Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats

Orriss,Isabel  R.; Hajjawi,Mark  O.R.; Huesa,Carmen; MacRae,Vicky  E.; Arnett,Timothy  R.

doi:10.3892/ijmm.2014.1926

November-2014 Volume 34 Issue 5

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November-2014 Volume 34 Issue 5

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Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats

Authors:
- Isabel R. Orriss
- Mark O.R. Hajjawi
- Carmen Huesa
- Vicky E. MacRae
- Timothy R. Arnett
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Affiliations: Department of Cell and Developmental Biology, University College London, London, UK, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, UK, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, UK

Copyright: © Orriss et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].
Pages: 1201-1208
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Published online on: September 8, 2014

https://doi.org/10.3892/ijmm.2014.1926
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Abstract

The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of bone formation. The widespread use of transgenic mice has created a particular need for a reliable, simple method that allows the differentiation and bone‑forming activity of murine osteoblasts to be studied. In the present study, we established such a method and identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells isolated from neonatal rodent calvariae by collagenase digestion were cultured for 14‑28 days before staining for tissue non-specific alkaline phosphatase (TNAP) and bone mineralisation (alizarin red). The reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of mineralised ‘trabecular‑shaped’ bone nodules, occurred only following culture in α minimum essential medium (αMEM) and took 21‑28 days. Dexamethasone (10 nM) inhibited bone mineralisation in the mouse osteoblasts. By contrast, TNAP expression and bone formation by rat osteoblasts were observed following culture in both αMEM and Dulbecco's modified Eagle's medium (DMEM) after approximately 14 days (although ~3‑fold more effectively in αMEM) and was strongly dependent on dexamethasone. Both the mouse and rat osteoblasts required ascorbate (50 µg/ml) for osteogenic differentiation and β‑glycerophosphate (2 mM) for mineralisation. The rat and mouse osteoblasts showed similar sensitivity to the well‑established inhibitors of mineralisation, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP; 1‑100 µM). The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former. These findings offer a reliable technique for inducing mouse osteoblasts to form bone in vitro and a more effective method for culturing bone‑forming rat osteoblasts.

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Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats

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