Skip to main content
Log in

Cryopreservation and Image Enhancement of Juvenile and Adult Dentine Mineral

  • Published:
The Histochemical Journal Aims and scope Submit manuscript

Abstract

The inorganic component of bone and related hard tissues is generally described as sheets of uniform needle- and plate-like crystals. However, cryofixation has become the method of choice for ultrastructural studies of bone mineral when ladder-like arrangements of filaments contained within deformable microspheres about 1 µm in diameter are apparently the prime structural feature and are consistent with the optical image. The same methodology has now been applied to mature human dentine in caries-free juvenile and adult teeth. These were fixed, sliced, stained for mineral and examined optically or were snap frozen, fragmented under liquid nitrogen, freeze-substituted with methanol or acetone and embedded without thawing in Lowicryl K4M for electron microscopy. Others were processed by traditional transmission electron microscopy methods. To obtain maximum resolution, the electron micrographs were photographically printed as negatives and image-enhanced by digitisation using a Polaroid Sprint Scan 45 and laser printer. In both optical and cryopreparations of juvenile and adult dentine, mineral microspheres up to 1 µm in diameter, were present in the dentinal tubules and peritubular dentine. Within these objects, the mineral was primarily in the form of sinuous electron dense filaments, 5 nm thick, which had a characteristic periodicity. In these preparations needle-like and plate-like structures were rare. In contrast, after traditional transmission electron microscopy preparation although similar filamentous structures remained, the mineral more generally had the familiar form of needles measuring approximately 50 nm in the long axis. The cryopreserved calcified filaments were apparently particularly densely distributed in the intertubular dentine where their parallel ladder-like arrays often formed highly orientated struts and stays. It was concluded that early dentine mineral has the form of filamentous microspheres and as in bone (and other calcifying tissues and cells) has no specific association with collagen. It was also concluded that these structures compact and deform with maturity into a sub-structural framework which may relate to powerful biomechanical forces transmitted through the tissue. Needle- or plate-like mineral is probably rare in vivo in dentine, only becoming commonplace after extensive chemical processing.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References cited

  • Aaron JE, Pautard FGE (1972) Ultrastructural features of phosphate in developing bone cells. Isrl J Med Sci 8: 625-629.

    Google Scholar 

  • Aaron JE (1973) Osteocyte types in the developing mouse calvarium. Calcif Tissue Res 12: 259-279.

    Google Scholar 

  • Aaron JE (1978) Histological aspects of vitamin D and bone. In: Lawson DEM, ed. Vitamin D. London: Academic Press, London, pp. 201-265.

    Google Scholar 

  • Aaron JE, Oliver B, Clarke N, Carter DH (1999) Calcified microspheres as biological entities and their isolation from bone. Histochem J 31: 455-470.

    Google Scholar 

  • Baskin TI, Miller DD, Vos JW, Wilson JE, Helpler PK (1996) Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy. J Microsc 182: 149-161.

    Google Scholar 

  • Boyde A (1974) TEM of ion beam thinned dentine. Cell Tiss Res 152: 543-550.

    Google Scholar 

  • Carter DH, Hatton PV, Aaron JE (1997) The microanalysis of slam-frozen bone mineral. Histochem J 29: 783-793.

    Google Scholar 

  • Carter DH, Scully AJ, Davies RM, Aaron JE (1998) Evidence for phosphoprotein microspheres in bone. Histochem J 30: 677-686.

    Google Scholar 

  • Edelmann L (1991) Freeze-substitution and the preservation of diffusible ions. J Microsc 161 217-228.

    Google Scholar 

  • Eisenmann DR, Glick PL (1972) Ultrastructure of initial crystal formation in dentine. J Anat 116: 285-302.

    Google Scholar 

  • Frank RM, Nalbandian J (1989) Structure and ultrastructure of dentine. In: Berkovitz BKB, Boyde A, Frank RM, Höhling HJ, Moxham BJ, Nalbandian J, Tonge CH, eds. Handbook of Microscopic Anatomy, Vol. 6, Teeth. Germany: Springer-Verlag, pp. 173-247.

    Google Scholar 

  • Gay CV (1977) The ultrastructure of the extracellular phase of bone as observed in frozen thin sections. Calcif Tissue Res 23: 215-223.

    Google Scholar 

  • Goldberg M, Escaig F (1984) Improved preservation of intramitochondrial granules in rat incisor odontoblasts by rapid freezing and freeze substitution fixation. Arch Oral Biol 29: 295-301.

    Google Scholar 

  • Hardwick JL, Martin CJ, Davies, TGH (1965) The microstructure of mature dental enamel as observed under the optical microscope. In: Stack MV, Fearnhead RW, eds. Tooth Enamel. Bristol: John Wright and Sons Ltd, pp. 168-171.

    Google Scholar 

  • Harvey DMR (1982) Freeze-substitution. J Microsc 127: 209-211.

    Google Scholar 

  • Hayat MA (1989) Principles and Techniques of Electron Microscopy. Biological Applications. 3rd Edition. London: Macmillan Press Ltd, pp. 395-397.

    Google Scholar 

  • Herold RC, Kaye H (1966) Mitochondria in odontoblastic processes. Nature 210: 108-109.

    Google Scholar 

  • Höhling HJ (1989) Special aspects of biomineralization of dental tissues. In: Berkovitz BKB, Boyde A, Frank RM, Höhling HJ, Moxham BJ, Nalbandian J, Tonge CH, eds. Handbook of Microscopic Anatomy, Vol. 6, Teeth. Germany: Springer-Verlag, pp. 475-524.

