Effects of Dexamethasone and Vitamin D₃on Cartilage Differentiation in a Clonal Chondrogenic Cell Population

Abstract

We have investigated the regulation of chondroblast/chondrocyte differentiation using a unique clonal cell population, designated RCJ 3.1C5 (C5), which differentiates into discrete three-dimensional cartilage nodules when grown in the presence of 15% fetal calf serum. Histologically, the nodules resembled hyaline cartilage; they contained large rounded chondrocytes surrounded by a refractile matrix which stained intensely with Alcian blue, exhibited metachromasia after Toluidine blue staining, and stained with an antibody against type II collagen. The cartilage nodules that formed did not mineralize, despite the presence of organic phosphate in the culture medium. The synthetic glucocorticoid dexamethasone (DEX) increased the number of cartilage nodules formed in a dose-dependent manner (ED50, approximately 10(-9) M), with a maximal stimulatory dose of 10(-8) M. DEX had no effect on the population doubling time and saturation density. The effects of DEX on the number of cartilage nodules were similar whether it was added from the beginning of the culture period (starting during exponential growth) or at confluence. In contrast, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited cartilage nodule formation in a dose-dependent manner (IC50, approximately 5 x 10(-10) M), with maximum inhibition at 10(-7) M. In addition, 1,25-(OH)2D3 decreased cell proliferation and saturation density. Equimolar doses of the vitamin D3 metabolites 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 had no effect. C5 cells treated with 1,25-(OH)2D3 in the absence of DEX during the exponential growth phase exhibited a reduced capacity to form cartilage nodules upon subsequent exposure to DEX. At confluence, before cartilage nodules had formed, C5 cells responded to PTH and prostaglandin-E2 with increases in intracellular cAMP of about 10- and 95-fold respectively. After cartilage nodules were present, prostaglandin-E2 responsiveness decreased to about 25-fold, whereas there was no significant change in PTH responsiveness. DEX decreased the population alkaline phosphatase levels at all times measured, whereas 1,25-(OH)2D3 had a biphasic effect: an increase at 5 days in culture, followed by a decrease at later times in culture. These data indicate that the clonal cell line RCJ 3.1C5 is a useful model system in which to investigate cartilage differentiation.

Optimal Combination of Soluble Factors for Tissue Engineering of Permanent Cartilage from Cultured Human Chondrocytes

“…These doses of individual factors were within the range between effective dose 75 and effective dose 100 of their proper effects on chondrocytes, according to dose-response curves in the previous reports (11,12,15,16,(21)(22)(23)(24)(25)(26)(27)(28) cells in 5 l of 0.8% atelocollagen gel) were cultured in Dulbecco's modified Eagle's medium/F-12 containing the combinations for 2 weeks, and the GAG accumulation was measured as described below. Parameter estimates of the GAG accumulation by one factor or two were calculated from the F values that represent the effects of the individual factors and the interaction terms of two factors by the analysis of variance using the software above.…”

“…IL-1RA, 20 ng/ml; growth hormone, 100 ng/ml; 17␤-estradiol, 100 nM; T3, 100 nM; 1␣-25-dihydroxy vitamin D3, 100 nM; FGF-2, 10 ng/ml; dexamethasone, 100 nM, based on previous reports by us and others (8,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). These doses of individual factors were within the range between effective dose 75 and effective dose 100 of their proper effects on chondrocytes, according to dose-response curves in the previous reports (11,12,15,16,(21)(22)(23)(24)(25)(26)(27)(28) cells in 5 l of 0.8% atelocollagen gel) were cultured in Dulbecco's modified Eagle's medium/F-12 containing the combinations for 2 weeks, and the GAG accumulation was measured as described below.…”

Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-β in mesenchymal chondroprogenitor cell growth

“…Cells plated at a density of 6 10 4 cells/well in six-well dishes, without requiring biochemical or oncogenic transformation undergo, a reproducible, time-dependent progression from MCCs, during the first 4 days of culture, to early (7-10 days of culture) and then terminally differentiated chondrocytes (14 days of culture) when fresh medium with 50 µg/ml ascorbic acid and 10 mM -glycerophosphate is added (Grigoriadis et al 1988, 1989, Lunstrum et al 1999, Spagnoli et al 2001. As previously reported, the morphology, the histochemical markers and the temporal sequential acquisition of the chondrocytic phenotype in the RCJ3·1C5·18 cell system mimics the chondrogenesis process that occurs in vivo (Lunstrum et al 1999, Spagnoli et al 2001.…”

Dexamethasone enhances SOX9 expression in chondrocytes

“…Glucocorticoids have been demonstrated to promote differentiation of chondrocytes and to maintain the integrity of the cartilaginous matrix in isolated primary cell populations (Jones & Addison 1975, Kato & Gospodarowicz 1985, Takano et al 1985, Horton et al 1987, Quarto et al 1992 as well as in chondrogenic cell lines (Calcagno et al 1970, Grigoriadis et al 1988, 1989. In addition, glucocorticoids suppress cartilage phenotype expression in vitro and in vivo depending on culture conditions (Tessler & Salmon 1975, Silbermann & Maor 1985.…”