Traumatic injuries to enamel formation in rat incisors

abstract – Twenty-three fullgrown white male rats were fitted with an inclined plane on the mandibular incisors with consequent raising of the bite by 2.5 mm. After an observation period of between 1 and 30 d the animals were killed. In all cases traumatic injuries to the enamel organs were seen. The defects were mainly of four different types. Type 1 occurred in the early maturation zone of the enamel as a local defect with dedifferentiated ameloblasts. Vascular injury was moderate and deformation of the dentin was seen. After 30 d there was still a local defect of the enamel organ. Type 2 occurred apically to the maturation zone and caused a two-layered ameloblastoma resembling a sandwich. Hemorrhages were common in the surrounding connective tissue, with accompanying appearance of phospholipids in the tissue. After 30 d some restitution of the enamel formation was seen. Type 3 was characterized by fractures of the dentin associated with total ameloblastic injury. Vascular necrosis was seen in the surrounding tissue. After 30 d a new ameloblastoma had developed in some areas. These three types appeared separately or combined. Type 4 occurred as a folding of newly formed apical dentin in areas without differentiated ameloblastoma and was seen in all of the experimental animals.     

Parathyroid hormone and enamel formation in rat maxillary incisors

Abstract – Parathyroid hormone (PTH) plays an important role in regulating calcium in serum. It is also known to affect bone and dentin formation. The purpose of this investigation was to evaluate enamel formation in normal rats receiving added PTH. It is in two parts: a pilot study where a known method was tested, followed by the main study where the rats were given different doses of PTH. The enamel was examined in both studies and in the main study the ameloblasts were also investigated. Contradictory results were seen. In the pilot study, severe enamel aberrations occurred, while no divergence from normal amelogenesis was noted in the main study. A factor causing the disparate results was the use of a hard tissue marker (oxytetracycline) in the pilot study. It can be concluded that injections of PTH in doses that affect bone and dentin did not cause any changes in normal enamel formation.     

Parathyroid hormone and enamel formation in rat maxillary incisors

Parathyroid hormone (PTH) plays an important role in regulating calcium in serum. It is also known to affect bone and dentin formation. The purpose of this investigation was to evaluate enamel formation in normal rats receiving added PTH. It is in two parts: a pilot study where a known method was tested, followed by the main study where the rats were given different doses of PTH. The enamel was examined in both studies and in the main study the ameloblasts were also investigated. Contradictory results were seen. In the pilot study, severe enamel aberrations occurred, while no divergence from normal amelogenesis was noted in the main study. A factor causing the disparate results was the use of a hard tissue marker (oxytetracycline) in the pilot study. It can be concluded that injections of PTH in doses that affect bone and dentin did not cause any changes in normal enamel formation.     

Spatially related amelogenin interactions in developing rat enamel as revealed by molecular cross-linking studies

A cleavable cross-linker (dithiobis[succinimidyl propionate], DTSP) was used to investigate the subunit structure of the developing enamel matrix. Intact matrix was cross-linked under conditions chosen to simulate those found in vivo. The cross-linked complexes were isolated by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their subunit composition determined by analytical SDS-PAGE following reductive cleavage of the cross-links. Western blotting using antiamelogenin antibodies was used to confirm the identity of the proteins involved. The results showed that nascent amelogenins tended to be cross-linked to other nascent amelogenins while amelogenin-processing products tended to be cross-linked to other processed molecules at the same stage of processing. The results suggest that nascent amelogenins are in close association after secretion and during extracellular processing, and that processed products are not free to associate with nascent molecules, presumably due to diffusion constraints in the tissue. This conclusion implies that individual amelogenin molecules within supramolecular aggregates (nanospheres) are processed in situ and remain in the same nanosphere while all the individual component amelogenins undergo processing. The biological function of amelogenin processing remains unclear but the fact that amelogenin-amelogenin associations are maintained during processing indicates that matrix stability is an important factor while the enamel layer is being deposited.     

Maintenance of amelogenin gene expression by transformed epithelial cells of mouse enamel organ.

Electroporation was used to introduce foreign genes into cells derived from the mouse enamel organ epithelia (EOE). Optimal conditions for this electroporation were established. The introduction of a plasmid construct bearing the coding region for the large T-antigen from polyoma virus into EOE cells permitted the establishment of a derivative cell line that has the following characteristics: (1) the cells could be passaged many times; (2) they expressed a keratin-containing cytoskeleton; and (3) approx. 60% of the cells expressed amelogenin, a tissue-specific gene product unique to ameloblasts. Potential uses for such a cell line include analysis of: (1) the upstream regulatory regions required for temporally and spatially restricted expression of amelogenin; (2) the post-translational modification of amelogenin in synchronized cells and (3) the organization and biomineralization of enamel extracellular matrix in monolayer culture.     

