Summary
To analyze the mechanism of initiation of cell-mediated calcification in hard tussue and its relationship to the frequency of gap junctions, enzymatically isolated cells from fetal rat calvaria cultured in collagen gels were observed ultrastructurally over a time course. Calcification was observed at 2–3 weeks after the initiation of culture when the seeding cellularity and the concentration of β-glycerophosphate were sufficiently high. In the collagen gels, round cells (R), spindle or stellate cells (S), and fat cells (F) were characterised morphologically. The ultrastructural features of initial calcification could be classified into 4 subtypes: 1) a large mass greater than 10 µm in diameter (Type I), 2) deposition associated with dead R cells or matrix vesicles (Type II), 3) intracellular deposition (Type III), and 4) other than Types I–III (Type IV). Type II was the most frequent (44.5%) and Type III was the least (6.8%). Gap junction was observed frequently between 1) R cells, 2) S cells, 3) between R cells and S cells. The frequency of gap junctions in collagen gels decreased statistically (X2-test; p<0.001), when calcification was initiated. This cell culture system can be regarded as a useful model to analyze the initiation of cell mediated calcification in hard tissue. Gap junctions might function in cell communication and a decrease in their numbers could lead to cell death and, subsequently to calcification.
Similar content being viewed by others
References
Pautard, F.G.E.: A biomolecular survey of calcification. In: Calcif. Tissues 1965, Proc. 3rd. Eur. Symp. (Fleisch, H., Blackwood, H.J.J., Owen, M., ed.), Springer-Verlag, Berlin-Hiedelberg-New York, pp108–122, 1966.
Anderson, H.C.: Calcific diseases. Arch. Pathol. Lab. Med. 107, 341–348, 1983.
Robinson, R.: The possible significance of hexosephosphoric esters in ossification. Biochem. J. 17, 286–293, 1923.
Ali, S.Y. (ed.): Cell mediated calcification and matrix vesicles. Excerpta Medica, Amsterdam-New York-Oxford, 1986.
Peck, W.A., Birge, S.J.Jr. and Fedak, S.A.: Bone cells: biochemical and biological studies after enzymatic isolation. Science, 146, 1476–1477, 1964.
Yagiela, J.A. and Woodbury, D.M.: Enzymatic isolation of osteoblasts from fetal rat calvaria. Anat. Rec. 188, 287–306, 1977.
Friedenstein, A.J.: Precursor cells of mechanocytes. Int. Rev. Cytol. 47, 327–355, 1976.
Ashton, B.A., Allen, T.D., Howlett, C.R., Eaglesom, C.C., Hattori, A. and Owen, M.: Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin. Orthop. & Rel. Res. 151, 294–307, 1980.
Owen, M.: Histogenesis of bone cells. Calcif. Tiss. Res. 25, 205–207, 1978.
Iguchi, T., Uchima, F-D.A., Ostrander, P.L. and Berr, H.A.: Growth of normal mouse vaginal epithelial cells in and on collagen gels. Proc. Natl. Acad. Sci. U.S.A. 80, 3743–3747, 1983.
Ecarot-Charier, B., Glorieux, F.H., van der Rest, M. and Pereira, G.: Osteoblast isolated from mouse calvaria initiate matrix mineralization in culture. J. Cell Biol. 96, 639–643, 1983.
Kodama, H-A., Amagai, Y., Sudo, H., Ohno, T. and Iijima, K-I.: Culture conditions affecting differentiation and calcification in the MC3T3-E1 osteogenic cell line. In: Cell mediated calcification and matrix vesicle. (Ali, S.Y. ed.), Excerpta Medica., Amsterdam, pp 297–302, 1986.
Ali, S.Y., Evans, L. and Ralph, J.R.: Chondrocyte culture in fibrin clots: Matrix vesicle formation and calcification in the presence of calcium-β-Glycerophosphate. In: Cell mediated calcification and matrix vesicles. (Ali, S.Y. ed.), Excerpta Medica. Amsterdam, pp253–262, 1986.
