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

Orriss IR, Hajjawi MO, Huesa C, MacRae VE, Arnett TR - Int. J. Mol. Med. (2014)

Bottom Line: 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.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.The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, London, UK.

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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Rat calvarial osteoblast cultures form bone more abundantly in αMEM. All cells were cultured in 50 μg/ml ascorbate and 2 mM β-glycerophosphate. (A) The images are representative, whole well reflective light scans of rat osteoblast cell layers cultured using DMEM or αMEM supplemented with 10% fetal calf serum (FCS) or heat-inactivated FCS (HI FCS), with or without 10 nM dexamethasone. Cell layers are either unstained (white), stained with alizarin red to show bone mineralisation (red) or for tissue non-specific alkaline phosphatase (TNAP) expression (purple). Scale bar, 5 mm. (B) The level of bone mineralisation was 3-fold higher in the cells cultured in αMEM compared to DMEM. In cultures without dexamethasone, bone mineralisation was 85% lower (αMEM) or completely absent (DMEM). Heat inactivation of FCS did not have an effect on the level of bone formation. (C) Levels of TNAP expression were 2.5-fold higher in the cells cultured in αMEM. The absence of dexamethasone had no effect on TNAP expression when the cells were grown in αMEM, but TNAP expression was reduced by 3- to 4-fold in the cells cultured in DMEM. Heat inactivation of FCS did not affect TNAP expression. Values are the means ± SEM (n=6 replicate wells), ***p<0.001.
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f1-ijmm-34-05-1201: Rat calvarial osteoblast cultures form bone more abundantly in αMEM. All cells were cultured in 50 μg/ml ascorbate and 2 mM β-glycerophosphate. (A) The images are representative, whole well reflective light scans of rat osteoblast cell layers cultured using DMEM or αMEM supplemented with 10% fetal calf serum (FCS) or heat-inactivated FCS (HI FCS), with or without 10 nM dexamethasone. Cell layers are either unstained (white), stained with alizarin red to show bone mineralisation (red) or for tissue non-specific alkaline phosphatase (TNAP) expression (purple). Scale bar, 5 mm. (B) The level of bone mineralisation was 3-fold higher in the cells cultured in αMEM compared to DMEM. In cultures without dexamethasone, bone mineralisation was 85% lower (αMEM) or completely absent (DMEM). Heat inactivation of FCS did not have an effect on the level of bone formation. (C) Levels of TNAP expression were 2.5-fold higher in the cells cultured in αMEM. The absence of dexamethasone had no effect on TNAP expression when the cells were grown in αMEM, but TNAP expression was reduced by 3- to 4-fold in the cells cultured in DMEM. Heat inactivation of FCS did not affect TNAP expression. Values are the means ± SEM (n=6 replicate wells), ***p<0.001.

Mentions: Osteoblasts isolated from rat calvariae formed ‘trabecular-shaped’ mineralised bone nodules that were clearly visible by the eye, and expressed significant amounts of TNAP when cultured in both DMEM and αMEM. However, bone formation and TNAP expression were 2- to 3-fold higher in the cells cultured in αMEM than in the cells cultured in DMEM (Fig. 1A–C). The onset of mineralisation in the DMEM cultures occurred at day 10, with optimal bone formation at days 14–17, whereas the αMEM cultures showed marked acceleration, with mineralisation commencing at day 7 and striking bone formation observed between days 10–14. The heat inactivation of the FCS added to the culture media had no effect on mineralised bone nodule formation and TNAP staining.


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

Orriss IR, Hajjawi MO, Huesa C, MacRae VE, Arnett TR - Int. J. Mol. Med. (2014)

Rat calvarial osteoblast cultures form bone more abundantly in αMEM. All cells were cultured in 50 μg/ml ascorbate and 2 mM β-glycerophosphate. (A) The images are representative, whole well reflective light scans of rat osteoblast cell layers cultured using DMEM or αMEM supplemented with 10% fetal calf serum (FCS) or heat-inactivated FCS (HI FCS), with or without 10 nM dexamethasone. Cell layers are either unstained (white), stained with alizarin red to show bone mineralisation (red) or for tissue non-specific alkaline phosphatase (TNAP) expression (purple). Scale bar, 5 mm. (B) The level of bone mineralisation was 3-fold higher in the cells cultured in αMEM compared to DMEM. In cultures without dexamethasone, bone mineralisation was 85% lower (αMEM) or completely absent (DMEM). Heat inactivation of FCS did not have an effect on the level of bone formation. (C) Levels of TNAP expression were 2.5-fold higher in the cells cultured in αMEM. The absence of dexamethasone had no effect on TNAP expression when the cells were grown in αMEM, but TNAP expression was reduced by 3- to 4-fold in the cells cultured in DMEM. Heat inactivation of FCS did not affect TNAP expression. Values are the means ± SEM (n=6 replicate wells), ***p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199408&req=5

f1-ijmm-34-05-1201: Rat calvarial osteoblast cultures form bone more abundantly in αMEM. All cells were cultured in 50 μg/ml ascorbate and 2 mM β-glycerophosphate. (A) The images are representative, whole well reflective light scans of rat osteoblast cell layers cultured using DMEM or αMEM supplemented with 10% fetal calf serum (FCS) or heat-inactivated FCS (HI FCS), with or without 10 nM dexamethasone. Cell layers are either unstained (white), stained with alizarin red to show bone mineralisation (red) or for tissue non-specific alkaline phosphatase (TNAP) expression (purple). Scale bar, 5 mm. (B) The level of bone mineralisation was 3-fold higher in the cells cultured in αMEM compared to DMEM. In cultures without dexamethasone, bone mineralisation was 85% lower (αMEM) or completely absent (DMEM). Heat inactivation of FCS did not have an effect on the level of bone formation. (C) Levels of TNAP expression were 2.5-fold higher in the cells cultured in αMEM. The absence of dexamethasone had no effect on TNAP expression when the cells were grown in αMEM, but TNAP expression was reduced by 3- to 4-fold in the cells cultured in DMEM. Heat inactivation of FCS did not affect TNAP expression. Values are the means ± SEM (n=6 replicate wells), ***p<0.001.
Mentions: Osteoblasts isolated from rat calvariae formed ‘trabecular-shaped’ mineralised bone nodules that were clearly visible by the eye, and expressed significant amounts of TNAP when cultured in both DMEM and αMEM. However, bone formation and TNAP expression were 2- to 3-fold higher in the cells cultured in αMEM than in the cells cultured in DMEM (Fig. 1A–C). The onset of mineralisation in the DMEM cultures occurred at day 10, with optimal bone formation at days 14–17, whereas the αMEM cultures showed marked acceleration, with mineralisation commencing at day 7 and striking bone formation observed between days 10–14. The heat inactivation of the FCS added to the culture media had no effect on mineralised bone nodule formation and TNAP staining.

Bottom Line: 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.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.The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, London, UK.

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.

Show MeSH
Related in: MedlinePlus