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Bone formation by osteoblast-like cells in a three-dimensional cell culture

  • Molecular and Cellular Biology
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Summary

Cells of the clonal osteogenic cell line MC3T3-E1 were seeded onto a three-dimensional matrix of denatured collagen type 1 and cultured for a period of up to 8 weeks. Specimens were analyzed by histological, enzyme histochemical, immunocytochemical, and ultrastructural methods and byin situ hybridization between day 7 and day 56 after seeding. In 56-day cultures, the MC3T3-E1 cells were arranged in a three-dimensional network and formation of bone-like tissue was indicated by calcification of a newly synthesized collagen type I matrix resembling osteoid and surrounding osteocyte-like cells. The differentiating culture showed high expression of osteocalcin and alkaline phosphatase activity. NIH3T3 fibroblasts used as control cells passed through the network of the substrate forming a confluent monolayer underneath. This culture system offers a potentially powerful model for bone formationin vitro and for investigating the osteogenic potential of bone-derived cells.

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References

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

    Article  PubMed  CAS  Google Scholar 

  2. Binderman I, Duksin D, Harell A, Katzir E, Sachs L (1974) Formation of bone tissue in culture from isolated bone cells. J Cell Biol 61:427–439

    Article  PubMed  CAS  Google Scholar 

  3. Wong GL, Cohn DV (1975) Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. Proc Natl Acad Sci USA 72:3167–3171

    Article  PubMed  CAS  Google Scholar 

  4. Nijweide PJ, van der Plas A, Scherft FP (1981) Biochemical and histological studies on various bone cell preparations. Calcif Tissue Int 33:529–540

    Article  PubMed  CAS  Google Scholar 

  5. Ecarot-Charrier B, Glorieux FH, van der Rest M, Pereira G (1983) Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture. J Cell Biol 96:639–643

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  7. Smith DM (1972) Studies of the metabolism of separated bone cells. Calcif Tissue Int 11:56–69

    Google Scholar 

  8. Williams DC, Boder GB, Toomey RE, Paul DC, Hillman CC Jr, King KL, van Frank RM, Johnston CC Jr (1980) Mineralization and metabolic response in serially passaged adult rat bone cells. Calcif Tissue Int 30:233–246

    Article  PubMed  CAS  Google Scholar 

  9. Whitson SW, Harrison W, Dunlap MK, Bowers DE Jr, Fisher LW, Robey PG, Termine JD (1984) Fetal bovine bone cells synthesize bone-specific matrix proteins. J Cell Biol 99:607–614

    Article  PubMed  CAS  Google Scholar 

  10. Robey PG, Termine JD (1985) Human bone cells in vitro. Calcif Tissue Int 37:453–460

    PubMed  CAS  Google Scholar 

  11. Auf'mkolk B, Hauschka PV, Schwartz ER (1985) Characterization of human bone cells in culture. Calcif Tissue Int 37:228–235

    PubMed  Google Scholar 

  12. Kodama H, Amagai Y, Sudo H, Kasai S, Yamamoto S (1981) Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Jpn J Oral Biol 23:899–901

    Google Scholar 

  13. Sudo H, Kodama H, Amagai Y, Yamamoto S, Kasai S (1983) In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191–198

    Article  PubMed  CAS  Google Scholar 

  14. Nishimoto SK, Stryker WF, Nimni ME (1987) Calcification of osteoblastlike rat osteosarcoma cells in agarose suspension cultures. Calcif Tissue Int 41:274–280

    PubMed  CAS  Google Scholar 

  15. Ernst M, Froesch ER (1987) Osteoblastlike cells in a serum-free methylcellulose medium form colonies: effects of insulin and insulinlike growth factor I. Calcif Tissue Int 40:27–34

    Article  PubMed  CAS  Google Scholar 

  16. Sudo H, Kodama H, Amagai Y, Itakura Y, Yamamoto S (1986) Mineralized tissue formation by MC3T3-E1 osteogenic cells embedded in three-dimensional gel matrix. In: Ali SY (ed) Cell-mediated calcification and matrix vesicles. Elsevier, Amsterdam, New York, pp 291–296

    Google Scholar 

  17. Shima M, Seino Y, Tanaka H, Kurose H, Ishida M, Yabuuchi H, Kodama H (1988) Microcarrier facilitates mineralization in MC3T3-E1 cells. Calcif Tissue Int 43:19–25

    PubMed  CAS  Google Scholar 

  18. Schmidt J, Casser-Bette M, Murray AB, Luz A, Erfle V (1987) Retrovirus-induced osteopetrosis in mice: effects of viral infection on osteogenic differentiation in skeletoblast cell cultures. Am J Pathol 129:503–510

    PubMed  CAS  Google Scholar 

  19. Celeste AJ, Rosen V, Buecker JL, Kriz R, Wang EA, Wozney JM (1986) Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones. EMBO J 5:1885–1889

