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Isolation and Generation of Osteoblasts

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Bone Research Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1914))

Abstract

This chapter describes the isolation, culture, and staining of osteoblasts. The key advantages of this assay are that it allows direct measurement of bone matrix deposition and mineralization, as well as yielding good quantities of osteoblasts at defined stages of differentiation for molecular and histological analysis. An additional focus of this chapter will be the culture of osteoblasts from less conventional animal species.

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References

  1. Peck WA, Birge SJJ, Fedak SA (1964) Bone cells: biochemical and biological studies after enzymatic isolation. Science 146:1476–1477

    Article  CAS  Google Scholar 

  2. Orriss IR, Taylor SE, Arnett TR (2012) Rat osteoblast cultures. Methods Mol Biol 816:31–41

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Taylor SE, Shah M, Orriss IR (2014) Generation of rodent and human osteoblasts. Bonekey Rep 3:85

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Shah M, Gburcik V, Reilly P, Sankey RA, Emery RJ, Clarkin CE, Pitsillides AA (2015) Local origins impart conserved bone type-related differences in human osteoblast behaviour. Eur Cell Mater 29:155–175

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Brandao-Burch A, Utting JC, Orriss IR, Arnett TR (2005) Acidosis inhibits bone formation by osteoblasts in vitro by preventing mineralization. Calcif Tissue Int 77:167–174

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Beresford JN, Gallagher JA, Poser JW, Russell RG (1984) Production of osteocalcin by human bone cells in vitro. Effects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids. Metab Bone Dis Relat Res 5:229–134

    Article  CAS  Google Scholar 

  13. Couchourel D, Aubry I, Delalandre A, Lavigne M, Martel-Pelletier J, Pelletier JP, Lajeunesse D (2009) Altered mineralization of human osteoarthritic osteoblasts is attributable to abnormal type I collagen production. Arthritis Rheum 60:1438–1450

    Article  Google Scholar 

  14. Ranzoni AM, Corcelli M, Hau KL, Kerns JG, Vanleene M, Shefelbine S, Jones GN, Moschidou D, Dala-Ali B, Goodship AE, De Coppi P, Arnett TR, Guillot PV (2016) Counteracting bone fragility with human amniotic mesenchymal stem cells. Sci Rep 6:39656

    Article  CAS  Google Scholar 

  15. Perpetuo IP, Meeson R, Pitsillides AA, Doube M, Orriss IR (2016) Primary osteoblast culture from domestic dog (Canis lupus familiaris). Bone Abstracts 5:P166

    Google Scholar 

  16. Perpetuo IP, Shah M, Parsons K, Orriss IR, Doube M, Pitsillides AA, Meeson R (2016) Canine osteoblasts from trabecular, cortical and subchondral bone present differences in alkaline phosphatase activity. Bone Abstracts 5:P165

    Google Scholar 

  17. Lajeunesse D, Martel-Pelletier J, Fernandes JC, Laufer S, Pelletier JP (2004) Treatment with licofelone prevents abnormal subchondral bone cell metabolism in experimental dog osteoarthritis. Ann Rheum Dis 63:78–83

    Article  CAS  Google Scholar 

  18. Perpetuo IP, Felder A, Pitsillides AA, Doube M, Orriss IR (2016) Primary osteoblast culture from red fox (Vulpes Vulpes). Bone Abstracts 5:P144

    Google Scholar 

  19. Bigi A, Panzavolta S, Sturba L, Torricelli P, Fini M, Giardino R (2006) Normal and osteopenic bone-derived osteoblast response to a biomimetic gelatin-calcium phosphate bone cement. J Biomed Mater Res 78:739–745

    Article  CAS  Google Scholar 

  20. Torricelli P, Fini M, Giavaresi G, Rocca M, Pierini G, Giardino R (2000) Isolation and characterization of osteoblast cultures from normal and osteopenic sheep for biomaterials evaluation. J Biomed Mater Res 52:177–182

