Characterization of human osteoblastic cells: Influence of the culture conditions

  • A. Rattner
  • O. Sabido
  • C. Massoubre
  • F. Rascle
  • J. Frey
Cellular Models

Summary

Human osteoblastic cells were isolated enzymatically from adult human spongy bone and grown in MEM-Ham F12 1:1 medium supplemented with 2% Ultroser (USM). They were subcultured and examined for osteoblast features by morphological, histological, and biochemical approaches. The cells had a characteristic polyhedral morphology and produced a high level of alkaline phosphatase (ALKP). Confluent cultures were uniformly stained for ALKP and flow cytometry analysis with fluorescein diphosphate gave a single peak signal, reflecting a highly positive population, distinct from cultures of fibroblasts. The ALKP activity was stimulated by 1,25 (OH)2 vitamin D3. CD 44 was strongly expressed in these cultures, although osteoblasts are negative in vivo and osteocytes are positive. The main collagen synthesized was type I collagen and osteocalcin was produced after stimulation by vitamin D3. 10 mM βGP induced mineralization and microprobe analysis of the crystals showed a composition close to hydroxyapatite.

Changing the culture conditions to MEM-10% calf serum acted on cell behavior: it reduced the production of these biochemical markers of osteoblasts and the morphology became fibroblastlike with more rapid cell multiplication. The parameter most affected by the change in culture medium was ALKP, which was selected as the determinant criterion for defining an osteoblast culture. ALKP activity was then used to characterize a culture of cells seeded in a collagen gel.

Key words

human osteoblastic cells collagen lattice mineral deposition collagen biosynthesis alkaline phosphatase osteocalcin 

