Skip to main content
SpringerLink
Log in

Isolation and Culture of Bone-Forming Cells (Osteoblasts) from Human Bone

  • Protocol
Human Cell Culture Protocols

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 2))

Abstract

The most conspicuous function of the osteoblast is the formation of bone. During phases of active bone formation, osteoblasts synthesize bone matrix and prime it for subsequent mineralization. Active osteoblasts are plump, cuboidal cells rich in organelles involved in the synthesis and secretion of matrix proteins. Unlike fibroblasts, they are obviously polarized, secreting matrix onto the underlying bony substratum which consequently grows by apposition. Some osteoblasts are engulfed in matrix during bone formation and are entombed in lacunae. These cells are described as osteocytes and remain in the bone matrix in a state of low metabolic activity. At the completion of a phase of bone formation, those osteoblasts which avoided entombment in lacunae lose their prominent synthetic function and become inactive osteoblasts, otherwise known as bone-lining cells. In mature bone, lining cells cover most of the bone surfaces. Osteocytes and bone-lining cells should not be considered as inactive cells since they play a major role in the regulation of bone modeling and remodeling and in calcium homeostasis (1).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gallagher, J. A., (1991) Human bone remodeling, in Encyclopaedia of Human Biology vol. 1 (Dulbecco, R., ed.), Academic, San Diego, CA, pp. 881–923.

    Google Scholar 

  2. Gallagher, J. A., Beresford, J. N., Caswell, A., and Russell, R. G. G., (1987) Subcellular investigation of skeletal tissue, in Subcellular Pathology of Systemic Disease (Peters, T. J., ed.), Chapman and Hall, London, pp. 377–397.

    Google Scholar 

  3. Bard, D. R., Dickens, M. J., Smith, A. U., and Zarek, J. M., (1972) Isolation of living cells from mature mammalian bone. Nature 236, 314,315.

    Article  PubMed  CAS  Google Scholar 

  4. Bard, D. R., Dickens, M. J., Edwards, J., and Smith, A. U., (1974) Ultra-structure, in vitro cultivation and metabolism of cells isolated from arthritic human bone. J. Bone Joint Surg. 56B, 352–360.

    Google Scholar 

  5. Mills, B. G., Singer, F. R., Weiner, L. P., and Hoist, P. A., (1979) Long term culture of cells from bone affected with Paget’s disease. Calcif. Tiss. Int. 29, 79–87.

    Article  CAS  Google Scholar 

  6. Beresford, J. N., Gallagher, J. A., Gowen, M., McGuire, M., Poser, J. W., and Russell, R. G. G., (1983) Human bone cells in culture. A novel system for the investigation of bone cell metabolism. Clin. Sci. 64, 38,39.

    Google Scholar 

  7. Gallagher, J. A., Beresford, J. N., McGuire, M., Ebsworth, N. M., Meats, J. E., Gowen, M., Elford, P., Wright P., Poser, J., Coulton, L. A., Sharrard, M., Imbimbo, Kanis, J. A., and Russell, R. G. G., (1983) Effects of glucocorticoids and anabolic steroids on cells derived from human skeletal and articular tissues in vitro, in Glucocorticoid Effects and their Biological Consequences (Imbimbo, B., and Avioli, L. V., eds.), Plenum, New York, pp. 279–292.

    Google Scholar 

  8. Beresford, J. N., Gallagher, J. A., Poser, J. W., and Russell, R. G. G., (1984) Production of osteocalcin by human bone cells in vitro. Effects of 1,25(OH)2D3 para-thyroid hormone and glucocorticoids. Metab. Bone Dis. Rel. Res. 5, 229–234.

