Elsevier

Steroids

Volume 69, Issue 4, April 2004, Pages 219-226
Steroids

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

https://doi.org/10.1016/j.steroids.2003.12.005Get rights and content

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.

Introduction

Regulation of bone turnover is a complex process involving many endocrine, paracrine, and autocrine factors, as well as direct interactions between bone-forming and -resorbing cells. Studying the biologic effect of bone turnover modulators on bone cells requires in vitro models of phenotypically, stable osteoblasts and osteoclasts, where the direct effect on the individual cell types can be investigated. Tumor-derived cell lines have long been used as in vitro models for osteoblasts [1], [2], as they express many osteoblast traits, such as high alkaline phosphatase (AP) activity, production of type I collagen and noncollagenic proteins, and responsiveness to PTH stimulation. Their neoplastic nature, which disrupts many cellular functions, and cell line-specific phenotypic differences complicate the interpretation of data obtained in tumoral cells. A few immortalized cell lines isolated from normal tissue have been developed and are believed to better represent “normal” osteoblasts, although they are not always phenotypically stable. Examples are the mouse calvarial MC3T3 cell line, which represents immature cells that can differentiate and produce mineralized matrix in culture [3], and the human HOBIT cell line, derived from human cells from long bones [4].

Cell lines derived from human osteoblasts are preferred by some investigators, as they are more likely to reflect human bone biology than animal cell models. Primary cultures of human osteoblasts are commonly obtained from collagenase-digested bone explants, or from osteoblasts outgrowing from bone fragments in culture [5]. These cells can be obtained using established procedures and yield enriched cultures of mature osteoblasts, but typically these cells proliferate slowly, and sometimes they stop proliferating even before reaching confluence, thus greatly limiting the possibility of expanding such differentiated cell populations from each bone explant. Another approach to producing osteoblastic cell cultures is to use the bone marrow, which harbors multipotent mesenchymal stem cells that, under appropriate conditions, can differentiate into osteoblasts, adipocytes, or even myoblasts [6]. Bone marrow-derived stromal cells are still undifferentiated, and thus are able to proliferate at higher rates than adult differentiated osteoblasts.

Bone marrow stromal cells are commonly isolated from the spongiosa of the epiphysis or metaphysis of long bones (femurs, ribs), usually obtained from surgical specimens. These cells can be induced to differentiate into mature osteoblasts by culturing them in the presence of a stimulator. The earliest and more readily available inducer of bone marrow stromal cell differentiation is dexamethasone (Dex), which reliably stimulates the development of many, although not all, phenotypic features of human osteoblasts [7], [8]. However, glucocorticoids have deleterious effects on bone in vivo, resulting in inhibition of osteoblast function. Other inducers of osteoblast differentiation in vitro include tranforming growth factor-β (TGF-β) [9] and bone morphogenetic protein-2 (BMP-2) [10], [11], both members of the TGF-β gene superfamily, and essential for normal postnatal bone formation. BMP-2 has been used to induce osteoblast differentiation in vitro in many human and animal models, and it is believed to provide a more physiologic stimulus than Dex [10], [11], [12]. We report herein the development of a technique to produce human osteoblastic cell cultures from bone marrow biopsies. We have compared Dex- and BMP-2-treated cells, and found that both stimulators give rise to cells of the osteoblast phenotype, although BMP-2 appears to enhance more mature osteoblast characteristics.

Section snippets

Materials

1α,25-Dihydroxycholecalciferol (Vit.D) was kindly provided by Leo Pharmaceuticals (Ballerup, Denmark). Human recombinant BMP-2 was obtained from R&D Systems European Ltd. (Abingdon, United Kingdom). Dex (water soluble), heparin, the Ficoll-Hypaque density gradient (Histopaque-1077), and fetal bovine serum were obtained from Sigma-Aldrich Denmark A/S (Vallensbaek Strand, Denmark). Phosphate-Buffered Saline with Ca and Mg (PBS+), or without Ca and Mg (PBS−), trypsin/EDTA, Minimum Essential

Alkaline phosphatase activity is increased by Dex, but not by BMP-2 treatment

Human bone marrow-derived stromal cells were treated for 7 days with either Vit.D, Dex, BMP-2 alone, or in different combinations. Vit.D or Dex alone increased AP activity, whereas BMP-2 alone was not as effective on this parameter. The combination of Dex with BMP-2 or Vit.D, or with both, showed an additive effect, whereas BMP-2 in combination with Vit.D did not significantly increase AP activity in these cells (Fig. 1). These results were corroborated by AP staining of the cells on chamber

Discussion

The main objective of this work was to validate an in vitro model of human osteoblastic cells obtained from iliac crest biopsies of bone marrow from young healthy volunteers. It is generally accepted that osteoblasts are derived from bone marrow stromal cell precursors. The data presented herein demonstrate that human cells isolated via standard biopsy procedures can be used to produce osteoblastic cells that may be suitable as a model for in vitro investigations. Our data also demonstrate that

Acknowledgements

We are grateful to Professor Thomas H. Steinberg for his critical review of and comments to the manuscript. We thank Michel Normark for kind technical assistance with the histochemical staining. This work was supported by grant no. 9502125 from the Danish Research Council, Copenhagen Hospital Corporation Research Foundation, Leo Pharmaceutical Company Research Foundation, Brødrene Hartmanns Foundation, The Foundation for the Advance of Medical Science (The A.P. Møller Foundation) to NRJ,

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