Calcified Tissue Research

, Volume 25, Issue 1, pp 179–190 | Cite as

The fine structure of decalcified cartilage and bone: A comparison between decalcification procedures performed before and after embedding

  • E. Bonucci
  • J. Reurink


The ultrastructure of calcifying cartilage and bone has been examined under the electron microscope after using three different methods of decalcification. The first was carried out before embedding (by soaking specimens in EDTA or formic acid), the second after embedding (by floating ultrathin sections on formic acid), and the third after embedding (by soaking embedded specimens in EDTA or formic acid), and with later re-embedding.

The first procedure invariably induces drastic changes in the fine structure of the cells and calcified matrix, probably as a results of the extraction of organic material along with extraction of mineral. The second and third procedures make it possible to preserve ultrastructural details perfectly in both cells and calcified matrix. Of the two, the third procedure is preferable because of its greater simplicity. In areas that are still calcifying, these post-embedding decalcification techniques reveal the presence of crystal-associated, filamentous organic structures which are not recognizable in specimens decalcified before embedding. These structures, which could have a key role in inducing and regulating crystal formation and growth, are less evident in fully calcified areas (but not at their borders). This may partly be due to the loss of glycan components in the matrix during calcification. The most important determinant, however, seems to be the fact that during calcification the components of the matrix, including collagen fibrils, are involved in an aggregation process which reduces the amounts of free chemical groups available for reaction with the stain solution. Because post-embedding decalcification does not disturb this state of aggregation, the stainability of the matrix and the electron microscopic evidence of its components remain very low.