    Article  CAS  Google Scholar 

  21. Schmitt SC, Wiedmann-Al-Ahmad M, Kuschnierz J, Al-Ahmad A, Huebner U, Schmelzeisen R, Gutwald R (2008) Comparative in vitro study of the proliferation and growth of ovine osteoblast-like cells on various alloplastic biomaterials manufactured for augmentation and reconstruction of tissue or bone defects. J Mater Sci Mater Med 19:1441–1450

    Article  CAS  Google Scholar 

  22. McDuffee LA, Anderson GI (2003) In vitro comparison of equine cancellous bone graft donor sites and tibial periosteum as sources of viable osteoprogenitors. Vet Surg 32:455–463

    Article  Google Scholar 

  23. Patel JJ, Utting JC, Key ML, Orriss IR, Taylor SE, Whatling P, Arnett TR (2012) Hypothermia inhibits osteoblast differentiation and bone formation but stimulates osteoclastogenesis. Exp Cell Res 318:2237–2244

    Article  CAS  Google Scholar 

  24. Perpetuo IP, Orriss IR, and Doube M (2017) Incubation at physiological temperature promotes ovine osteoblast proliferation and activity. Bone research society annual meeting, p. 76

    Google Scholar 

  25. Orriss IR, Hajjawi MO, Huesa C, MacRae VE, Arnett TR (2014) Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats. Int J Mol Med 34:1201–1208

    Article  CAS  Google Scholar 

  26. Chen TL, Cone CM, Feldman D (1983) Glucocorticoid modulation of cell proliferation in cultured osteoblast-like bone cells: differences between rat and mouse. Endocrinology 112:1739–1745

    Article  CAS  Google Scholar 

  27. Banerjee C, McCabe LR, Choi JY, Hiebert SW, Stein JL, Stein GS, Lian JB (1997) Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex. JCell Biochem 66:1–8

    Article  CAS  Google Scholar 

  28. Li H, Qian W, Weng X, Wu Z, Li H, Zhuang Q, Feng B, Bian Y (2012) Glucocorticoid receptor and sequential P53 activation by dexamethasone mediates apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells. PLoS One 7:e37030

    Article  CAS  Google Scholar 

  29. Li J, Zhang N, Huang X, Xu J, Fernandes JC, Dai K, Zhang X (2013) Dexamethasone shifts bone marrow stromal cells from osteoblasts to adipocytes by C/EBPalpha promoter methylation. Cell Death Dis 4:e832

    Article  CAS  Google Scholar 

  30. Esmail MY, Sun L, Yu L, Xu H, Shi L, Zhang J (2012) Effects of PEMF and glucocorticoids on proliferation and differentiation of osteoblasts. Electromagn Biol Med 31:375–381

    Article  CAS  Google Scholar 

  31. Orriss IR, Key ML, Hajjawi MO, Millan JL, Arnett TR (2015) Acidosis is a key regulatior of osteoblast ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity. J Cell Physiol 230:3049–3056

    Article  CAS  Google Scholar 

  32. Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636

    Article  CAS  Google Scholar 

  33. Orriss IR, Arnett TR (2012) Rodent osteoclast cultures. Methods Mol Biol 816:103–117

    Article  CAS  Google Scholar 

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Acknowledgment

The authors gratefully acknowledge the support of The Leverhulme Trust and Arthritis Research UK.

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Authors and Affiliations

  1. Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK

    Inês P. Perpétuo, Lucie E. Bourne & Isabel R. Orriss

Authors
  1. Inês P. Perpétuo
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  2. Lucie E. Bourne
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  3. Isabel R. Orriss
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Corresponding author

Correspondence to Isabel R. Orriss .

Editor information

Editors and Affiliations

  1. Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK

    Aymen I. Idris

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Perpétuo, I.P., Bourne, L.E., Orriss, I.R. (2019). Isolation and Generation of Osteoblasts. In: Idris, A. (eds) Bone Research Protocols. Methods in Molecular Biology, vol 1914. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8997-3_2

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  • DOI: https://doi.org/10.1007/978-1-4939-8997-3_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8996-6

  • Online ISBN: 978-1-4939-8997-3

  • eBook Packages: Springer Protocols

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