Advertisement

Embryonic Limb Mesenchyme Micromass Culture as an In Vitro Model for Chondrogenesis and Cartilage Maturation

  • Anthony M. De Lise
  • Emanuela Stringa
  • Wendy A. Woodward
  • Maria Alice Mello
  • Rocky S. Tuan
Part of the Methods in Molecular Biology™ book series (MIMB, volume 137)

Abstract

In vitro techniques for the study of chondrogenic differentiation of embryonic limb mesenchymal cells have been available for some time. Early methods require highdensity confluent monolayer cell cultures (1,2). The micromass culture method developed by Ahrens et al. (3) represented a convenient system for the observations and analysis of the differentiative processes and phenomena analogous to those exhibited by the limb cartilage anlagen in situ. In these cultures, limb mesenchymal cells first undergo condensation giving rise to aggregates that later become cartilage nodules (3,4), thereby mimicking the differentiative phenomena occurring during embryonic limb development in vivo, i.e., mesenchymal condensation preceding cartilage differentiation (5, 6, 7, 8, 9). The micromass limb mesenchymal culture system has gained great popularity for the analysis of the regulatory steps and differentiative processes that result in the condensation of the mesenchyme and the formation and maturation of the cartilage anlagen.

Keywords

Amyl Acetate Chick Embryo Extract Embryonic Limb Cartilage Nodule Differentiative Process 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Umansky, R. (1966) The effect of cell population density on the developmental fate of reaggregating mouse limb bud mesenchyme. Dev. Biol. 13, 31–56.PubMedCrossRefGoogle Scholar
  2. 2.
    Caplan, A. (1970) Effects of the nicotinomide-sensitive teratogen 3-acetylpyridine on chick limb cells in culture. Exp. Cell Res. 62, 341–355.PubMedCrossRefGoogle Scholar
  3. 3.
    Ahrens, P. B., Solursh, M., and Reiter, R. S. (1977) Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60, 69–82.PubMedCrossRefGoogle Scholar
  4. 4.
    Solursh, M., Ahrens, P., and Reiter, R. (1978) A tissue culture analysis of the steps in limb chondrogenesis. In Vitro 14, 41–61.CrossRefGoogle Scholar
  5. 5.
    Kosher, R., Savage, M., and Chan, S. (1979) In vitro studies on the morphogenesis and differentiation of the mesoderm subjacent to the apical ectodermal ridge of the embryonic chick limb bud. J. Embryol. Exp. Morphol. 50, 75–97.PubMedGoogle Scholar
  6. 6.
    Newman, S., Pautou, M. P., and Kierney, M. (1981) The distal boundary of morphogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells. Dev. Biol. 84, 440–448.PubMedCrossRefGoogle Scholar
  7. 7.
    San Antonio, J. D. and Tuan, R. S. (1986) Chondrogenesis of limb bud mesenchyme in vitro: stimulation by cations. Dev. Biol. 115, 313–324.CrossRefGoogle Scholar
  8. 8.
    Jiang, H., Soprano, D. R., Li, S. W., Soprano, K. J., Penner, J. D., Gyda, M., III, and Kochhar, D. M. (1995) Modulation of limb bud chondrogenesis by retinoic acid and retinoic acid receptors. Int. J. Dev. Biol. 39(4), 617–627.PubMedGoogle Scholar
  9. 9.
    Thorogood, P. V. and Hinchcliffe, J. R. (1975) An analysis of the condensation process during chondrogenesis in the embryonic chick hind limb. J. Embryol. Exp. Morphol. 33, 581–606.PubMedGoogle Scholar
  10. 10.
    Tuan, R. S., Scott, W. A., and Cohn, Z. A. (1978) Calcium-binding protein of the chick chorioallantoic membrane. II. Vitamin K-dependent expression. J. Cell Biol. 77, 752–761.PubMedCrossRefGoogle Scholar
  11. 11.
