During amelogenesis, elements and compounds are transported to and from the mineralizing areas. The tissue to be calcified is at first a soft matrix that gradually transforms into a fully mineralized tissue. During the period of hard-tissue formation the ameloblasts undergo a series of differentiation stages. The enamel organ, which is of ectodermal origin, has as its major function the production of dental enamel. During amelogenesis, the ameloblasts pass through several phases of differentiation. First, there is a proliferative phase, where preameloblasts form by ectodermal–mesodermal interactions. Next comes a presecretory phase where the cells differentiate, ending with the loss of their mitotic capacity. During the following secretory phase, the enamel matrix is secreted. Between the secretory and the final maturation stages, there is a short transition zone. During the maturation phase the proteinaceous matrix is almost completely resorbed in parallel with the growth of hydroxyapatite crystals.

In previous analyses of the inorganic composition of dental enamel, various techniques have been utilized. The SIMS technique has been used for more than 20 years for the analysis of dental hard tissues. This technique is very suitable for the elemental analysis of mineralized tissues, such as dental enamel, which have a high content of inorganic elements. SIMS has shown consistent patterns for some inorganic elements from the enamel–dentine border towards the enamel surface. During the differentiation of the ameloblasts, the elements form gradients expressed during the subsequent formation of enamel. Elemental gradients in the outermost enamel surface, however, might have a posteruptive origin, explained by the exposure of the enamel to various elements in saliva. In the continuously erupting rat incisor, all stages of differentiation appear in a sequence along the same tooth. Within the same tooth all the stages of cellular differentiation can be found i.e., both newly formed and mature hard tissue. For this reason, the rat incisor is a convenient and suitable model for studies of amelogenesis.

In most earlier studies on the elemental composition pattern of dental enamel, analyses have been made in sites corresponding to one, or only a few, phases of ameloblast differentiation. Here, estimations of the incorporation of some critical elements into enamel are correlated with the differentiation stages of the ameloblasts through out the whole enamel.

Ten Sprague–Dawley rats were fed a standard diet (R3; EWOS AB Södertälje, Sweden) and given tap water (0.1 parts/10⁶ F) ad libitum. The animals were killed with an overdose of sodium pentobarbital intraperitoneally. The experiments were approved by the Ethical Committee for Animal Experiments at the Göteborg University.

In one group, the maxillary incisors were extracted after death. Each tooth sample was mounted on an aluminium holder and immersed in liquid N₂ in a lyophilizer vessel. The nitrogen was not in contact with the sample. The vessel was then evacuated, allowing the nitrogen to evaporate. Lyophilization was continued for 4 days, after which conventional embedding in benzoylperoxide-catalysed methylmethacrylate followed (see below). The sections were polished with aluminium powder and, after mounting with carbon and silver glue on sample holders, gold-coated by vacuum deposition.

In another group, whole crania were dissected out and split sagitally. The specimens were dehydrated in 70% ethanol for 24 h and in absolute ethanol for 24 h with several changes. After pre-embedding in methylmethacrylate/absolute ethanol (50/50) for 1 h, the samples were embedded in benzoylperoxide-catalysed methylmethacrylate. From each animal one maxillary incisor was cut sagitally, using a Leitz 1600 low-speed saw.

For orientation, a series of thickness measurements were made on dentine in conventionally prepared, haematoxylin/eosin-stained, light-microscopic sections, cut sagitally. As dentine grows continuously thicker in the incisal direction, its thickness was used to orientate the samples in the apico-incisal direction. The different ameloblast phases were also discriminated in the samples. The occurrence of Tomes’ processes determined the beginning of the secretory phase, and their gradual disappearance the transitional zone. The maturation phase started when the processes were no longer discernible. The secretory phase corresponds to line scan B, C and D in the graphs. Scan E corresponds to the transitional phase, and scan F, G and H the maturation phases.

The high-resolution scanning ion microscope used in this study is described in detail elsewhere. For this experiment, the instrument was set to use a liquid gallium ion source producing 40 keV, 30 pA primary ion probe, focused on a spot of about 50 nm dia. A high-transmission secondary-ion analyser and transport system in conjunction with a RF quadropole mass filter were used. The low-magnification images presented in this paper come from scans containing 1024×1024 probe settings and the detected signal was displayed as individual counts on a television screen. A series of secondary ion images of ²³Na, ²⁶CN, ³⁵Cl, ³⁹K was taken in the secretory-phase region of the incisors. A complementary series was taken in the maturation-phase region. When interpreting the images, light areas were referred to a high-signal areas and dark areas as low-signal areas. The contrast of the SIMS images was enhanced in a Teragon 4000 image-analysis computer.

The secondary ion mass spectrometer (Cameca IMS-3F, Paris, France) was used in the quantitative step-scan mode according to previously described procedures. The specimens were bombarded by an O^? primary ion beam, approx. 25 ?m dia., 0.1–0.5 ?A, accelerated through 15 kV. Sputtered positive secondary ions were extracted into two separation fields of an electrostatic and a magnetic sector. The mass-separated and energy-focused ion currents were recorded as mass spectra. The following mass peaks were studied: ¹²C, ¹⁹F, ²³Na, ³¹P, ³⁹K and ⁷⁷CaCl. All signals were normalized against the signal of ⁴⁴Ca, a naturally occurring calcium isotope, in order to compensate for intensity fluctuations due to possible irregularities on the sample surface.

The ²³Na images exhibited higher counts in secretory enamel matrix than in underlying dentine. In predentine, higher counts were also seen. In the maturation-phase enamel, considerably lower counts were found, with a slight rise towards the surface. In the ³⁵Cl images from the secretory enamel matrix, the counts were overall low, both in enamel and dentine. In the maturation-phase enamel, a higher signal was seen on the surface.

Fig. 1. (a) The ²³Na images exhibited higher counts for ²³Na in secretory enamel matrix (arrows S) than in underlying dentine (D); (b) in the maturation phase enamel (arrows M) considerably lower counts were found. Light areas=high signal; dark areas=low signal.

Fig. 2. (a) In the ³⁵Cl images the counts were low over the entire area sampled, both in secretory enamel matrix (arrows S) and in dentine; (b) in the maturation-phase enamel (arrows M) the counts had decreased somewhat.
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The ³⁹K signal from enamel matrix was similar to that from the underlying dentine. In mature enamel the highest ion yield was obtained from the surface.

Fig. 3. (a) ³⁹K exhibited high signals in secretory enamel matrix (arrows S), as in dentine; (b) in maturation-phase enamel (arrows M) the count was low over the entire area sampled.

In enamel matrix, the counts for ²⁶CN were high, as also in predentine. Conversely, in maturation-phase enamel the ²⁶CN signals were very low.