    Article  CAS  Google Scholar 

  9. MacDonald, B. R., Gallagher, J. A., Ahnfelt-Ronne, I., Beresford, J. N., Gowen, M., and Russell, R. G. G., (1984) Effects of bovine parathyroid hormone and 1,25(OH)2D3 on the production of prostaglandins by cells derived from human bone. FEBS Lett. 169, 49–52

    Article  PubMed  CAS  Google Scholar 

  10. Beresford, J. N., Gallagher, J. A., Gowen, M., Couch, M., Poser, J., Wood, D. D., and Russell, R. G. G., (1984) The effects of monocyte-conditioned medium and interleukin 1 on the synthesis of collagenous and non-collagenous proteins by mouse bone and human bone cells in vitro. Biochim. Biophysics. Acta Gen. Subj. 801, 58–65.

    Article  CAS  Google Scholar 

  11. Skjodi, H., Gallagher, J. A., Beresford, J. N., Couch, M., Poser, J. W., and Russell, R. G. G., (1985) Vitamin D metabolites regulate osteocalcin synthesis and proliferation of human bone cells in vitro. J. Endocrinol. 105, 391–396.

    Article  Google Scholar 

  12. Beresford, J. N., Gallagher, J. A., and Russell, R. G. G., (1986) 1,25-dihydroxy-vitamin D3 and human bone derived cells in vitro: effects on alkaline phosphatase, type I collagen and proliferation. Endocrinology 119, 1776–1785.

    Article  PubMed  CAS  Google Scholar 

  13. MacDonald, B. R., Gallagher, J. A., and Russell, R. G. G., (1986) Parathyroid hormone stimulates the proliferation of cells derived from human bone. Endocrinology 118, 2445–2449.

    Article  PubMed  CAS  Google Scholar 

  14. Vaishnav, R., Gallagher, J. A., Beresford, J. N., Poser, J. W., and Russell, R. G. G., (1984) Direct effects of stanozolol and oestrogen on human bone cells in culture, in Osteoporosis, Proceedings of Copenhagen international Symposium (Christiansen, C, ed.), Glostrup Hospital, Copenhagen, Denmark, pp. 485–488.

    Google Scholar 

  15. Treble, N. J., Dorgan, J. and Gallagher, J. A. (1990) Maintenance of cell viability in stored bone. Spine 15, 830–832.

    Article  PubMed  CAS  Google Scholar 

  16. Maurizi, M., Binaglia, L., Donti, E., Ottaviani, F., Paludetti, G., and Venti Donti, G. (1983) Morphological and functional characterisation of human temporal-bone cultures. Cell Tiss. Res. 229, 505–513.

    Article  CAS  Google Scholar 

  17. Wergedal, J. E. and Baylink, D. J. (1984) Characterization of cells isolated and cultured from human trabecular bone. Proc. Sot. Exp. Biol. Med. 176, 60–69.

    CAS  Google Scholar 

  18. Crisp, A. J., McGuire-Goldring, M., and Goldring, S. R., (1984) A system for culture of human trabecular bone and hormone response profiles of derived cells. Br. J. Exp. Path. 65, 645–654.

    CAS  Google Scholar 

  19. Auf”mkolk, B., Hauschka, P. V., and Schwartz, R., (1985) Characterisation of human bone cells in culture. Calcif. Tiss. Int. 37, 228–235.

    Article  CAS  Google Scholar 

  20. Gehron Robey, P., and Termine, J. D., (1985) Human bone cells in vitro. Calcif Tiss. Int. 37, 453–460.

    Article  Google Scholar 

  21. Ashton, B. A., Abdullah, F., Cave, J., Williamson, M., Sykes, B. C., Couch, M., and Poser, J. W., (1985) Characterization of cells with high alkaline phosphatase activity derived from human bone and marrow: preliminary assessment of their osteogenicity. Bone 6, 313–319.

    Article  PubMed  CAS  Google Scholar 

  22. Marie, P. J., Lomri, A., Sabbagh, A., and Basle, M., (1989) Culture and behaviour of osteoblastic cells isolated from normal trabecular bone surfaces. In Vitro Cell Dev. Biol. 25, 373–380.

