In vivo and in vitro human osteoblast morphology and Cyclosporin A treatment

Introduction Osteoporosis and osteomalacia are both characterized by an imbalance between synthesis, degradation and mineralization of extracellular matrix (ECM). Cyclosporin A (CyA) is able to modify the ECM components such as collagen and proteoglycans. Cell activity is dependent on cell morphology and substrate cell attachment. Moreover, cell morphology and polarization are dependent on cytoskeletal organization. In this work, we have treated normal human osteoblasts with CyA and analysed the gene expression related to cell cytoskeleton and polarization by microarray and immunofluorescent antibody methods. Materials and Methods Cells obtained from iliac crest of 5 healthy patients without immune and bone pathologies, were grown in culture flasks with 199 medium supplemented with 10% fetal calf bovine serum (FBS) and antibiotics, at 37°C and 5% CO2. At the 4th passage, fully confluent cultures were maintained in 199 medium containing 10% FBS alone or 800 mg/ml CyA for 24 hours. After this time, the gene expression related to actin polymerization, focal adhesion and Wnt signaling pathways were analyzed by microarray methods, cytoskeletal components by fluorescent antibodies and cellular organules by transmission electron microscope. Results The microarray analysis shows that the CyA inhibits or stimulates the gene related to actin polimerization, focal adhesion and Wnt signaling pathways. The immunofluorescent observations show that the CyA decreases actin (P≤0.01), while the tubulin of cytoskeleton does not change. TEM observations of osteoblasts cultures show that rough endoplasmic reticulum and secretory vesicles are increased in treated osteoblasts compared to controls. The optical microscope observations do not show morphological osteoblasts changes between treated and control cultures, while in vivo the osteoblasts show different morphology. Discussion Our data show that in vitro CyA modifies the cytoskeletal components and gene expression related to osteoblast morphology and polarity, in accordance with in vivo data, showing that osteoblasts have different morphology and ECM synthesis in osteoporosis and osteomalacia. Since CyA-treated patients develop different bone pathologies, it suggests a possible genetic sensitivity according to Tipon et al. (1991) that observed inter-individual heterogeneity in the collagenolytic responses of gingival fibroblasts after CyA treatment. Moreover, in vivo the bone physiology is the result of the relation between several cells and stimuli that are omissis in vitro. These data seem suggest that in CyA-treated patients the bone pathologies could be the result of multiple effects at cellular level according to literature.