Summary
The fine structure of the extracellular phase of avian medullary bone and embryonic chick femur was examined in thin sections prepared by ultracryotomy and ultramicroincineration. Since contact with solutions was completely avoided, little or no loss or dislocation of mineral constituents could occur. Amorphous bone mineral (ABM) was present in two forms: as 15–30 nm spheres and as a structure-free haze. Removal of all organic material by low temperature ashing left the ABM intact. Crystals were usually associated with the ABM. In newly ossifying regions clusters or nodules of randomly oriented crystals and ABM appeared to coalesce when they reached approximately 1 μm in diameter. In highly calcified regions crystals appeared to be oriented along collagen fibers. ABM did not appear to be associated with collagen. Unmineralized collagen was visible in osteoid after staining with dry OsO4 vapor and it appeared to be diverted around nodules. Structures which resembled matrix vesicles were present. Selected area electron diffraction patterns indicated the presence of hydroxyapatite.
Similar content being viewed by others
References
Anderson, H.C.: Calcium accumulating vesicles in the intercellular matrix of bone. In: Hard tissue growth, repair and remineralization (K. Elliott and D.W. Fitzsimons, eds.), pp. 213–246. Amsterdam: Associated Scientific Publishers 1973
Anderson, H.C.: Matrix vesicles of cartilage and bone. In: The biochemistry and physiology of bone (G.H. Bourne, ed.), Vol. 4, pp. 135–157. New York: Academic Press 1976
Anderson, H.C., Reynolds, J.J.: Pyrophosphate stimulation of calcium uptake into cultured embryonic bone. Fine structure of matrix vesicles and their role in calcification. Develop. Biol.34, 211–227 (1973)
Appleton, T.C.: Cryoultramicrotomy, possible applications in cytochemistry. In: Electron microscopy and cytochemistry (E. Wisse et al., eds.), pp. 229–241. Amsterdam: North-Holland 1973
Appleton, T.C.: A cryostat approach to ultrathin dry frozen sections for electron microscopy: A morphological and x-ray analytical study. J. Microsc.100, 49–74 (1974)
Arnott, H.J., Pautard, F.G.E.: Osteoblast function and fine structure. Israel J. Med. Sci.3, 657–670 (1967)
Ascenzi, A., Francois, C., Bocciarelli, D.S.: On the bone induced by oestrogens in birds. J. Ultrastruct. Res.8, 491–505 (1963)
bernard, G.W., Pease, D.C.: An electron-microscopic study of initial intramembranous osteogenesis. Am. J. Anat.125, 271–290 (1969)
Betts, F., Posner, A.S.: An x-ray radial dsitribution study of amorphous calcium phosphate. Mat. Res. Bull.9, 353–360 (1974)
Bocciarelli, D.S.: Morphology of crystallites in bone. Calcif. Tiss. Res.5, 261–269 (1970)
Bonucci, E.: The locus of initial calcification in cartilage and bone. Clin. Orthop.78, 108–139 (1971)
Boothroyd, B.: Observations on embryonic chick-bone crystals by high resolution transmission electron microscopy. Clin. Orthop.106, 290–310 (1975)
Cameron, D.A.: The ultrastructure of bone. In: The biochemistry and physiology of bone (G.H. Bourne, ed.), Vol. 1, pp. 191–236. New York: Academic Press 1972
Christensen, A.K.: Frozen thin sections of fresh tissue for electron microscopy, with a description of pancreas and liver. J. Cell Biol.51, 772–804 (1971)
Decker, J.D.: An electron microscopic investigation of osteogenesis in the embryonic chick. Am. J. Anat.118, 591–618 (1966)
Eanes, E.D.: The interaction of supersaturated calcium phosphate solutions with apatitic substrates. Calcif. Tiss. Res.20, 75–89 (1976)
Eanes, E.D., Posner, A.S.: Kinetics and mechanism of conversion of non-crystalline calcium phosphate to crystalline hydroxyapatite. Trans. N.Y. Acad. Sci.28, 233–241 (1965)
Eanes, E.D., Posner, A.S.: Structure and chemistry of bone mineral. In: Biological calcification: Cellular and molecular aspects (H. Schraer, ed.), pp. 1–26. New York: Appleton-Century-Crofts 1970
Eanes, E.D., Termine, J.D., Nylen, M.U.: An electron microscope study of the formation of amorphous calcium phosphate and its transformation to crystalline apatite. Calcif. Tiss. Res.12, 143–158 (1973)