    Google Scholar 

  • Höhling HJ, Arnold S, Barckhaus RH, Plate U, Weismann HP (1995) Structural relationship between the primary crystal formations and the matrix macromolecules in different hard tissues. Discussion of a general principle. Connect Tissue Res 33: 171-178.

    Google Scholar 

  • Kashiwa HK, House CM (1964) The glyoxal bis-(2-hydroxyanil) method modified for localizing insoluble calcium salts. Stain Technol 39: 359-367.

    Google Scholar 

  • Kashiwa HK (1970) Calcium phosphate in osteogenic cells. Clin Orthop 70: 200-211.

    Google Scholar 

  • Kashiwa HK, Sigman DM (1966) Calcium localized in odontogenic cells of rat mandibular teeth by the glyoxal bis-(2-hydroxyanil) method. J Dent Res 45: 1796-1799.

    Google Scholar 

  • La Flèche R, Frank RM, Steuer P (1985) The extent of the human odontoblast process as determined by transmission electron microscopy: the hypothesis of a retractable suspensor system. J Biol Buccale 13: 293-305.

    Google Scholar 

  • Livesey SA, Del Campo AA, McDowall AW, Stasney JT (1991) Cryo-fixation and the ultra-low-temperature freeze-drying as a preparative technique for TEM. J Microsc 161: 205-215.

    Google Scholar 

  • Lepault J, Erk I, Nicolas G (1991) Time-resolved cryo-electron microscopy of vitrified muscular components. J Microsc 161: 47-57.

    Google Scholar 

  • Lepault J, Ranck J-L, Erk I (1992) Electron cryomicroscopy of frozenhydrated biological specimens: analysis of freezing artefacts by X-ray cryo-crystallography. Ultramicroscopy 46: 19-24.

    Google Scholar 

  • Lester KS, Boyde A (1968) The surface morphology of some crystalline components of dentine. In: Symons NBB, ed. Dentine and Pulp: Their Structure and Reactions. London: Livingstone, pp. 197-219.

    Google Scholar 

  • Morgan AJ (1979) Non-freezing techniques of preparing biological specimens for electron microprobe analysis. Scan Elec Microsc 11: 635-648.

    Google Scholar 

  • Oyajobi BO, Frazer A, Hollander AP, Graveley RM, Xu C, Houghton A, Hatton PV, Russell RGG, Stringer BMJ (1998) Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature sensitive chondrocytes derived from adult human articular cartilage. J Bone Min Res 13: 432-442.

    Google Scholar 

  • Pautard FGE (1975) The structure and genesis of calcium phosphate in vertebrates and invertebrates. In: Physico-chemie et Crystallographie des Apatites d'Interêt Biologique. Colloques Internationaux CNRS 230: 93-100.

    Google Scholar 

  • Pautard FGE (1981) Calcium phosphate microspheres. Prog Biol Crystal Growth Charact 4: 89-98.

    Google Scholar 

  • Plate U, Tkotz T, Wiesmann HP, Stratmann U, Joos U, Hohling HJ (1996) Early mineralization of matrix vesicles in the epiphyseal growth plate. J Microsc 183: 102-107.

    Google Scholar 

  • Robards AW, Sleytr UB (1985) Low temperature methods in biological electron microscopy. In: Glauert AM, ed. Practical Methods in Electron Microscopy Vol. 10. Amsterdam, New York, Oxford: Elsevier, pp. 1-551.

    Google Scholar 

  • Schraer H, Gay CV (1977) Matrix vesicles in newly synthesised bone observed after ultracryotomy and ultramicroincineration. Calcif Tissue Res 23: 185-188.

    Google Scholar 

  • Stratmann U, Schaarschmidt K, Weismann HP, Plate U, Höhling HJ, Szuwart T (1997) The mineralization of mantle dentine and circumpulpal dentine in the rat: an ultrastructural and element-analytical study. Anat Embryol 195: 289-297.

    Google Scholar 

  • Takano Y, Yamamoto T, Domon T, Wakta M (1990) Histochemical, ultrastructural, and electron microprobe analytical studies on the localisation of calcium in rat incisor ameloblasts at early stage amelogenesis. Anat Rec 228: 123-131.

    Google Scholar 

  • Takuma S, Eda N (1971) Ultrastructure of rat odontoblasts in various stages of their development and maturation. Arch Oral Biol 16: 993-1011.

    Google Scholar 

  • Takuma S, Yanagisawa T, Lin WL (1977) Ultrastructural and microanalytical aspects of developing osteodentin in rat incisors. Calcif Tissue Res 24 215-222.

    Google Scholar 

  • Termine, J.D. (1972) Discussion. In: Slavkin, H.C., ed. The Comparative Biology of Extracellular Matrices. New York, London: Academic Press, pp. 443-448.

    Google Scholar 

  • Van Harrevald A, Crowell J (1964) Electron microscopy after rapid freezing on a metal surface and substitution fixation. J Anat Res 149: 381-385.

    Google Scholar 

  • Wroblewsi R, Wroblewski J, Roomans GM (1987) Low temperature techniques in biomedical microanalysis. Scan Microsc 1: 1225-1240.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Carter, D.H., Scully, A.J., Hatton, P.V. et al. Cryopreservation and Image Enhancement of Juvenile and Adult Dentine Mineral. Histochem J 32, 253–261 (2000). https://doi.org/10.1023/A:1004059219242

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1004059219242

Keywords

Navigation