    Boskey, A. L., Stiner, D., Doty, S. B., Binderman, I., and Leboy, P. (1991) Studies of mineralization in tissue culture: optimal conditions for cartilage calcification. J. Bone Min. Res. 16, 11–36.Google Scholar
  12. 12.
    Aulthouse, A. A. and Solursh, M. (1987) The detection of a precartilage, blastema specific marker. Dev. Biol. 84, 440–448.Google Scholar
  13. 13.
    Milaire, J. (1991) Lectin binding sites in developing mouse limb buds. Anat. Embryol. 184, 479–488.PubMedCrossRefGoogle Scholar
  14. 14.
    Zimmermann, B. and Thies, M. (1984) Alterations of lectin binding during chondrogenesis of mouse limb bud. Histochemistry 81, 353–361.PubMedCrossRefGoogle Scholar
  15. 15.
    Stringa, E. and Tuan, R. S. (1996) Chondrogenic cell subpopulation of chick embryonic calvarium: isolation by peanut agglutinin affinity chromatography and in vitro characterization. Anat. Embryol. 223, 1–11.Google Scholar
  16. 16.
    Stringa, E., Love, J. M., McBride, S. C., Suyama, E., and Tuan, R. S. (1997) In vitro characterization of chondrogenic cells isolated from chick embryonic muscle using peanut agglutinin affinity chromatography. Exp. Cell Res. 232(2), 287–294.PubMedCrossRefGoogle Scholar
  17. 17.
    Kiernan, J. A. (1990) Histological and Histochemical Methods: Theory and Practice, 2nd ed. Pergamon Press, New York.Google Scholar
  18. 18.
    Jacenko, O. and Tuan, R. S. (1986) Calcium deficiency induces expression of cartilagelike phenotype in chicken embryo calvaria. Dev. Biol. 115, 215–232.PubMedCrossRefGoogle Scholar
  19. 19.
    Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A., (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119(3), 493–501.PubMedCrossRefGoogle Scholar
  20. 20.
    Ray, S., Ponnathpur, V., Huang, Y., Tang, C., Mahoney, M. E., Ibrado, A. M., et al. (1994) 1-β-D-Arabinofuranosylcytosine-, mitoxantrone-, and paclitaxel-induced apoptosis in HL-60 cells: improved method for detection of internucleosomal DNA fragmentation. Cancer Chemother. Pharmacol. 34, 365–371.PubMedCrossRefGoogle Scholar
  21. 21.
    Tilly, J. and Hsueh, A. J. W. (1993) Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J. Cell Phys. 154, 519–526.CrossRefGoogle Scholar
  22. 22.
    Hamburger, V. and Hamilton, H. L. (1951) A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49–92.CrossRefGoogle Scholar
  23. 23.
    Lev, R. and Spicer, S. S. (1964) Specific staining of sulfate groups with Alcian blue at low pH. J. Histochem. 12, 309.Google Scholar
  24. 24.
    Mello, M. A. and Tuan, R. S. (1999) High density micromass cultures of embryonic limb bud mesenchymal cells: an in vitro model of endochondral skeletal development. In Vitro Cell Dev. Biol. 35, 262–269.CrossRefGoogle Scholar
  25. 25.
    Mello, M. A. and Tuan, R. S. (1998) Cartilage maturation in vitro is influenced by TGF-β1 and T3. Trans. Ortho. Res. Soc. 44, 495.Google Scholar
  26. 26.
    Reginato, A. M., Tuan, R. S., Ono, T., Jimenez, S. A., and Jacenko, O. (1993) Effects of calcium deficiency on chondrocyte hypertrophy and type X collagen expression in chick embryonic sternum. Dev. Dyn. 189, 284–295.Google Scholar
  27. 27.
    Arends, M. J., Morris, R. G., and Wyllie, A. H. (1990) Apoptosis: the role of the endonuclease. Am. J. Pathol. 136, 593–608.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Anthony M. De Lise
    • 1
  • Emanuela Stringa
    • 1
  • Wendy A. Woodward
    • 1
  • Maria Alice Mello
    • 1
  • Rocky S. Tuan
    • 1
  1. 1.Department of Orthopaedic SurgeryThomas Jefferson UniversityPhiladelphia

Personalised recommendations