    Article  PubMed  CAS  Google Scholar 

  23. Eriksen, E. F., Colvard, D. S., Berg, N. J., Graham, M. L., Mann, K. G., Spelsberg, T. C., and Riggs, B. L., (1988) Evidence of estrogen receptors in normal human osteoblast-like cells. Science 241, 84–86.

    Article  PubMed  CAS  Google Scholar 

  24. Schoefl C. Cuthbertson K. S. R. Walsh A. Cobbold P. Mayne N. Von zur Muhlen A. Hesch R. D. and Gallagher J. A. 1992 Evidence for P2-puri-ceptors on osteoblast-like cells. J. Bone Min. Res. 7 485–449

    Article  CAS  Google Scholar 

  25. Bowler, W., Gallagher, J. A., and Bilbe, G. (1995) Identification and cloning of human P2U purinoceptor present in osteoclastoma, bone and osteoblasts. J. Bone Miner. Res. 10, 1137–1145.

    Article  PubMed  CAS  Google Scholar 

  26. Birch, M. A., Ginty, A. F., Walsh, C. A., Fraser, W. D., Gallagher, J. A., and Bilbe, G. (1993) PCR detection of cytokines in normal human and pagetic osteo-blast-like cells. J. Bone Miner. Res. 8, 1155–1162.

    Article  PubMed  CAS  Google Scholar 

  27. Birch, M. A., Taylor, W., Fraser, W. D., Ralston S. H., Hart, A., and Gallagher, J. A. (1994) Absence of paramyxovirus RNA in cultures of pagetic bone cells and in pagetic bone. J. Bone Miner. Res. 9, 11–16.

    Article  PubMed  CAS  Google Scholar 

  28. Walsh, A., Birch, M. A., Fraser, W. D., Lawton, R., Dorgan, J., Walsh, S., Beresford, J. N., Sansom, D., and Gallagher, J. A. (1995) Expression and secretion of parathyroid hormone-related protein by human osteoblasts in vitro: effects of glucocorticoids. J. Bone Miner. Res. 10, 17–25.

    Article  PubMed  CAS  Google Scholar 

  29. Walsh, C.A, Birch, M.A, Fraser, W.D, Robinson, J, Lawton, R, Dorgan, J, Klenerman L, and Gallagher, J.A. (1994) Primary cultures of human osteoblasts produce parathyroid hormone-related protein Bone Mzner 27, 43–51

    Article  CAS  Google Scholar 

  30. Chaudhary, L.R, Spelsberg, T.C,and Riggs, B.L (1992) Production of various cytokmes by normal human osteoblast-like cells in response to interleukin-1β and tumour necrosis factor-α lack of regulation by 17β-estradiol. Endocrznology 130, 2528–2534

    Article  CAS  Google Scholar 

  31. Brittberg, M, Lmdahl, A, Nelson, A, Ohlsson, C, Isaksson, O, and Peterson, L (1994) Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation New Engl J Med 331, 889–895.

    Article  PubMed  CAS  Google Scholar 

  32. Goldring, S. R (1979) PTH inhibitors comparison of biological activity in bone and skin derived tissue J Clan Endocrznol Metab 49, 655–659

    Article  Google Scholar 

  33. Beresford, J. N, Taylor, G.T, and Triffitt, J.T (1990) Interferons and bone A comparison of the effects of Interferon-α and Interferon-α in cultures of human bone-derived cells and an osteosarcoma cell line Eur J Biochem 193, 589–597

    Article  PubMed  CAS  Google Scholar 

  34. Beresford, J. N, Graves, S. E, and Smoothy, C.A (1993) Formation of mineralized nodules by bone derived cells in vitro a model of bone formation? Am J Med Genet 45, 163–178

    Article  PubMed  CAS  Google Scholar 

  35. Chavassieux, P.M, Chenu, C, Valentm-Opran, A, Merle, B, Delmas, P.D, Hartmann, D.J, Saez, S, and Meumer, P.J (1990) Infiuence of experimental conditions on osteoblast activity in human primary bone cell cultures J Bone Miner Res 5, 337–343