    PubMed  CAS  Google Scholar 

  20. Wolf H, Haus M, Wilmes E (1984) Persistence of Epstein-Barr virus in the parotid gland. J Virol 51:795–798

    PubMed  CAS  Google Scholar 

  21. Deschepper CF, Mellon SH, Cumin F, Baxter JD, Ganong WF (1986) Analysis by immunocytochemistry and in situ hybridization of renin and its mRNA in kidney, testis, adrenal, and pituitary of the rat. Proc Natl Acad Sci USA 83:7552–7556

    Article  PubMed  CAS  Google Scholar 

  22. Brahic M, Haase AT (1978) Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc Natl Acad Sci USA 75:6125–6129

    Article  PubMed  CAS  Google Scholar 

  23. Price PA, Lothringer JW, Baukol SA, Reddi AH (1981) Developmental appearance of the vitamin K-dependent protein of bone during calcification. J Biol Chem 256:3781–3784

    PubMed  CAS  Google Scholar 

  24. Rodan GA, Rodan SB (1984) Expression of the osteoblastic phenotype. In: Peck WA (ed) Bone and mineral research, annual 2. Elsevier, Amsterdam, pp 244–285

    Google Scholar 

  25. Gerstenfeld LC, Chipman SD, Kelly CM, Hodgens KJ, Lee DD, Landis WJ (1988) Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts. J Cell Biol 106:979–989

    Article  PubMed  CAS  Google Scholar 

  26. Tenenbaum HC, Heersche JNM (1986) Differentiation of osteoid-producing cells in vitro: possible evidence for the requirement of a microenvironment. Calcif Tissue Int 38:262–267

    Article  PubMed  CAS  Google Scholar 

  27. Livne E, Schmidt J, Closs EI, Silbermann M, Erfle V (1989) Effects of leukemogenic retroviruses on condylar cartilage in vitro: an ultrastructural study. Calcif Tissue Int 44:25–35

    PubMed  CAS  Google Scholar 

  28. Schmidt J, Livne E, Erfle V, Gössner W, Silbermann M (1986) Morphology and in vivo growth characteristics of an atypical murine proliferative osseous lesion induced in vitro. Cancer Res 46:3090–3098

    PubMed  CAS  Google Scholar 

  29. Tenenbaum HC, Heersche JNM (1981) Mineralization of osteoid produced in vitro by the addition of β-glycerophosphate. In: Cohn DV, Talmage RV, Matthews JL (eds) Hormonal control of calcium metabolism. Proc 7th Intl Conf on Calcium-Regulating Hormones, p 415

  30. Tenenbaum HC, Heersche JNM (1982) Differentiation of osteoblasts and formation of mineralized bone in vitro. Calcif Tissue Int 34:76–79

    Article  PubMed  CAS  Google Scholar 

  31. Kodama H, Amagai Y, Sudo H, Ohno H, Iijima K (1986) Culture conditions affecting differentiation and calcification in the MC3T3-E1 osteogenic cell line. In: Ali SY (ed) Cell-mediated calcification and matrix vesicles. Elsevier, Amsterdam, New York, pp 297–302

    Google Scholar 

  32. Nefussi J, Boy-Lefevre ML, Boulekbache H, Forest N (1985) Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion. Differentiation 29:160–168

    Article  PubMed  CAS  Google Scholar 

  33. Gerstenfeld LC, Chipman SD, Glowacki J, Lian JB (1987) Expression of differentiated function by mineralizing cultures of chicken osteoblasts. Dev Biol 122:49–60

    Article  PubMed  CAS  Google Scholar 

  34. Williams DC, Boder GB, Toomey RE, Paul DC, Hillman CC Jr, King KL, van Frank RM, Johnston CC Jr (1980) Mineralization and metabolism response in serially passaged adult rat bone cells. Calcif Tissue Int 30:233–246

    Article  PubMed  CAS  Google Scholar 

  35. Glimcher MJ (1981) On the form and function of bone: from molecules to organs. Wolff's law revisited 1981. In: Veis A (ed) The chemistry and biology of mineralized connective tissues. Elsevier, New York, pp 618–673

    Google Scholar 

  36. Climcher MJ (1984) Recent studies of the mineral phase in bone and its possible linkage to the phosphate bonds. Phil Trans R Soc Lond B 304:479–508

    Google Scholar 

  37. Tenenbaum HC, Palangio KG, Holmyard DP, Pritzker KPH (1986) An ultrastructural study of osteogenesis in chick periosteum in vitro. Bone 7:295–302

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Institut für Pathologie, Gesellschaft für Strahlen- und Umweltforschung (GSF), Neuherberg, Federal Republic of Germany

    A. B. Murray

  2. Abteilung für Molekulare Zellpathologie, Gesellschaft für Strahlen-und Umwelt forschung, D-8042, Neuherberg, FRG

    M. Casser-Bette, E. I. Closs, V. Erfle &  J. Schmidt

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  1. M. Casser-Bette
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  2. A. B. Murray
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  3. E. I. Closs
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  4. V. Erfle
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  5. J. Schmidt
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Casser-Bette, M., Murray, A.B., Closs, E.I. et al. Bone formation by osteoblast-like cells in a three-dimensional cell culture. Calcif Tissue Int 46, 46–56 (1990). https://doi.org/10.1007/BF02555824

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  • DOI: https://doi.org/10.1007/BF02555824

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