Cell Tissue Res (1993) 273:175 182 Cell&Tissue Research 9 Springer-Verlag 1993 Human amelogenesis: high resolution electron microscopy of nanometer-sized particles F.J.G. Cuisinier, P. Steuer, B. Senger, J.C. Voegel, R.M. Frank Centre de Recherches Odontologiques, INSERM CJF 92-04, 1, Place de l'H6pital, F-67000 Strasbourg, France Received: 9 November 1992/Accepted: 15 January 1993 Abstract. Growth of inorganic crystals of enamel is de- scribed as a two-stage process with growth of ribbon-like crystals in length and width, followed by their develop- ment in thickness. In early stages of crystal growth dur- ing human amelogenesis nanometer-sized particles with a mean diameter of 1.1 nm were described between rib- bon-like crystals. These small particles had a crystalline structure but their lattice parameters did not seem to be directly related to those of calcium phosphates. The nanometer-sized particles appear to correspond to initial stages of apatite crystal growth. Their localization close to ribbon-like crystals and their progressive increase in size and number may indicate that they represent a pre- cursor phase for these crystals. Nucleation areas at both extremities, of elongated ribbon-like crystals could be in- volved in the two-directional growth of ribbons and/or in nanometer-sized particle nucleation. Key words: Dental enamel - Mineralization - Develop- ment - Nanometer-sized particles - Hydroxyapatite - Human Introduction Enamel crystal formation begins in the secretory stage of amelogenesis within the extracellular matrix very close to the apical membrane of the ameloblast. Conventional transmission electron-microscopic studies revealed at this stage the presence of ribbon-like crystals (R6nnholm 1962; Daculsi and Kerebel 1978; Weiss et al. 1981). The inorganic material then grows progressively in thickness during the maturation stage until final size has been reached. High resolution electron microscopy (HREM) of ribbon-like crystals showed that they can be described by a structure closely related to hydroxyapatite (HA) mineral (Cuisinier et al. 1992a), and that they present numerous structural defects (Cuisinier et al. 1990). Infra- Correspondence to: F.J.G. Cuisinier red spectroscopic studies demonstrated important changes in the mineral composition (variation of carbon- ate content) during the secretory stage (Aoba and Mor- eno 1990; Rey et al. 1990). By the indirect pyrolysis method the in vivo presence of acidic calcium phosphate during enamel mineral deposition was noted with acidic calcium phosphate content failing off with maturation time (Siew et al. 1992). Acidic calcium phosphate could act as a precursor phase for ribbon-like crystals and could either be related to octacalcium phosphate (OCP) (Brown 1965) or to dicalcium phosphate dihydrate (DCPD) (Young and Brown 1982). The detection of acidic calcium phosphates by nuclear magnetic reso- nance in bone did not suggest that the former represent precursor phases; such findings indicate the presence of imperfect apatite with surface adsorption of HPO4 z- in a DCPD-like configuration (Roberts et al. 1992). These observations in bone suggested indirectly that an initial mineral phase different from HA could be formed in enamel. The present work was devoted to the study of the very early stages of enamel mineralization by use of HREM. This technique is a powerful tool for the study of crystalline structure at the atomic level (with a resolu- tion of 0.19 nm) as well as for the detection of small particles and for the observation of amorphous material. Whereas other investigation techniques such as X-ray diffraction and infrared spectroscopy allow the study of the composition and the structure over larger areas and thus lead to averaged data, HREM seems to be an appropriate method for the study of the earliest min- eral phases of enamel. Materials and methods Germs of primary incisors were collected from two 5-month-old human aborted fetuses (Gynecological Department, Universit~ Louis Pasteur, Strasbourg, France) and fixed, for 3 h, in a 2% glutaraldehyde and 2% paraformaldehyde solution in 0.1 M caco- dylate buffer at pH 7.4. Specimens were dissected and secretory zones of enamel were selected_ After 2 h of post-fixation in a 1% OsO4 sotution in the same buffer, samples were embedded in epoxy