    Article  PubMed  CAS  Google Scholar 

  36. Mohan, S and Baylmk, D.J (1991) Bone growth factors Clin Orthop 263, 30–48.

    PubMed  Google Scholar 

  37. Canalis E, Pash, J, and Varghese, S (1993) Skeletal growth factors Crit Rev Eukaryotic Gene Express 3, 155–166

    CAS  Google Scholar 

  38. Franceschi, R.T and Iyer, B.S (1992) Relationship between collagen syntheses and expression of the osteoblast phenotype in MC3T3-El cells J Bone Miner Res 7, 235–246

    Article  PubMed  CAS  Google Scholar 

  39. Prockop, D.J. and Kivivrikko, K. I (1984) Heritable diseases of collagen. New Engl J Med 311, 376–386

    Article  PubMed  CAS  Google Scholar 

  40. Graves, S E, Francis, M.J.O, Gundle, R, and Beresford, J.N (1994) Ascorbate increases collagen synthesis and promotes differentiation in human bone derived cell cultures Bone 15, 133 (abstract)

    Article  Google Scholar 

  41. Winkler, B.S. (1987) In vitro oxidation of ascorbic acid and its preservation by GSH Blochim Biophys Acta 925, 258–264

    CAS  Google Scholar 

  42. Friedrtch, W (1988) Vitamin C in Vitamins (Frtedrtch, W, ed), Walter de Gruyter, Berlin, pp. 931–1021

    Google Scholar 

  43. Nomura, H., Ishiguro, T, and Morimoto, S (1969) Studies on L-ascorbic acid derivatives III Bis(L-ascorbtc actd-3,3′)phosphate and L-ascorbic acid 2-phosphate Chem Pharm Bull 17, 387–393

    PubMed  CAS  Google Scholar 

  44. Hata, R.-I. and Senoo, H (1989) L-ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation and formation of a three-dimensional tissue-like substance by skin fibroblasts J Cell Physiol 138, 8–16

    Article  PubMed  CAS  Google Scholar 

  45. Oxford Textbook of Medicine, 2nd ed (1987) Oxford University Press, Oxford, UK

    Google Scholar 

  46. Graves, S.E, Francis, M.J.O, Gundle, R.G, and Beresford, J.N (1994) Primary culture of human trabecular bone. effects of L-ascorbate-2-phosphate Bone 15, 132 (abstract)

    Article  Google Scholar 

  47. Graves, S.E (1991)Studies on Human Bone-Dewed Cells In Vitro D Phil Thesis, Oxford University, Oxford, UK

    Google Scholar 

  48. Gundle, R (1995) Microscopical and Blochemlcal Studies of Mineralised Mattrix Production by Human Bone-Dewed Cells. D Phil Thesis, Oxford Umversity, Oxford, UK

    Google Scholar 

  49. Bellows, C.G, Heersche, J.N.M, and Aubin, J.E. (1992) Inorgainc phosphate added exogenously or released from β-glycerophosphate intiates mineralization of osteoid nodules in vitro. Bone Miner 17, 15–29

    Article  CAS  Google Scholar 

  50. Beresford, J.N, Joyner, C.J, Devlin, C, and Triffitt, J.T (1994) Osteogenic differentiation in human marrow stromal cell cultures effects of dexamethasone and 1,25-dihydroxyvitamin D3 Arch Oral Blol 39, 941–947

    Article  CAS  Google Scholar 

  51. Falla, N., Van Vlasselaer, P., Bierkens, J, Borremans, J, Schoeters, G, and Van Gorp, U (1993) Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow Blood 82, 358–3591

    Google Scholar 

  52. Benayahu, D, Kletter, Y., Zipori, D., and Weintroub, S (1989) Bone marrow-derived stromal cell line expressing osteoblastic phenotype In Vitro and osteo-genic capacity in vivo J Cell Physzol 140, 1–7.