Ralph, J.R and Ali, S.Y.: Induction of calcification in cultures of epiphyseal chondocytes with calcium β-glycerophosphate. In: Cell mediated calcification and matirx vesicles. (Ali, S.Y. ed.), Excerpta Medica, Amsterdam, pp263–268, 1986.
Kim, Y.S., Birge, S.J., Avioli, L.V. and Miller, R.: Cell density-dependent vitamin D effects on calcium accumulation in rat osteogenic sarcoma cells (ROS 17/2). Calcif. Tissue. Int. 41, 218–222, 1987.
Sudo, H., Kodama, H-A., Amagai, Y., Itakura, Y. and Yamamoto, S.: Mineralized tissue formation by MC3T3-E1 osteogenic cells embedded in three-dimensional gel matrix. In: Cell mediated calcification and matrix vesicles. (Ali, S.Y. ed.), Excerpta Medica. Amsterdam, pp 291–296, 1986.
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and Watson, J.D.: Molecular Biology of the Cell. 2nd ed. Garland Publishing. New York, 1989.
Loewenstein, W.R.: Junctional Intercellular communication: The cell-to-cell membrane channel. Physiol. Rev. 61, 829–913, 1981.
Yamazaki, K.: Sl/Sld mice have an increased number of gap junctions in their stromal cells. Blood Cells. 13, 421–432, 1988.
Doty, S.B.: Morphological evidence of gap junctions between bone cells. Calcif. Tissue. Int. 33, 509–512, 1981.
Piez, K.A., Eigner, E.A. and Lewis, M.S.: The chromatographic separation and amino acid composition of the subunit of several collagens. Biochemistry 2, 58–66, 1963.
Luna, L.G.: Manual of histologic staining methods of the Armed Forces Institute of Pathology. McGraw-Hill, New York, 1960.
von Kossa, J.: Ueber die im Organismus küstlich erzeugbaren Verkalkungen. Beitr. Pathol. Anat. Alleg. Pathol. 29, 163–202, 1901.
Burstone, M.S.: Enzyme histochemistry and its application in the study of neoplasma. Academic Press, London, 1962.
Anderson, H.C.: Vesicles Associated with calcification in the matrix of epiphyseal cartilage,. J. Cell Biol. 41, 59–72, 1969.
Nishimoto, S.K., Stryker, W.F. and Nimni, M.E.: Calcification of osteoblast like rat osteosarcoma cells in agarose suspension cultures. Calcif. Tissue Int. 41, 274–280, 1987.
Yasui, N., Osawa, S., Ochi, T., Nakashima, H. and Ono, K.: Primary culture of chondrocytes embedded in collagen gels. Exp. Cell Biol. 50: 92–100, 1982.
Allen, T.D., Schor, S.L. and Schor, A.M.: An ultrastructural review of collagen gels, a model system for cell-matrix, cell-basement membrane and cell-cell interactions. In: Scanning electron microscopy/1984/I SEM Inc. AME O Hare, pp 357–390, Chicago.
Gronowicz, G., Woodiel, F.N., McCarthy, M.-B. and Raisz, L.G.: In vitro mineralization of fetal rat parietal bones in defined serum-free medium: Effect of β-glycerol phosphate. J. Bone Mineral Res. 4, 313–324, 1989.
Holtrop, M.E.: The ultrastructure of the epiphyseal plate. II. The hypertrophic chondrocyte. Calcif. Tiss. Res. 9, 140–151, 1972.
Yamada, M.: Ultrastructural and cytochemical studies on the calcification of the tendon-bone joint. Arch. histol. jap. 39, 347–378, 1976.
Weigner, J.M. and Holtrop, M.E.: An ultrastructural study of bone cells: the occurrence of microtubles, microfilaments and tight junctions, Calcif. Tiss. Res. 14, 15–29, 1974.
Author information
Authors and Affiliations
About this article
Cite this article
Yamazaki, K., Ichimura, S., Allen, T.D. et al. Cell mediated calcification in collagen gel cultures of fetal rat calvaria cells. Loss of gap junctions precedes calcification. J Bone Miner Metab 7, 6–17 (1989). https://doi.org/10.1007/BF02399048
Issue Date:
DOI: https://doi.org/10.1007/BF02399048