by heating at 56° for 30 min. The addition of phosphatidylcholine abolished some of this inhibition, suggesting that phospholipids may be a part of the SF receptor. Therefore, the isolated chondrocyte preparation is a highly sensitive and responsive system for studying hormonal regulation. The addition of normal human serum in this system results in a remarkable stimulation of DNA synthesis. Delivery of identical populations of chondrocytes to over 100 incubation tubes has the potentiality of greatly facilitating study of SF action and providing a more convenient quantitative assay. (Endocrinology90: 1086, 1972)