CLONAL ANALYSIS OF RAT TIBIA GROWTH-PLATE CHONDROCYTES IN SUSPENSION-CULTURE - DIFFERENTIAL-EFFECTS OF GROWTH-HORMONE AND INSULIN-LIKE GROWTH-FACTOR-I

The number of growth hormone (GH) receptors in cultured rat epiphyseal chondrocytes are increased with numbers of cell divisions in monolayer. We wanted to study if increased number of cell divisions in monolayer influence GH or insulin-like growth factor I(IGF-I) response in a subsequent suspension culture. Primary isolated chondrocytes from rat tibia growth plates were cultured in monolayer at different seeding densities (4000, 8000 and 24 000 cells/cm(2)). After a culture period of 7 days, cells were trypsinized, counted and subcultured at 50 000 cells per dish in suspension stabilized with 0.5% agarose. 14 days later the agarose cultures were dried, stained and the number of clones with a diameter exceeding 50 microns was counted. Individual clones were classified as undifferentiated or differentiated according to the following criteria: cell clusters with a diameter of 50 microns and without matrix staining were classified as undifferentiated; cell clusters with a diameter over 50 microns consisting of 4 cells or more and with matrix stained by Alcian Blue were classified as differentiated clones. Human growth hormone (hGH) added to the suspension culture medium increased the number of undifferentiated clones if cells had been precultured at 4000 and 8000 cells/cm(2) but hGH had no stimulatory effect on either clone type at 24 000 cells/cm(2). IGF-I significantly increased the number of differentiated clones at all seeding densities while no effect was demonstrated on the number of undifferentiated clones. The results from the present study suggest that an increased number of cell divisions during primary monolayer culture increases GH responsiveness in a subsequent suspension culture. Furthermore, GH and IGF-I exerted differential stimulatory effects in this culture system; IGF-I primarily stimulated the growth of differentiated chondrocyte cell clones while GH mainly stimulated the growth of undifferentiated cell clones. These results demonstrate that the culture conditions are important for the GH effect and data are presented indicating that GH is a primary and IGF-I a secondary stimulatory effector in cartilage growth and maturation.