The dissolution rate of enamel in acid in developing rat incisors

This study was undertaken to determine the changes in this dissolution rate at different developmental stages after different fluoride dietary regimes. Four groups of Wistar rats received water with 0, 25, 50 and 100 parts/10(6) fluoride respectively for 10 weeks. Six exposed windows of 2 mm2 were prepared on the enamel surface of the upper incisors, corresponding to six different developmental stages. The acid-dissolution rates were determined at each window by using 1.4 M sodium acetate-hydrochloric acid buffer (pH 2.3). The rate of enamel dissolution was highest in the matrix-formation stage and dropped sharply in a step-wise fashion towards the stages of secondary mineralization and iron deposition. The dissolution rate in the maturation stage decreased significantly with increasing intake of fluoride. However, in the pigmented enamel, the opposite occurred. The iron pigmentation or the porosity in this region of fluorosed enamel might be responsible for the change in the dissolution rate of the pigmented enamel.     

Ultrastructural study of the proteoglycans in enamel from rat incisors during late enamel maturation.

The distribution pattern of proteoglycans in the enamel matrix stained by Alcian Blue or by bismuth nitrate was similar to that of its protein components, suggesting that the two are closely associated. Only the most superficial aprismatic zone of enamel was low in these components, whereas they were abundant in prism sheaths. The lack of correlation between the distribution of fibrous structures and that of both crystals and inter-crystalline spaces could be explained by the possibility that, in enamel, proteoglycans exist as amorphous structures.     

Effect of hypocalcemic state on enamel formation in rat maxillary incisors

Several authors have proposed that hypocalcemia can interfere with amelogenesis, resulting in enamel aberrations. Therefore, the purpose of this investigation was to study the effects of a diet-induced hypocalcemic state in young rats on enamel formation of the maxillary incisors. The experimental rats were fed a special diet, free from vitamin D and very low in calcium. The control rats were fed a normal diet. The experimental period was 3 wk. After termination, the blood analysis showed that the experimental rats had developed hypocalcemia with very low values of both total and ionized blood calcium. The experimental rats were smaller than their controls after 3 wk, with smaller skulls and teeth. At the light microscope level, the enamel and the ameloblasts did not seem to be affected, except in one rat, the smallest, which showed enamel hypoplasias in both maxillary incisors and a delayed increase of the mineral content during the maturation stage process. It is concluded that the hypocalcemic state induced did not greatly affect enamel formation. However, occasional enamel aberrations may occur.     

Distribution of fluoride in the enamel of rat incisors examined by an abrasive microsampling technique

This study was undertaken to reveal detailed changes in fluoride distribution at different developmental stages of upper incisor enamel under various fluoride administration regimes. Four groups of Wistar rats received water containing 0, 25, 50 and 100 parts/10(6) fluoride respectively for 10 weeks. Five different enamel specimens were removed from the developing enamel, excluding the matrix-formation stage. Fluoride distribution in each specimen was analysed from the surface to the enamel-dentine junction using an abrasive microsampling technique. Fluoride concentration was invariably highest at the surface and decreased sharply towards the interior at every site in both control and experimental groups. The concentration throughout the tissue increased with fluoride intake at each stage of development. The fluoride-gradient curves were similar at each of the different sites of tooth development. However, the fluoride concentration of the enamel interior was significantly higher at early maturation than at the other four sites.     

Micelle structure of amelogenin in porcine secretory enamel

Even during the secretory stage of amelogenesis, enamel crystals thicken as amelogenins (the major protein component) decrease. To explain this phenomenon, we propose a model for amelogenin structure and function based upon the hypothesis that amelogenin forms micelles. Solubility and hydrophobicity analyses suggest that all but the hydrophilic amelogenin C-terminal regions aggregate via hydrophobic bonds to form a micelle core. Amelogenin micelles may form super-assemblies via their C-termini (KTKREEVD), which contain complementary positive (KTKR) and negative (EEVD) elements. Disassembly of the micelles through controlled proteolysis provides space for crystal growth. Initial cleavage (by enamelysin) removes the surface-accessible amelogenin C-terminus, exposing the middle portion to cleavage (by EMSP1). As a result, the 13-kDa amelogenin, a rod-shaped domain based upon ultrafiltration and transmission electron microscopy studies, is released. This model explains how amelogenin is able to 'space' and support the ribbon-like crystals and continuously yield space as the crystals thicken, until they are sufficiently mature to support themselves.     