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References

  1. 1.
    Allen, T. D.; Schor, S. L. The contraction of collagen matrices by dermal fibroblasts. J. Ultrastruct. Res. 83:205–219; 1983.PubMedCrossRefGoogle Scholar
  2. 2.
    Auf’mkolk, B. M.; Hauschka, P. V.; Schwartz, E. R. Characterization of human bone cells in culture. Calcif. Tissue Int. 37:228–235; 1985.PubMedCrossRefGoogle Scholar
  3. 3.
    Bancroft, J. D.; Stevens, A. Theory and practice of histological techniques. 2nd ed. Edinburg, Scotland: Churchill Livingstone; 1982.Google Scholar
  4. 4.
    Bell, E.; Ivarsson, B.; Merrill, C. Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. USA 76:1274–1278; 1979.PubMedCrossRefGoogle Scholar
  5. 5.
    Bellows, C. G.; Aubin, J. E.; Heersche, J. N. M., et al. Mineralized bone modules formed in vitro from enzymatically released rat calvaria cell population. Calcif. Tissue Int. 38:143–147; 1986.PubMedCrossRefGoogle Scholar
  6. 6.
    Beresford, J. N.; Gallagher, J. A.; Russel, R. G. G. 1.25 dihydroxyvitamin D3 and human bone derived cells in vitro: effects of alkaline phosphatase, type I collagen and proliferation. Endocrinology 119:1776–1785; 1986.PubMedGoogle Scholar
  7. 7.
    Bonis, M. P.; Germain, N.; Frey, J. A direct fluorimetric DNA assay on cell suspensions. J. Tissue Cult. Meth. 13:285–288; 1991.CrossRefGoogle Scholar
  8. 8.
    Chamson, A.; Rattner, A.; Raby, N., et al. Characterization of human osteoblast-like cells cultivated in vitro. In: Development and diseases of cartilage and bone matrix. N.Y.: Alan R. Liss. 1987:257–263.Google Scholar
  9. 9.
    Clover, J.; Gowen, M. Are MG-63 and HOS TE 85 human osteosarcoma cell lines representative models of the osteoblastic phenotype? Bone 15:585–591; 1994.PubMedCrossRefGoogle Scholar
  10. 10.
    Flanagan, B. F.; Dalchau, R.; Allen, A. K., et al. Chemical composition and tissue distribution of the human CDW44 glycoprotein. Immunology 67:167–175; 1989.PubMedGoogle Scholar
  11. 11.
    Franceschi, R. T.; James, W. M.; Zerlauth, G. 1a, 25- dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line. J. Cell. Physiol. 123:401–409; 1985.PubMedCrossRefGoogle Scholar
  12. 12.
    Frey, J.; Chamson, A.; Raby, N. Separation of amino-acids using ion-paired reversed phase high performance liquid chromatography with special reference to collagen hydrolysate. Amino Acids 4:45–51; 1993.CrossRefGoogle Scholar
  13. 13.
    Gerstenfeld, L. C.; Chipman, S. D.; Kelley, C. M., et al. Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts. J. Cell Biol. 106:979–989; 1988.PubMedCrossRefGoogle Scholar
  14. 14.
    Heldin, C. H.; Johnsson, A.; Wennergren, S., et al. A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains. Nature 319:511–514; 1986.PubMedCrossRefGoogle Scholar
  15. 15.
    Hughes, D. E.; Salter, D. M.; Simpson, R. CD44 expression in human bone: a novel marker of osteocytic differentiation. J. Bone Miner. Res. 9:39–44; 1994.PubMedGoogle Scholar
  16. 16.
    Kirstein, M.; Baglioni, C. Tumor necrosis factor stimulates proliferation of human osteosarcoma cells and accumulation of c- myc messenger RNA. J. Cell. Physiol. 134:479–484; 1988.PubMedCrossRefGoogle Scholar
  17. 17.
    Koutsilieris, M.; Sourla, A.; Pelletier, G., et al. Three-dimensional type I collagen gel system for the study of osteoblastic metastases produced by metastatic prostate cancer. J. Bone Miner. Res. 9:1823–1832; 1994.PubMedGoogle Scholar
  18. 18.
    Labarca, C.; Paigen, K. A simple rapid and sensitive DNA assay procedure. Anal. Biochem. 102:344–352; 1980.PubMedCrossRefGoogle Scholar
  19. 19.
    Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685; 1970.PubMedCrossRefGoogle Scholar
  20. 20.
    Lian, J. B.; Coutts, M. C.; Cannalis, E. Studies of hormonal regulation of osteocalcin synthesis in cultured fetal rat calvaria. J. Biol. Chem. 260:8706–8710; 1985.PubMedGoogle Scholar
  21. 21.
    Lowry, O. H.; Rosebrough, N. J.; Farr, A. L., et al. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.PubMedGoogle Scholar
  22. 22.
    Manolagas, S. C.; Burton, D. W.; Deftos, L. J. 1,25 dihydroxyvit. D3 stimulates the alkaline phosphatase activity of osteoblast-like cells. J. Biol. Chem. 256:7115–7117; 1981.PubMedGoogle Scholar
  23. 23.
    Owen, T. A.; Aranow, M. S.; Barone, L. M., et al. Pleiotropic effects of vitamin D on osteoblast gene expression are related to the proliferative and differentiated state of the bone cell phenotype: dependency upon basal levels of gene expression, duration of exposure, and bone matrix competency in normal rat osteoblast cultures. Endocrinology 128:1496–1504; 1991.PubMedCrossRefGoogle Scholar
  24. 24.
    Partridge, N. C.; Alcorn, D.; Michelangeli, V. P., et al. Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells. Endocrinology 108:213–219; 1981.PubMedGoogle Scholar
  25. 25.
    Rattner, A.; Frey, J. Supermolecular complexes formed in lattices prepared with fibroblasts embedded in type I collagen. Biomed. Res. 13:95–105; 1992.Google Scholar
  26. 26.
    Rieck, P.; Peters, D.; Hartmann, C., et al. A new, rapid colorimetric assay for quantitative determination of cellular proliferation, growth inhibition and viability. J. Tissue Cult. Meth. 15:37–42; 1993.CrossRefGoogle Scholar
  27. 27.
    Robey, R. G.; Termine, J. D. Human bone cells in vitro. Calcif. Tissue Int. 37:453–460; 1985.PubMedCrossRefGoogle Scholar
  28. 28.
    Rodan, S. B.; Imai, Y.; Thiede, M. A., et al. Characterization of a human cell line (Saos-2) with osteoblastic properties. Cancer Res. 47:4961–4996; 1987.PubMedGoogle Scholar
  29. 29.
    Sudo, M.; Kodama, H.; Amagai, Y., et al. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 96:191–198; 1983.PubMedCrossRefGoogle Scholar
  30. 30.
    Talley-Ronsholdt, D. J.; Lajiness, E.; Nagodawithana, K. Transforming growth factor-beta inhibition of mineralization by neonatal rat osteoblasts in monolayer and collagen gel culture. In Vitro Cell. Dev. Biol. 31:274–282; 1995.CrossRefGoogle Scholar
  31. 31.
    Whitson, S. W.; Whitson, M. A.; Bowers, D. E., et al. Factors influencing synthesis and mineralization of bone matrix from fetal bovine bone cells grown in vitro. J. Bone Miner. Res. 7:727–741; 1992.PubMedCrossRefGoogle Scholar
  32. 32.
    Zilversmit, D. B.; Davis, K. J. Boehringer Mannheim colorimetric method. J. Lab. Clin. Meth. 35:155; 1950.Google Scholar

Copyright information

© Society for In Vitro Biology 1997

Authors and Affiliations

  • A. Rattner
    • 1
  • O. Sabido
    • 1
  • C. Massoubre
    • 1
  • F. Rascle
    • 1
  • J. Frey
    • 1
  1. 1.Laboratoire de BiochimieFaculté de MédecíneSaint-Etienne Cedex 2France

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