Inhibition of chondrogenic differentiation in chick limb-bud mesenchyme microcultures treated with cyclosporine

Objectives: To explore the effects of cyclosporine (CsA) on skeletal development (chondrogenesis). Materials and Methods: Mesenchymal cells obtained from stage-23 to stage-24 chick-embryo limb buds were grown in 96-well plates using chemically defined tissue-culture medium. Cultures were treated with CsA (0.01-5.0 µg/ml) and incubated (37ºC, 5% CO2) with daily medium changes for 4 days. After incubation of the cells in multiwell plate, cartilage differentiation (chondrogenesis) was assessed by selectively staining sulfated glycosaminoglycans (GAGs) in the cartilage matrix with Alcian blue, extracting the GAGs with 4 M guanidinium HCl, and spectrophotometric analysis of the extracts. Results: CsA treatment had concentration-dependent effects on chick limb-bud mesenchymal cell cultures. At 5 µg/ml, CsA caused cell loss, as judged microscopically by the paucity of cells remaining at the end of the culture period. CsA concentrations between 0.1 and 1 µg/ml caused a marked, dose-dependent decrease in chondrogenesis. At 0.01 µg/ml, CsA had no significant effect on chondrogenesis. At concentrations above 0.01 µg/ml, normalized data showed significant chondrogenic inhibition at 0.5 and 1.0 µg/ml CsA. Conclusions: The findings suggest a possible biological basis for CsA-associated effects on mesenchyme-derived tissues and provide a model system for further studies.