Summary
Chick limb mesenchymal cells differentiate into muscle, cartilage, fibrous, and bone tissue. Previous reports show that when stage 24 limb mesenchymal cells are cultured in vitro, chondrocytes, myocytes, fibrocytes, and osteoblasts can be identified on the basis of morphological and biochemical parameters. The study reported here demonstrates that phenotypic expression in culture seems to be dependent on the initial plating density, Scanning electron microscopic observations indicate that when stage 24 limb mesenchymal cells are initially seeded at high densities (5 × 106 cells per 35 mm culture dish), mounds of cells appear in culture. These mounds represent cartilage nodules composed of a fine fibrous matrix and chondrocytes, surrounded by a loose fibrous connective tissue matrix. Cultures initially plated at intermediate densities (2.0–2.5 × 106 cells/35 mm culture dish) produce a flattened layer of fibrocytes overlying a matrix of collagen fibers and calcium phosphate deposits as determined by electron-microprobe analysis; these observations are indicative of osteoblast expression. Cells seeded at this intermediate density appear larger and possess greater surface area than cells seeded at high density. It is suggested that conditions that permit such increased cell surface area coupled with a relative compaction due to cell crowding may provide conditions permissive for osteogenesis. Based on morphological criteria, it appears that chick limb mesenchymal cell osteogenesis in vitro is not associated with chondrogenesis but represents a separate route of phenotypic expression.
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Miller, E., Matukas, G., Piez, K., Powers, M.: Characterization of chick bone collagen and compositional changes occurring with maturation, J. Biol. Chem.242:5481–5489, 1967
Hascall, V.C., Oegema, T.R., Browns, M., Caplan, A.I.: Isolation and characterization of proteoglycans from avian embryonic limb bud chondrocytes grownin vitro, J. Biol. Chem.251:3511–3519, 1976
Osdoby, P., Caplan, A.I.: The possible differentiation of osteogenic elementsin vitro from chick limb mesodermal cells, Dev. Biol.52:283–299, 1976
von der Mark, K., von der Mark, H.: Immunological and biochemical studies of collagen type transitions duringin vitro chondrogenesis of chick limb mesenchymal cells, J. Cell Biol.73:736–747, 1977
Zull, J.E., Krug, S., Abel, D., Caplan, A.I.: Development of parathyroid hormone- and calcitonin-activated adenylate cyclases in embryonic chicken limb and in cultured cells from embryonic chicken limb, Proc. Natl. Acad. Sci. U.S.A.75:3871–3875, 1978
Osdoby, P., Caplan, A.I.: Osteogenesis in cultures of limb mesenchymal cells, Dev. Biol.73:84–102, 1979
Hamburger, V., Hamilton, H.S.: A series of normal stages in the development of the chick embryo, J. Morphol.88:49–92, 1951
Caplan, A.I.: Effect of nicotinamide-sensitive teratogen 3-acetyl pyridine on chick limb cells in culture, Exp. Cell Res.62:341–355, 1970
Caplan, A.I.: Muscle, cartilage, and bone development and differentiation from chick limb mesenchymal cells. In D.A. Ede, J.R. Hinchliffe, and M. Balls (eds): Vertebrate Limb and Somite Morphogenesis, pp. 199–213. Cambridge University Press, London, 1977
Anderson, T.: Techniques for the preservation of the three-dimensional structure in preparing specimens for the electron microscope, Trans. N.Y. Acad. Sci.13:130–134, 1951
Jones, S.J., Boyde, A., Pawley, J.B.: Osteoblast and collagen orientation, Cell Tissue Res.159:73–80, 1975
Vertel, B.M.: Limb bud developmentin vitro. An SEM study. In: Scanning Electron Microscropy 1976. Part V. Proceedings of the Workshop on Advances in Biomedical Applications of the SEM, pp. 291–298. ILT Research Institute, Chicago
Vertel, B.M., Dorfman, A.: An immunohistochemical study of extracellular matrix formation during chondrogenesis, Dev. Biol.62:1–12, 1978
Levitt, D., Ho, P.L., Dorfman, A.: Effect of 5-bromodeoxyuridine on ultrastructure of developing limb bud cellsin vitro, Dev. Biol.43:75–90, 1975
Boyde, A., Jones, S.J., Binderman, I., Harrel, A.: Scanning electron microscopy of bone cells in culture, Cell Tissue Res.166:65–70, 1976
Binderman, I., Duskin, D., Harrel, A., Katzir, E., Sachs, L.: Formation of bone tissue in culture from isolated bone cells, J. Cell Biol.61:427–439, 1974
Ahrens, P.B., Solursh, M., Reiter, R.S.: Stage related capacity for limb chondrogenesis in cell culture, Dev. Biol.60:69–82, 1977
Umansky, R.: The effect of cell population density on the developmental fate of reaggregating mouse limb bud mesenchyme, Dev. Biol.13:31–56, 1966
Fell, H.: The histogenesis of cartilage and bone in the long bones of the embryonic fowl, J. Morphol.40:417–459, 1925
Thorogood, P., Hinchliffe, J.: An analysis of the condensation process during chondrogenesis in the embryonic hind limb, J. Embryol. Exp. Morphol.33:581–606, 1975
Solursh, M., Reiter, R.S.: Determination of limb bud chondrocytes during a transient block of the cell cycle, Cell Differentiation4:131–137, 1975
Caplan, A., Koutroupas, S.: The control of muscle and cartilage development in the chick limb: The role of differential vascularization, J. Embryol. Exp. Morphol.29:571–583, 1973
Ali, S.Y., Sajdera, S.W., Anderson, H.C.: Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage, Proc. Natl. Acad. Sci. U.S.A.67:1513–1520, 1970
Anderson, H.C.: Vesicles associated with calcification in the matrix of epiphyseal cartilage, J. Cell Biol.41:59–72, 1969
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Osdoby, P., Caplan, A.I. A scanning electron microscopic investigation of in vitro osteogenesis. Calcif Tissue Int 30, 43–50 (1980). https://doi.org/10.1007/BF02408605
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DOI: https://doi.org/10.1007/BF02408605