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

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

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)1201-8
Number of pages8
JournalInternational Journal of Molecular Medicine
Volume34
Issue number5
DOIs
Publication statusPublished - 8 Sep 2014
Externally publishedYes

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Osteoblasts
Osteogenesis
Alkaline Phosphatase
Physiologic Calcification
Eagles
Dexamethasone
Rodentia
Adenosine Triphosphate
In Vitro Techniques
Glycerophosphates
Bone and Bones
Collagenases
Skull
Transgenic Mice
Digestion
Staining and Labeling

Keywords

  • Alkaline Phosphatase
  • Animals
  • Ascorbic Acid
  • Calcification, Physiologic
  • Cell Differentiation
  • Culture Media
  • Dexamethasone
  • Glycerophosphates
  • Mice
  • Osteoblasts
  • Osteogenesis
  • Primary Cell Culture
  • Rats
  • Skull
  • Comparative Study
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Orriss, Isabel R ; Hajjawi, Mark O R ; Huesa, Carmen ; MacRae, Vicky E ; Arnett, Timothy R. / Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats. In: International Journal of Molecular Medicine. 2014 ; Vol. 34, No. 5. pp. 1201-8.
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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.

In: International Journal of Molecular Medicine, Vol. 34, No. 5, 08.09.2014, p. 1201-8.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats

AU - Orriss, Isabel R

AU - Hajjawi, Mark O R

AU - Huesa, Carmen

AU - MacRae, Vicky E

AU - Arnett, Timothy R

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AB - 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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KW - Animals

KW - Ascorbic Acid

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KW - Cell Differentiation

KW - Culture Media

KW - Dexamethasone

KW - Glycerophosphates

KW - Mice

KW - Osteoblasts

KW - Osteogenesis

KW - Primary Cell Culture

KW - Rats

KW - Skull

KW - Comparative Study

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

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DO - 10.3892/ijmm.2014.1926

M3 - Article

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SP - 1201

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