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

Article English OPEN
Orriss, I R ; Hajjawi, M O R ; Huesa, C ; Macrae, V E ; Arnett, T R ; Royal (Dick) School of Veterinary Studies ; UCL
  • Publisher: D.A. Spandidos
  • Journal: volume 34, issue 5, pages 1,201-1,208 (issn: 1107-3756, eissn: 1791-244X)
  • Related identifiers: pmc: PMC4199408, doi: 10.3892/ijmm.2014.1926
  • Subject: mineralisation | bone formation | alkaline phosphatase | osteoblast | Articles

<p>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.</p>
  • References (18)
    18 references, page 1 of 2

    1. Orriss IR, Taylor SE and Arnett TR: Rat osteoblast cultures. Methods Mol Biol 816: 31-41, 2012.

    2. Dillon JP, Waring-Green VJ, Taylor AM, Wilson PJ, Birch M, Gartland A and Gallagher JA: Primary human osteoblast cultures. Methods Mol Biol 816: 3-18, 2012.

    3. Bakker AD and Klein-Nulend J: Osteoblast isolation from murine calvaria and long bones. Methods Mol Biol 816: 19-29, 2012.

    4. Semeins CM, Bakker AD and Klein -Nulend J: Isolation of primary avian osteocytes. Methods Mol Biol 816: 43-53, 2012.

    5. Peck WA, Birge SJ Jr and Fedak SA: Bone cells: biochemical and biological studies after enzymatic isolation. Science 146: 1476-1477, 1964.

    6. Wong G and Cohn DV: Separation of parathyroid hormone and calcitonin‑sensitive cells from non‑responsive bone cells. Nature 252: 713-715, 1974.

    7. Bellows CG, Aubin JE, Heersche JN and Antosz ME: Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue Int 38: 143-154, 1986.

    8. Orriss IR, Utting JC, Brandao-Burch A, Colston K, Grubb BR, Burnstock G and Arnett TR: Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate. Endocrinology 148: 4208-4216, 2007.

    9. BrandaoB‑urch A, Utting JC, Orriss IR and Arnett TR: Acidosis inhibits bone formation by osteoblasts in vitro by preventing mineralization. Calcif Tissue Int 77: 167-174, 2005.

    10. Utting JC, Robins SP, Brandao -Burch A, Orriss IR, Behar J and Arnett TR: Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts. Exp Cell Res 312: 1693-1702, 2006.

  • Metrics
    No metrics available
Share - Bookmark