title: Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts.
abstract: In vitro models of bone cells are important for the study of bone biology, including the regulation of bone formation and resorption. In this study, we have validated an in vitro model of human osteoblastic cells obtained from bone marrow biopsies from healthy, young volunteers, aged 20-31 years. Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro.
authors.affiliation: Osteoporosis and Bone Metabolic Unit, Copenhagen University Hospital Hvidovre, Kettdgaard Allé 30, Hvidovre DK-2650, Denmark. niklas@dadlnet.dk
authors.forename: N R
authors.initials: NR
authors.lastname: Jørgensen
authors.forename: Z
authors.initials: Z
authors.lastname: Henriksen
authors.forename: O H
authors.initials: OH
authors.lastname: Sørensen
authors.forename: R
authors.initials: R
authors.lastname: Civitelli
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D015519:Bone Density; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis; D000242:Cyclic AMP / Q000096:biosynthesis; D003907:Dexamethasone / Q000494:pharmacology*; D005260:Female; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D008297:Male; D008954:Models, Biological; D010006:Osteoblasts / Q000187:drug effects* / Q000502:physiology; D015675:Osteocalcin / Q000096:biosynthesis; D010281:Parathyroid Hormone / Q000096:biosynthesis; D010641:Phenotype; D015203:Reproducibility of Results; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology*
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D005938:Glucocorticoids; D010281:Parathyroid Hormone; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D014807:Vitamin D; C097949:1,25-dihydroxyvitamin D; D003907:Dexamethasone; D000242:Cyclic AMP; D000469:Alkaline Phosphatase