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

Objectives: To explore the effects of cyclosporine (CsA) on skeletal development (chondro­ genesis). Anatomy Department, Shiraz Materials and Methods: Mesenchymal cells obtained from stage-23 to stage-24 chickUniversity of Medical embryo limb buds were grown in 96-well plates using chemically defined tissue-culture Sciences, Shiraz, Iran. medium. Cultures were treated with CsA (0.01–5.0 μg/ml) and incubated (37oC, 5% CO 2 ) with Departments of Anatomy & daily medium changes for 4 days. After incubation of the cells in multiwell plate, cartilage Neurobiology, Morehouse School of Medicine, Atlanta, differentiation (chondrogenesis) was assessed by selectively staining sulfated GA, USA. glycosaminoglycans (GAGs) in the cartilage matrix with Alcian blue, extracting the GAGs with 4M guanidinium HCl, and spectrophotometric analysis of the extracts. Received: 14.5.2005 Results: CsA treatment had concentration-dependent effects on chick limb-bud mesenchyRevised: 6.10.2005 mal cell cultures. At 5 μg/ml, CsA caused cell loss, as judged microscopically by the paucity of Accepted: 30.10.2005 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 Correspondence to: significant effect on chondrogenesis. At concentrations above 0.01 μg/ml, normalized data Soghra Bahmanpour showed significant chondrogenic inhibition at 0.5 and 1.0 μg/ml CsA. E-mail: [email protected] Conclusions: The findings suggest a possible biological basis for CsA-associated effects on mesenchyme-derived tissues and provide a model system for further studies.