Eanes, E.D., Termine, J.D., Posner, A.S.: Amorphous calcium phosphate in skeletal tissues. Clin. Orthop.53, 223–235 (1967)
Fitton-Jackson, S., Randall, J.T.: Fibrogenesis and the formation of matrix in developing bone. In: Bone structure and metabolism (G.E.W. Wolstenholme and C.M. O'Connor, eds.), pp. 47–64. Boston: Little, Brown 1956
Frazier, P.D., Brown, F.J., Rose, L.S., Fowler, B.O.: Radiofrequency oxygen excitation apparatus for low-temperature ashing. J. dent. Res.46, 1098–1101 (1967)
Gay, C.V., Schraer, H.: Ultrastructure of rapidly forming bone prepared by frozen thin-sectioning. Fed. Proc.34, 936a (1975a)
Gay, C., Schraer, H.: Frozen thin-sections of rapidly forming bone: Bone cell ultrastructure. Calcif. Tiss. Res.19, 39–49 (1975b)
Gersh, I.: Relation of the walls of large matrix compartments of epiphyseal cartilage to the formation of calcium crystals. In: Submicroscopic cytochemistry. Membranes, mitochondria and connective tissues (I. Gersh, ed.), Vol. 2, pp. 187–205. New York: Academic Press 1973
Hancox, N.M., Boothroyd, B.: Electron microscopy of the early stages of osteogenesis. Clin. Orthop.40, 153–161 (1965)
Harper, R.A., Posner, A.S.: Measurement of non-crystalline calcium phosphate in bone mineral. Proc. Soc. Exp. Biol. Med.122, 137–142 (1966)
Höhling, H.J., Ashton, B.A., Köster, H.D.: Quantitative electron microscopic investigations of mineral nucleation in collagen. Cell Tiss. Res.148, 11–26 (1974)
Hohman, W., Schraer, H.: Low temperature ultramicro-incineration to thin-sectined tissue. J. Cell Biol.55, 328–354 (1972)
Jackson, S.F.: The fine structure of developing bone in the embryonic fowl. Proc. Roy. Soc. (London)B146, 270–280 (1957)
Kato, Y., Ogura, H.: Low-temperature ashing of bovine dentine. Calcif. Tiss. Res.18, 141–148 (1975)
Miller, A.L., Schraer, H.: Ultrastructural observations of amorphous bone mineral in avian bone. Calcif. Tiss. Res.18, 311–324 (1975)
Molnar, Z.: Development of the parietal bone of young mice. I. Crystals of bone mineral in frozen-dried preparations. J. Ultrastruct. Res.3, 39–45 (1959)
Nylen, M.U., Eanes, E.D., Termine, J.D.: Molecular and ultrastructural studies of non-crystalline calcium phosphates. Calcif. Tiss. Res.3, 95–108 (1972)
Posner, A.S., Betts, F.: Synthetic amorphous calcium phosphate and its relation to bone mineral structure. Accts. Chem. Res.8, 273–281 (1975)
Pugliarello, M.C., Vittur, F., deBernard, B., Bonucci, E., Ascenzi, A.: Analysis of bone composition at the microscopic level. Calcif. Tiss. Res.12, 209–216 (1973)
Rebhun, L.I.: Freeze-substitution and freeze-drying. In: Principles and techniques of electron microscopy (M.A. Hayat, ed.), Vol. 2, pp. 1–49. New York: Academic Press 1972
Robinson, R.A., Cameron, D.A.: Electron microscopy of cartilage and bone matrix at the dital epiphyseal line of the femur in the newborn infant. J. Biophys. Biochem. Cytol.2 (suppl.), 253–260 (1956)
Robinson, R.A., Watson, M.L.: Crystal collagen relationships in bone as observed in the electron microscope. III. Crystal and collagen morphology as a function of age. Ann. N.Y. Acad. Sci.60, 596–628 (1955)
Schraer, H., Gay, C.V.: Matrix vesicles in newly synthesizing bone observed after ultracryotomy and ultramicroincineration. Calcif. Tiss. Res. (in press)
Tannenbaum, P.J., Schraer, H., Posner, A.S.: Crystalline changes in avian bone as related to the reproductive cycle. Calcif. Tiss. Res.14, 83–86 (1974)
Termine, J.D.: Mineral chemistry and skeletal biology. Clin. Orthop.85, 207–241 (1972)
Termine, J.D., Posner, A.S.: Amorphous/crystalline interrelationships in bone mineral. Calcif. Tiss. Res.1, 8–23 (1967)
Turner, R.T., Schraer, H.: Estrogen-induced cyclic changes in avian bone metabolism. Calcif. Tiss. Res. (in press)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Gay, C.V. The ultrastructure of the extracellular phase of bone as observed in frozen thin sections. Calc. Tis Res. 23, 215–223 (1977). https://doi.org/10.1007/BF02012788
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02012788