Key words

Decalcification Electron microscopy Calcified matrices 


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  1. Baird, I.L., Winborn, W.B., Bockman, D.E.: A technique of decalcification suited to electron microscopy of tissues closely associated with bone. Anat. Record159, 281–290 (1967)Google Scholar
  2. Bernard, G.W., Pease, D.C.: An electron microscopic study of initial intramembranous osteogenesis. Am. J. Anat.125, 271–290 (1969)PubMedGoogle Scholar
  3. Bernardi, G.: Interactions between hydroxyapatite and biological macromolecules (protein, nucleic acids). In: Physico-chimie et cristallographie des apatites d'intérêt biologique; p. 463–465 Colloque 230 du Centre Nat. de la Recherche Scientifique, Paris: Centre Nat. Rech. Sci. 1975Google Scholar
  4. Bernardi, G., Kawasaki, T.: Chromatography of polypeptides and proteins on hydroxyapatite columns. Biochim. Biophys. Acta160, 301–310 (1968)PubMedGoogle Scholar
  5. Bonucci, E.: Fine structure of early cartilage calcification. J. Ultrastruct. Res.20, 33–50 (1967)PubMedGoogle Scholar
  6. Bonucci, E.: Further investigation on the organic-inorganic relationships in calcifying cartilage. Calcif. Tiss. Res.3, 38–54 (1969)Google Scholar
  7. Bonucci, E.: The locus of initial calcification in cartilage and bone. Clin. Orthop.78, 108–139 (1971)PubMedGoogle Scholar
  8. Bonucci, E.: The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption. Calcif. Tiss. Res.16, 13–36 (1974)Google Scholar
  9. Bonucci, E.: The organic-inorganic relationships in calcified organic matrices. In: Physico-chimie et cristallographie des apatites d'intérêt biologique; p. 231–246. Colloque 230 du Centre Nat. de la Recherche Scientifique, Paris: Centre Nat. Rech. Sci. 1975Google Scholar
  10. Bonucci, E.: Gherardi, G.: Histochemical and electron microscope investigations on medullary bone. Cell Tiss. Res.163, 81–97 (1975)Google Scholar
  11. Bonucci, E., Derenzini, M., Marinozzi, V.: The organic-inorganic relationships in calcified mitochondria. J. Cell Biol.59, 185–211 (1973)PubMedGoogle Scholar
  12. Boothroyd, B.: The problem of demineralization in thin sections of fully calcified bone. J. Cell Biol.20, 165–173 (1964)PubMedGoogle Scholar
  13. Charman, J., Reid, L.: The effect of decalcifying fluids on the staining of epithelial mucins by Alcian blue. Stain Technol.47, 173–178 (1972)PubMedGoogle Scholar
  14. De Bernard, B., Stagni, N., Colautti, I., Vittur, F., Bonucci, E.: Glycosaminoglycans and endochondral calcification. Clin. Orthop.126, 285–291 (1977)PubMedGoogle Scholar
  15. Engfeldt, B., Hjerpe, A.: Glycosaminoglycans of dentine and predentine. Calcif. Tiss. Res.10, 152–159 (1972)Google Scholar
  16. Hirschman, A., Dziewiatkowski, D.D.: Protein-polysaccharide loss during endochondral ossification: immunochemical evidence. Science154, 393–395 (1966)PubMedGoogle Scholar
  17. Howell, D.S., Carlson, L.: Alterations in the composition of growth cartilage septa during calcification studied by microscopic x-ray elemental analysis. Exp. Cell Res.51, 185–195 (1968)PubMedGoogle Scholar
  18. Howell, D.S., Pita, J.C.: Calcification of growth plate cartilage with special reference to studies on micropuncture fluids. Clin. Orthop.118, 208–229 (1976)PubMedGoogle Scholar
  19. Jibril, A. O.: Proteolytic degradation of ossifying cartilage matrix and the removal of acid mucopolysaccharides prior to bone formation. Biochim. Biophys. Acta136, 162–165 (1967)PubMedGoogle Scholar
  20. Landis, W.J., Paine, M.C., Glimcher, M.J.: Electron microscopic observations of bone tissue prepared anhydrously in organic solvents. J. Ultrastruct. Res.59, 1–30 (1977)PubMedGoogle Scholar
  21. Leduc, E.H., Bernard, W.: Recent modifications of the glycol methacrylate embedding procedure. J. Ultrastruct. Res.19, 196–199 (1967)PubMedGoogle Scholar
  22. Lohmander, S., Hjerpe, A.: Proteoglycans of mineralizing rib and epiphyseal cartilage. Biochim. Biophys. Acta404, 93–109 (1975)PubMedGoogle Scholar
  23. Luft, J.H.: Improvements in epoxy resin embedding media. J. Biophys. Biochem. Cytol.9, 409–414 (1961)PubMedGoogle Scholar
  24. Marinozzi, V.: Silver impregnation of ultrathin sections for electron microscopy. J. Biophys. Biochem. Cytol.9, 121–133 (1961)PubMedGoogle Scholar