    CAS  Google Scholar 

  53. Wong, M.M, Rao, L.G, Ly, H, Hamilton, L, Tong, J, Sturtridge, W, McBroom, R, Aubin, J.E, and Murray, T.M (1990) Long-term effects of physiologic concentrations of dexamethasone on human bone-derived cells. J Bone Miner Res 5, 803–813

    Article  PubMed  CAS  Google Scholar 

  54. Gundle, R, Graves, S. E, Francis, M.J.O, and Beresford, J. N (1994) Osteogenic and adipogeinc differentiation in human bone derived cell cultures. Bone 15, 114 (abstract)

    Article  Google Scholar 

  55. Gundle, R, Bradley, J, Joyner, C.J., Francis, M. J.O, Triffitt, J.T, and Beresford, J. N(1993) Bone formation in vitro and in vzvo by cultured adult human bone derived cells J Bone Miner Res. 8(Suppl.), 1031

    Google Scholar 

  56. Davzdson’s Prlnclples and Practice of Medlczne, 13th ed (1981) Churchill Livingstone, London

    Google Scholar 

  57. Bancroft, J.D. and Stevens, A.C (1977) Theory and Practice of Hzstological Technzques Churchill Livingstone, London

    Google Scholar 

  58. Gundle, R and Beresford, J.N (1995) The isolation and culture of cells from explants of human trabecular bone. Calcif Tzss Int 56Suppl.), 8–10.

    Google Scholar 

  59. Gundle, R, Joyner, C.J, and Trtffitt, J. T (1995) Human bone tissue formation in diffusion chamber culture in vlvo by bone derived cells and marrow stromal cells Bone 16, 597–601

    Article  PubMed  CAS  Google Scholar 

  60. Simmons, P.J and Torok Storb, B (1991) Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1 Blood 78, 55–62

    PubMed  CAS  Google Scholar 

  61. Gronthos, S, Graves, S.E, Ohta, S., and Simmons, P. J. (1994) The stro-l(+) fraction of adult human bone-marrow contains the osteogenic precursors Blood 84, 4164–4173

    PubMed  CAS  Google Scholar 

  62. Bmderman, I and Somjen, D (1992) Serum factors and calcium modulate the growth of osteoblast-like cells in culture, in Current Advances in Skeletogenesis Development, Biomineralisation, Mediators and Metabolic Bone Disease (Stlbermann, M and Slavkm, H.C, eds.), Elsevier, Amsterdam, pp 338–342

    Google Scholar 

  63. Gehron Robey, P (1995) Collagenase-treated trabecular bone fragments— a reproducrble source of cells In the osteoblastic lineage Calcif Tiss Int 56(Suppl. l), 11,12

    Google Scholar 

  64. Fedarko, N.S, Vetter, U.K, and Gehron Robey, P (1995) Age-related-changes in bone-matrix structure in-vitro Calcif Tiss Int 56(Suppl. l), 41–43

    Google Scholar 

  65. Fedarko, N.S, Vetter, U, Weinstein, S, and Robey, P.G (1992) Age-related changes in the in hyaluronan, proteoglycan, collagen and osteonectm synthesis by human bone cells J Cell Physiol 151, 215–227.

    Article  PubMed  CAS  Google Scholar 

  66. Fedarko, N.S, Moertke, M, Brenner, R, Gehron Robey, P, and Vetter, U (1992) Extracellular matrix formation by osteoblasts from patrents with osteogenesis imperfecta J Bone Mmer Res 7, 921–930

    Article  CAS  Google Scholar 

  67. Fedarko, N.S, Bianco, P, Vetter, U, and Gehron Robey, P (1990) Human bone cell enzyme expression and cellular heterogeneity correlation of alkaline phosphatase enzyme activrty with cell cycle J Cell Physiol 144, 115–121

