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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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Tissue culture conditions are more important to the success of the culture than the initial method of obtaining cells. In order to determine how important the method of isolation is in the success of the culture, rodent cells were obtained using both the trypsin-collagenase-collagenase (TCC) and collagenase-collagenase-EDTA-collagenase (CCEC) digestion protocols. Cells were cultured in αMEM with heat inactivated FCS (HI FCS), 50 μg/ml ascorbate and 2 mM β-glycerophosphate (plus 10 nM dexamethasone, rat only) for up to 28 days. Mature, mineralising osteoblasts were generated from rat and mouse calvarial cells using both isolation methods. (A) The level of bone formation and tissue non-specific alkaline phosphatase (TNAP) expression were 25 and 35% lower, respectively, in rat cells obtained using CCEC digestion. (B) The method of isolation did not affect the level of bone mineralisation or TNAP expression in mouse osteoblasts. Values are the means ± SEM (n=6 replicate wells), **p<0.01.
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f6-ijmm-34-05-1201: Tissue culture conditions are more important to the success of the culture than the initial method of obtaining cells. In order to determine how important the method of isolation is in the success of the culture, rodent cells were obtained using both the trypsin-collagenase-collagenase (TCC) and collagenase-collagenase-EDTA-collagenase (CCEC) digestion protocols. Cells were cultured in αMEM with heat inactivated FCS (HI FCS), 50 μg/ml ascorbate and 2 mM β-glycerophosphate (plus 10 nM dexamethasone, rat only) for up to 28 days. Mature, mineralising osteoblasts were generated from rat and mouse calvarial cells using both isolation methods. (A) The level of bone formation and tissue non-specific alkaline phosphatase (TNAP) expression were 25 and 35% lower, respectively, in rat cells obtained using CCEC digestion. (B) The method of isolation did not affect the level of bone mineralisation or TNAP expression in mouse osteoblasts. Values are the means ± SEM (n=6 replicate wells), **p<0.01.

Mentions: Rodent osteoblasts were isolated using both TCC and CCEC digestion protocols and cultured in αMEM with HI FCS, 50 μg/ml ascorbate and 2 mM β-glycerophosphate (plus 10 nM dexamethasone for rat cells). We found that mature, bone-forming osteoblasts that expressed TNAP were generated from rat and mouse calvarial cells using both methods, although the CCEC protocol appeared somewhat less efficient for rat cells (Fig. 6). The optimal conditions determined in this study for culturing bone-forming rodent osteoblasts are summarised in Table I.


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)

Tissue culture conditions are more important to the success of the culture than the initial method of obtaining cells. In order to determine how important the method of isolation is in the success of the culture, rodent cells were obtained using both the trypsin-collagenase-collagenase (TCC) and collagenase-collagenase-EDTA-collagenase (CCEC) digestion protocols. Cells were cultured in αMEM with heat inactivated FCS (HI FCS), 50 μg/ml ascorbate and 2 mM β-glycerophosphate (plus 10 nM dexamethasone, rat only) for up to 28 days. Mature, mineralising osteoblasts were generated from rat and mouse calvarial cells using both isolation methods. (A) The level of bone formation and tissue non-specific alkaline phosphatase (TNAP) expression were 25 and 35% lower, respectively, in rat cells obtained using CCEC digestion. (B) The method of isolation did not affect the level of bone mineralisation or TNAP expression in mouse osteoblasts. Values are the means ± SEM (n=6 replicate wells), **p<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6-ijmm-34-05-1201: Tissue culture conditions are more important to the success of the culture than the initial method of obtaining cells. In order to determine how important the method of isolation is in the success of the culture, rodent cells were obtained using both the trypsin-collagenase-collagenase (TCC) and collagenase-collagenase-EDTA-collagenase (CCEC) digestion protocols. Cells were cultured in αMEM with heat inactivated FCS (HI FCS), 50 μg/ml ascorbate and 2 mM β-glycerophosphate (plus 10 nM dexamethasone, rat only) for up to 28 days. Mature, mineralising osteoblasts were generated from rat and mouse calvarial cells using both isolation methods. (A) The level of bone formation and tissue non-specific alkaline phosphatase (TNAP) expression were 25 and 35% lower, respectively, in rat cells obtained using CCEC digestion. (B) The method of isolation did not affect the level of bone mineralisation or TNAP expression in mouse osteoblasts. Values are the means ± SEM (n=6 replicate wells), **p<0.01.
Mentions: Rodent osteoblasts were isolated using both TCC and CCEC digestion protocols and cultured in αMEM with HI FCS, 50 μg/ml ascorbate and 2 mM β-glycerophosphate (plus 10 nM dexamethasone for rat cells). We found that mature, bone-forming osteoblasts that expressed TNAP were generated from rat and mouse calvarial cells using both methods, although the CCEC protocol appeared somewhat less efficient for rat cells (Fig. 6). The optimal conditions determined in this study for culturing bone-forming rodent osteoblasts are summarised in Table I.

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