  25. Marinozzi, V.: Phosphotungstic acid (PTA) as a stain for polysaccharides and glycoproteins in electron microscopy. In: Electron Microscopy 1968 (Bocciarelli, D.S., ed.) v. 2, p. 55–56, Rome: Tipografia Poliglotta Vaticana 1968Google Scholar
  26. Miller, E.J., Martin, G.R.: The collagen of bone. Clin. Orthop.59, 195–232 (1968)PubMedGoogle Scholar
  27. Mollenhauer, H.H.: Plastic embedding mixtures for use in electron microscopy. Stain Technol.39, 111–114 (1964)PubMedGoogle Scholar
  28. Nylen, M.U., Ommell, K.-A.: The relationship between the apatite crystals and the organic matrix of rat enamel. In: Electron Microscopy (Breese, S.S., Jr., ed.), v. 2, p. QQ 4–5. New York and London: Academic Press 1962Google Scholar
  29. Pugliarello, M.C., Vittur, F., de Bernard, B., Bonucci, E., Ascenzi, A.: Chemical modifications in osteones during calcification. Calcif. Tiss. Res.5, 108–114 (1970)Google Scholar
  30. Rambourg, A.: Localisation ultrastructurale et nature du matériel coloré au niveau de la surface cellulaire par le mélange chromique-phosphotungstique. J. Microscopie8, 325–342 (1969)Google Scholar
  31. Robinson, R.A.: Chemical analysis and electron microscopy of bone. In: Bone as a Tissue (Rodahl, K., Nicholson, J.T., Brown, E.M., eds.), p. 186–250. New York: McGraw-Hill Book Co. 1960Google Scholar
  32. Rölla, G.: Adsorption of salivary glycoproteins and bacteria to hydroxyapatite. In: Physico-chimie et cristallographie des apatites d'intérêt biologique; p. 459–462, Colloque 230 du Centre Nat. de la Recherche Scientifique, Paris: Centre Nat. Rech. Sci. 1975Google Scholar
  33. Rönnholm, E.: III. The structure of the organic stroma of human enamel during amelogenesis. J. Ultrastruct. Res.3, 368–389 (1962)Google Scholar
  34. Schajowicz, F., Cabrini, R.L.: The effect of acids (decalcifying solutions) and enzymes on the histochemical behaviour of bone and cartilage. J. Histochem. Cytochem.3, 122–129 (1955)PubMedGoogle Scholar
  35. Scherft, J.P.: The Lamina Limitans of the organic matrix of calcified cartilage and bone. J. Ultrastruct. Res.38, 318–331 (1972)PubMedGoogle Scholar
  36. Scott, J.E.: Phosphotungstate: a “universal” (non specific) precipitant of polar polymers in acid solution. J. Histochem. Cytochem.19, 689–690 (1971)PubMedGoogle Scholar
  37. Smales, F.C.: Structural subunit in prisms of immature rat enamel. Nature258, 772–774 (1974)Google Scholar
  38. Smith, J.W.: The disposition of proteinpolysaccharide in the epiphyseal plate cartilage of the young rabbit. J. Cell Sci.6, 843–864 (1970)PubMedGoogle Scholar
  39. Sundström, B., Takuma, S.: A further contribution on the ultrastructure of calcifying cartilage. J. Ultrastruct. Res.36, 419–424 (1971)PubMedGoogle Scholar
  40. Tiselius, A., Hjertén, S., Levin, Ö.: Protein chromatography on calcium phosphate columns. Arch. Biochem. Biophys.65, 132–155 (1956)PubMedGoogle Scholar
  41. Travis, D.F., Glimcher, M.J.: The structure and organization of, and the relationship between the organic matrix and the inorganic crystals of embryonic bovine enamel. J. Cell Biol.23, 447–497 (1964)PubMedGoogle Scholar
  42. Vigliani, F., Marotti, F.: Modificazioni microradiografiche ed istochimiche del tessuto osseo nella decalcificazione con Na4EDTA. Clin. Ortopedica15, 521–534 (1963)Google Scholar
  43. Vittur, F., Pugliarello, M.C., de Bernard, B.: Chemical modifications of cartilage matrix during endochondral calcification. Experientia27, 126–127 (1971)PubMedGoogle Scholar
  44. Warshawsky, H., Moore, G.: A technique for the fixation and decalcification of rat incisors for electron microscopy. J. Histochem. Cytochem.15, 542–549 (1967)PubMedGoogle Scholar
  45. Weatherford, T.W., Mann, W.V., Jr.: A method of microscopic evaluation of effects of demineralizers on complex carbohydrates of rat tissues. J. Microscopy99, 91–100 (1973)Google Scholar
  46. Wuthier, R.E.: A zonal analysis of inorganic and organic constituents of the epiphysis during endochondral calcification. Calcif. Tiss. Res.4, 20–38 (1969)Google Scholar

Copyright information

© Springer-Verlag 1978

Authors and Affiliations

  • E. Bonucci
    • 1
  • J. Reurink
    • 2
  1. 1.First Institute of Pathological Anatomy and Laboratory of HistochemistryUniversity of RomeRomaItaly
  2. 2.Department of Orthodontics, Faculty of MedicineVrije UniversiteitAmsterdamThe Netherlands

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