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21 February 2009

Cyclosporine A, but not tacrolimus, is associated with impaired proliferation and differentiation of human osteoblast-like cells in vitro

Rodrigo Oliveira MoreiraABCDEF, Leandro Souza ThiagoABDE, Felipe Leite OliveiraABDE, Alex BalduinoABDEF, Radovan BorojevicDEG, Maria Eugenia Leite DuarteADEFG, Maria Lucia Fleiuss FariasADEFG

Med Sci Monit 2009; 15(3): BR65-70 :: ID: 869575

Abstract

Background
The prevention of graft rejection after liver transplant with cyclosporine A (CyA) and tacrolimus has been associated to decreased bone mineral density. The aim of this study was to investigate the in vitro effects of increasing concentrations of CyA and tacrolimus on human osteoblasts.
Material and Method
Human osteoblast-like cells obtained by the outgrowth of cells from bone chips were exposed to tacrolimus (10-1000 ng/mL) or CyA (1000-50,000 ng/mL). Alkaline phosphatase (ALP) activity was determined on days 2, 4, 6, and 8, and cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell counting on days 2, 4, 6, and 8. Cell death was investigated by flow cytometry with propidium iodine, and apoptosis was assessed by flow cytometry with fluorescently conjugated annexin V.
Results
No effects of tacrolimus were detected with respect to ALP activity or cell proliferation. In CyA-treated cultures, concentrations greater than 10 000 ng/mL were associated with decreased ALP activity after 8 days (P<0.05). In addition, a dose-dependent reduction in cell proliferation was detected after 6 days (P<0.05). This decreased cell proliferation was associated with increased apoptosis in response to 50 000 ng/mL CyA.
Conclusions
Our results showed that CyA reduced, in a time- and concentration-dependent manner, the number of metabolically active cells via a decrease in proliferation and an increase in cell death, and induced impairment of osteoblast differentiation. These negative effects of CyA on human osteoblast-like cells might contribute to the bone loss reported in vivo.

Keywords: Staining and Labeling, Osteoblasts - enzymology, Cyclosporine - pharmacology, Dose-Response Relationship, Drug, Cells, Cultured, Cell Survival - drug effects, Cell Proliferation - drug effects, Cell Differentiation - drug effects, Cell Count, Annexins - metabolism, Alkaline Phosphatase - metabolism, Tacrolimus - pharmacology

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