The spectrochemical analysis of metals in rat molar enamel,femurs and incisors

A quantitative a.c. are salt cap method using 20 mg of molar enamel has been devised for the determination of cobalt, copper, iron, manganese, nickel and zinc in the range of 1–300 p.p.m. Rat molar enamel in powder form is dissolved in HCl; the resulting solution is dried on graphite electrodes and introduced into an a.c. are discharge. Femurs and incisors are oxidized with nitric acid and treated similarly. Use of an Ilford Q2 ultra-violet sensitive plate has demonstrated greatly improved sensitivity for zinc in this matrix to 0.5 p.p.m. using the 2138.56 Å line. Average precision is within 10 per cent of the amount present. Analytical results are indicated and correlation of metal uptake in enamel with metal-rich diet demonstrated.     

Ultrastructural examination of dentine formation in rat incisors following multiple fluoride injections

Rats were divided into two experimental and one control groups. In the first experimental group, two doses were injected each day at 6 hr intervals. The high dosages of sodium fluoride were administered daily for 5 days. These fluorotic animals were killed as the fluoride was exerting an effect on mineralization. In the other experimental group, the rats were allowed a recovery period by killing the rats 3 days after the last fluoride injections. Control animals received equivalent dosages of sodium chloride and were killed on the 5th day.Each day's injections induced a consistent paired response in the dentine in both experimental groups—a zone of hypermineralization followed by a zone of hypomineralization. The hypermineralized component was consistently visible only on microradiographs as bands of increased density, while the hypomineralized component was evident only on electron micrographs. The fine structure of the hypomineralized zone consisted of patchy areas with the appearance of predentine devoid of crystals and interspersed with calcospherites of mineralized dentine. These globules acquired more mineral with time to fill most areas, leaving unmineralized patches adjacent to odontoblast processes. In the recovery animals, a few small regions of unmineralization persisted and a layer of normal dentine formed over the fluoride response zones.Neither fluorotic nor recovery odontoblasts manifested visible ultrastructural alterations.     

Calcium movement in vivo and in vitro in secretory-stage enamel of rat incisors.

The lower incisors of young rats were dissected, immersed in physiological saline containing 45Ca under various conditions, and processed for autoradiography. The data were compared with those from in vivo 45Ca autoradiography. In secretory-stage enamel, wiped free of the enamel organ and immediately immersed in radioactive saline, there was intense labelling in the surface layers. The labelled area expanded only gradually into the deeper layers at a rate similar to that observed in vivo. Labelling in the enamel was similar in pattern but much weaker in intensity when the incisor was identically treated in vitro with the enamel organ attached. Glutaraldehyde pretreatment of the exposed enamel abolished expansion of the labelled area, whereas a hypochlorite pretreatment allowed a rapid diffusion of the isotope into the deeper layers of the secretory-stage enamel. The findings confirm the role of the enamel organ as a diffusion barrier to the penetration of calcium from the extracellular fluid to the secretory-stage enamel, and suggest an intimate correlation between physicochemical properties of the organic enamel matrix and the rate of surface-to-interior diffusion of calcium within the secretory-stage enamel of rat incisors.     

Demonstration by staining and radioautography of cyclical distributions of protein at the enamel surface in rat incisors

Staining patterns in the enamel during the maturation stage of amelogenesis reflect the banded distribution of ruffle-ended and smooth-ended ameloblasts. This study investigated the possibility that proteins at the enamel surface may be distributed cyclically according to cyclical changes in ameloblast morphology. Dissected lower rat incisors were wiped free of their enamel organs and immediately immersed in fixative containing one of the following heavy metal and histological stains: uranyl acetate, lead citrate, Coomassie blue, alcian blue and ruthenium red. Other animals were injected with [35S]methionine to label newly-formed enamel proteins. Their incisors were dissected, the enamel organs were wiped from the enamel surface, and the teeth were processed as whole mounts for radioautography. Teeth stained by heavy metals were also viewed by back-scattered electron imaging. The in-situ staining revealed that proteins were distributed in bands and stripes across maturing enamel. Radioautography revealed that the proteins in the stripes were newly-synthesized and secreted into the enamel by certain maturation ameloblasts. We conclude that the enamel organ expresses cyclical activity in part through secretion of proteins.     

Effect of prolonged iron deficiency on enamel pigmentation and tooth structure in rat incisors

The effect of iron deficiency maintained for over 6 months on the pigmentation and structure of incisors was investigated in female white rats. Gross loss of pigmentation was observed. Sections revealed enamel hypoplasia and aplasia. Dentinogenesis was much less affected, but there were areas of altered dentine with abnormal tubules and increased thickness of predentine with cellular and vascular inclusions.