Calcified Tissue Research

, Volume 26, Issue 1, pp 173–179 | Cite as

Effect of solubilized bone matrix components on cultured fibroblasts derived from neonatal rat tissues

  • T. Anastassiades
  • O. Puzic
  • R. Puzic


Decalcified rat bone matrix was extracted with 4M guanadinium chloride. Extensive dialysis of the solution yielded material which could be, in part, further solubilized in isotonic salt solution and which was particularly enriched in glycoprotein compared to whole bone matrix. The addition of this solubilized material to tissue culture media in concentrations of 25 to 100 μg dry weight per milliliter of medium resulted in characteristic morphologic and metabolic alterations of fibroblasts from neonatal rats. These alterations in glycosaminoglycan synthesis were similar to those we had previously reported, resulting from the addition of whole, particulate bone matrix to such cultures. The most prominent change was an increase in hyaluronic acid synthesis and a smaller increase in chondroitin sulfate synthesis. Both intracellular synthesis and secretion into the medium were stimulated, and the magnitude of this stimulation was related to the concentration of the solubilized material in the medium. However, the age of the rat bone appeared to be also an important determinant of the amount of biological activity that could be solubilized. Bone appears to be greatly enriched in this biological activity compared to skin, and fibroblasts derived from neonatal rat muscle are relatively more responsive than fibroblasts derived from neonatal rat skin.

Key words

Bone matrix Glycosaminoglycans Tissue culture Fibroblasts 


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  1. 1.
    Sacerdotti, C., Frattin, G.: Sulla produzione eteroplastica di osso, Giorn. Acad. Med. Torino64:825–828, 1901Google Scholar
  2. 2.
    Huggins, C.: The formation of bone under the influence of epithelium of the urinary tract, Arch. Surg.22:377–381, 1931Google Scholar
  3. 3.
    Lacroix, P.: In Desser (ed.): L'Organization des Os, pp. 148–154. Liege, Belgium, 1949Google Scholar
  4. 4.
    Urist, M.R.: Bone formation by autoinduction, Science150:893–899, 1965PubMedGoogle Scholar
  5. 5.
    Huggins, C., Wiseman, S., Reddi, A.H.: Transformation of fibroblasts by allogeneic and xenogeneic transplants of demineralized tooth and bone, J. Exp. Med.132:1250–1258, 1970PubMedGoogle Scholar
  6. 6.
    Urist, M.R.: A morphogenetic matrix for differentiation of bone tissue, Calcif. Tissue Res. (Suppl.)4:98–101, 1970Google Scholar
  7. 7.
    Kosher, R.A., Church, R.L.: Stimulation of in vitro somite chondrogenesis by procollagen and collagen, Nature258:327–330, 1975PubMedGoogle Scholar
  8. 8.
    Ainsworth, T., Puzic, O., Anastassiades, T.: The effect of bone matrix on young connective tissue cells in culture, J. Lab. Clin. Med.89:781–792, 1977PubMedGoogle Scholar
  9. 9.
    Gardell, S.: Separation on Dowex-50 ion exchange resin of glucosamine and galactosamine and their quantitative determination, Acta Chem. Scand.7:201–206, 1953Google Scholar
  10. 10.
    Anastassiades, T., Dziewiatkowski, D.: The effect of cortisone on the metabolism of connective tissues in the rat, J. Lab. Clin. Med.75:826–839, 1970PubMedGoogle Scholar
  11. 11.
    Lowry, O.H., Rosebrough, N., Farr, A.L., Randall, R.J.: Protein measurement with the folin phenol reagent, J. Biol. Chem.193:265–275, 1951PubMedGoogle Scholar
  12. 12.
    Neuman, R.E., Logan, M.A.: The determination of hydroxyproline, J. Biol. Chem.184:299–305, 1950PubMedGoogle Scholar
  13. 13.
    Bitter, T., Muir, H.M.: A modified uronic acid carbazole reaction, Anal. Biochem.4:330–334, 1962PubMedGoogle Scholar
  14. 14.
    Newton, D.J., Scott, J.E., Whiteman, P.: The estimation of acid glycosaminoglycans-alcian blue complexes eluted from electrophoretic strips, Anal. Biochem.62:268–273, 1974PubMedGoogle Scholar
  15. 15.
    Schmidt, M.: Fractionation of acid mucopolysaccharides on DEAE-sephadex anion exchange, Biochem. Biophys. Acta63:346–348, 1962PubMedGoogle Scholar
  16. 16.
    Winzler, R.J.: Determination of serum glycoproteins, Meth. Biochem. Anal.2:278–311, 1955Google Scholar
  17. 17.
    Porter, W.H.: Application of nitrous acid deamination of hexosamines to the simultaneous GLC determination of neutral sugars and aminosugars in glycoproteins, Anal. Biochem.63:27–43, 1975PubMedGoogle Scholar
  18. 18.
    Toole, B.P.: Hyaluronate turnover during chondrogenesis in the developing chick limb and axial skeleton, Dev. Biol.29:321–329, 1972PubMedGoogle Scholar
  19. 19.
    Iwata, H., Urist, M.R.: Hyaluronic acid production and removal during bone morphogenesis in implants of bone matrix in rats, Clin. Orthop.90:236–245, 1973PubMedGoogle Scholar
  20. 20.
    Toole, B.P.: Hyaluronate inhibition of chondrogenesis: antogonism of thyroxine, growth hormone and calcitonin, Science180:302–303, 1973PubMedGoogle Scholar
  21. 21.
    Nevo, Z., Dorfman, A.: Stimulation of chondromucoprotein synthesis in chondrocytes by extracellular chondromucoprotein, Proc. Natl. Acad. Sci. U.S.A.69:2069–2072, 1972PubMedGoogle Scholar
  22. 22.
    Wiebkin, O.W., Muir, H.: The inhibition of sulfate incorporation in isolated adult chondrocytes by hyaluronic acid, FEBS Lett.37:42–46, 1973PubMedGoogle Scholar
  23. 23.
    Handey, C.J., Lowther, D.A.: Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture, Biochim. Biophys. Acta444:69–74, 1976PubMedGoogle Scholar
  24. 24.
    Piez, K., Eigner, R., Lewis, M.: The chromatographic separation and amino acid composition of subunits of several collagens, Biochemistry2:58–66, 1963Google Scholar
  25. 25.
    Hronowski, L. and Anastassiades, T.: Quantitation and Interaction of Glycosaminoglycans with Alcian Blue in Dimethyl Sulfoxide Solutions, Anal. Biochem. In pressGoogle Scholar

Copyright information

© Springer-Verlag 1978

Authors and Affiliations

  • T. Anastassiades
    • 1
  • O. Puzic
    • 1
  • R. Puzic
    • 1
  1. 1.Rheumatic Disease UnitQueen's UniversityKingstonCanada

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