    Article  PubMed  CAS  Google Scholar 

  68. Beresford, J.N, Fedarko, N.S, Fisher, L.W, Midura, R.J., Yanagashtta, M, Termine, J.D, and Gehron Robey, P (1987) Analysts of the proteoglycans synthesised by human bone cells in vztro J Biol Chem 262, 17,164–17,172

    CAS  Google Scholar 

  69. Wergedal, J.E, Matsuyama, T, and Strong, D.D (1992) Differentration of normal human bone cells by transforming growth factor-beta and 1,25(OH)2D3 Metabolism 41, 42–48

    Article  PubMed  CAS  Google Scholar 

  70. Matsuyama, T, Lau, K.H. W, and Wergedal, J.E (1990) Monolayer cultures of normal human bone cells contain multiple subpopulations of alkaline phosphatase positive cells Calcif Tiss Int 47, 276–283

    Article  CAS  Google Scholar 

  71. Wergedal, J.E, Mohan, S, Lundy, M, and Baylink, D. J. (1990) Skeletal growth factor and other growth factors known to be present in bone matrix stimulate prohferation and protein syntheses in human bone cells J Bone Miner Res 5, 179–186

    Article  PubMed  CAS  Google Scholar 

  72. Howard, G.A, Turner, R.T, Sherrard, D.J, and Baylink, D.J (1981) Human bone cells in culture metabolize 25(OH)D3 to 1,25(OH)2D3 and 24,25(OH)2D3 J Biol Chem 256, 7738–7740

    PubMed  CAS  Google Scholar 

  73. Lomri, A and Marie, P.J (1990) Bone cell responsiveness to transforming growth factor β, parathyrord hormone and prostaglandin E2 in normal and postmenopausal osteoporotic women J Bone Miner Res 5, 1149–1155

    Article  PubMed  CAS  Google Scholar 

  74. Marie, P.J, Sabbagh, A, De VerneJoul, M and Lomri, A (1988) Osteocalcin and deoxyribonucleic acid synthesis in vitro and histomorphometric indrces of bone formation in postmenopausal osteoporosrs J Clin Invest 69, 272–279

    Google Scholar 

  75. Mane, P.J, De VerneJoul, M.C, and Lomri, A (1992) Stimulation of bone formation in osteoporosrs patients treated wrth fluoride associated with Increased DNA synthesis by osteoblastic cells in vitro J Bone Miner Res 7, 103–113

    Google Scholar 

  76. Mane, P.J, Hott, M, Launay, J.M, Graulet, A.M, and Gueris, J (1993) In vitro production of cytokines by bone surface-derived osteoblastic cells in normal and osteoporotic postmenopausal women relationship with cell proliferation J Clin Endocrinol Metab 77, 824–830

    Article  Google Scholar 

  77. Mane, P.J (1995) Human endosteal osteoblastic cells—relationship with boneformation. Calcif Tiss Int 56(Suppl.), 13–16

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Human Anatomy and Cell Biology, Bone Cell Research Group, University of Liverpool, UK

    James A. Gallagher

  2. Department of Orthopaedic Surgery, The Nuffield Orthopaedic Centre, Headington, Oxford, UK

    Roger Gundle

  3. School of Pharmacy and Pharmacology, University of Bath, Avon, UK

    Jon N. Beresford

Authors
  1. James A. Gallagher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roger Gundle
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jon N. Beresford
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. King’s College, University of London, UK

    Gareth E. Jones

Rights and permissions

Reprints and permissions

Copyright information

© 1996 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Gallagher, J.A., Gundle, R., Beresford, J.N. (1996). Isolation and Culture of Bone-Forming Cells (Osteoblasts) from Human Bone. In: Jones, G.E. (eds) Human Cell Culture Protocols. Methods in Molecular Medicine, vol 2. Humana Press. https://doi.org/10.1385/0-89603-335-X:233

Download citation

  • DOI: https://doi.org/10.1385/0-89603-335-X:233

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-335-1

  • Online ISBN: 978-1-59259-586-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation