The structure and composition of deciduous enamel affected by local hypoplastic autosomal dominant amelogenesis imperfecta resulting from an ENAM mutation.

In a group of families in northern Sweden, a mutation in the ENAM gene (predicted to produce a highly truncated protein) results in the local hypoplastic form of autosomal dominant amelogenesis imperfecta. In this study, sections of deciduous teeth from members of 3 of these families were examined by scanning electron microscopy (SEM) and the enamel mineral was analysed by energy dispersive X-ray spectroscopy (EDX). The sections were also probed with antibodies raised to a conserved sequence of the enamelin protein. Selected intact teeth were first analysed by digital imaging and ascribed with an 'Enamel Defects Index' (EDI) score. SEM of tooth sections revealed disrupted prism morphology and the prisms had a glass-like appearance in some areas. These areas of dysplasia were sometimes irregular but formed regular arrays in others. Comparison of EDI scores with SEM indicated that in one tooth the surface had no measurable defects but significant defects were present in the underlying enamel microstructure. SEM immunohistochemistry with the antibody raised to a fragment of the enamelin protein produced positive, but light, labelling throughout normal enamel. In dysplastic areas, however, the labelling intensity appeared to be reduced. The results indicate that the presence of functional enamelin in the correct amounts is necessary for correct prism morphogenesis. In addition, a combination of EDI and structural analysis indicate that defects in enamel microstructure are not necessarily visible as defects on the surface of the tooth, suggesting the possibility, at least, that some instances of under-diagnosis may occur.

Self-assembling peptide scaffolds promote enamel remineralization.

Rationally designed beta-sheet-forming peptides that spontaneously form three-dimensional fibrillar scaffolds in response to specific environmental triggers may potentially be used in skeletal tissue engineering, including the treatment/prevention of dental caries, via bioactive surface groups. We hypothesized that infiltration of caries lesions with monomeric low-viscosity peptide solutions would be followed by in situ polymerization triggered by conditions of pH and ionic strength, providing a biomimetic scaffold capable of hydroxyapatite nucleation, promoting repair. Our aim was to determine the effect of an anionic peptide applied to caries-like lesions in human dental enamel under simulated intra-oral conditions of pH cycling. Peptide treatment significantly increased net mineral gain by the lesions, due to both increased remineralization and inhibition of demineralization over a five-day period. The assembled peptide was also capable of inducing hydroxyapatite nucleation de novo. The results suggest that self-assembling peptides may be useful in the modulation of mineral behavior during in situ dental tissue engineering.

Surface chemistry of enamel apatite during maturation in relation to pH: implications for protein removal and crystal growth.

Apatite crystal growth rate and morphology in mineralized tissues are considered to be controlled by surface interaction with extracellular matrix proteins. During enamel maturation where protein is finally removed from crystal surfaces to permit massive crystal growth, pH oscillates between approximately 5.8 and approximately 7.2. With this in mind, a study of enamel apatite surface chemistry in terms of local environmental pH was undertaken. Using atomic force microscopy adhesion force measurements were made between hydroxylated or carboxylated cantilever tips and maturation stage crystals between pH 2 and 10. Adhesion force increased from pH 10 to a maximum at pH 6.6 presumably due to increased hydrogen bonding due to replacement of surface cations (Na, Ca, Mg) with protons and/or protonation of phosphate per se. Below pH 6.6 adhesion force decreased and became very variable indicating that the surface had become unstable probably due to removal of fully protonated phosphate from the surface by adherence to the cantilever tip. Frictional force measurements also revealed 2-3, approximately 30 nm diameter high friction domains in bands across the crystal long axis. Their location mirrored the binding pattern of similarly sized amelogenin aggregates seen in vitro. The data suggests that specific protein binding sites may exist on crystal surfaces and may be released at lower pH by protonation which would lower cationic charge on both crystal surface and ionic charge on the protein. Instability of the crystal surface could also play a role.

Morphology and elemental composition of subgingival calculus in two ethnic groups.

BACKGROUND: The aim of the present study was to compare the morphology and elemental composition of subgingival calculus between Indo-Pakistani and Caucasian patient groups. METHODS: Extracted teeth from 14 Indo-Pakistani and 19 Caucasian subjects were collected. Of these, 12 Indo-Pakistani and 10 Caucasian teeth had sufficient subgingival calculus for analysis. Subgingival calculus present on the 22 teeth was classified into six morphological types: 1) crusty/spiny/nodular; 2) ledge/ ring; 3) thin, smooth veneers; 4) finger/fern-like; 5) individual islands/spots; or 6) supramarginal on submarginal. Subgingival calculus was zoned: coronal, mid, and apical. A sample obtained from each zone was subdivided to allow 3 separate analyses: transmission electron microscopic (TEM) x-ray microanalysis for elemental composition, fluoride analysis, and carbonate analysis. RESULTS: Crusty/spiny/nodular, ledge/ring, and thin, smooth veneers were more commonly found in the Indo-Pakistani group; individual islands were more prevalent in the Caucasian subjects. Supramarginal on submarginal calculus was found only in the Indo-Pakistani group. No finger/fern-like deposits were found. No differences within or between the two ethnic groups were found with regard to calcium:phosphate ratios, fluoride, or carbonate content. However, the Indo-Pakistani group showed significantly lower levels of sodium in apical samples than in coronal samples (ANOVA, F1,16 = 5.98, P= 0.03), and significantly lower levels of sodium (ANOVA, F1,12 = 4.75, P= 0.05) and magnesium (ANOVA, F1,12 = 5.16, P= 0.04) in apical samples than in those from Caucasians. After adjusting for smoking, the magnesium results remained significant (ANOVA, F2,11 = 4.64, P= 0.05). CONCLUSIONS: Subgingival calculus demonstrated differences in morphology between these two ethnic groups and differences in elemental composition, which may influence its overall solubility and contribute to its greater accretion in the Indo-Pakistani subjects.

A three-dimensional histological method for direct determination of the number of trabecular termini in cancellous bone.

Osteoporotic fractures occur frequently in aging populations. Established methods for analyzing microarchitecture indicate that cancellous bone loss in the elderly is associated with progressive reduction in the connectivity of the trabecular network. This disconnection may explain the increased skeletal fragility that is sometimes out of proportion to the amount of bone lost. Connectivity, however, is difficult to measure and usually requires indirect methods. We describe development of a simple, inexpensive and direct procedure for counting sites of trabecular disconnection. The method is based upon preparation of 300-500 microm thick slices of methylmethacrylate embedded material rather than the more usual thin 8 microm histological sections. The marrow tissue is retained within the thick slice; this is essential for conservation of any detached bone fragments. In such preparations differential superficial staining of the upper and lower surfaces with alizarin red and light green, respectively, allows the two-dimensional image to be viewed at the same time as its three-dimensional counterpart. In this way, "real" (i. e., unstained) trabecular termini can be distinguished from "apparent" (i. e., stained red or green) termini that are artifacts of the plane of section. Partly polarized light enhances the microscope image. The method does not destroy the material for subsequent bone histomorphometry and, therefore, may be a useful adjunct to iliac bone biopsy analysis in studies of metabolic bone disease.

Trabecular architecture in women and men of similar bone mass with and without vertebral fracture: II. Three-dimensional histology.

We recently developed a simple and inexpensive method that complements established bone histomorphometry procedures by enabling the two-dimensional imaging of cancellous bone to be viewed within its three-dimensional context with the marrow tissue in place and without detriment to the material for other histological purposes. The method, based on the preparation and superficial staining of slices 300 microm thick, enables "real" (i.e., unstained) trabecular termini to be separated from "artifactual" (i.e., stained) termini, providing a direct measure of cancellous connectivity in osteopenic bone. The technique was applied to osteopenic age-matched, white, postmenopausal women (31 with and 22 without vertebral compression fractures) with a similar bone status, as measured at the spine by absorptiometry and at the iliac crest by histology (see part I of this study). Despite the similarity in the mass of trabecular bone at either site, the results showed a significant difference (p < 0. 05) in the number of "real" trabecular termini between the groups, such that the fracture group had almost four times as many termini (mean +/- SE: 1.98 +/- 0.51/30 mm(2)) at the iliac crest as the nonfracture group (mean +/- SE: 0.53 +/- 0.31/30 mm(2)). Previous histomorphometry of the same material failed to detect a structural distinction between the two groups using established variables. It was concluded that a mass-independent trabecular discontinuity contributes to skeletal failure and that determination of the number of "real" disconnections (i.e., unstained termini) by the direct method proposed may provide a more sensitive discriminant of fracture than the present indirect procedures. A group of fracture and nonfracture men (see part I) suggested a similar distinction (fracture: 0.69 +/- 0.30/30 mm(2); nonfracture: 0.18 +/- 0.18/30 mm(2)), although the difference was not significant.

The developing enamel matrix: nature and function.

The hydroxyapatite crystals of mature enamel are unusually large, uniform and regularly disposed within the tissue, implying that their development is a highly controlled process. The organic matrix of developing enamel is presumed to play an important role in the modulation of mineral deposition and growth during tooth morphogenesis but the precise functions of individual matrix proteins remain unclear. The aim of this review was to survey the current knowledge of enamel matrix proteins with a view to suggesting possible functions. The organic matrix is highly heterogeneous, comprising proteins derived from a number of different genes, including amelogenin, enamelin, ameloblastin (amelin/sheathlin), tuftelin, dentine sialophosphoprotein, enzymes and serum proteins such as albumin. Each of these classes appears to undergo post-secretory sequential degradation which contributes further towards matrix heterogeneity. Possible functions of these proteins include de novo mineral nucleation/initiation (dentine sialophosphoprotein, tuftelin), mineral ion binding as crystal precursors (amelogenin, enamelin), control of crystal growth (amelogenin, enamelin, ameloblastin), support of growing crystals (amelogenin, enamelin), determination of prismatic structure (ameloblastin), cell signalling (tuftelin, ameloblastin), control of secretion (breakdown products) and protection of the mineral phase (amelogenin, enamelin). Failure of these mechanisms could lead to incomplete maturation of the enamel and the eruption of dysplastic tissue.

Enamel maturation.

Enamel maturation is characterized by massive crystal growth in both width and thickness, resulting in the most highly mineralized of all mammalian skeletal tissues. The control of this process is mediated via a carefully orchestrated series of events that are temporally and spatially regulated, and it requires the co-ordinated degradation and removal of the endogenous enamel matrix. This is affected by both neutral metalloproteases and serine proteases, which are developmentally restricted and may be further modulated by changes in the chemistry of the enamel crystals themselves. Failure of these mechanisms, or the adventitious entry of mineral-binding proteins during the later stages of maturation, may result in the incomplete maturation of the enamel crystals and the eruption of dysplastic tissue.

The effect of glycosylaminoglycans on the mineralization of sheep periodontal ligament in vitro.

The effect of removal of glycosylaminoglycans on the mineralization of sheep periodontal ligament was determined using enzyme digests followed by incubation in solutions supersaturated with respect to hydroxyapatite at pH 7.4. TEM revealed that control periodontal ligament remained unmineralized. However, tissue from which glycosylaminoglycans had been removed contained plate-like crystals arranged parallel to and within the collagen fibrils. Electron probe and electron diffraction studies suggested that the crystals were apatitic with a similar order of crystallinity to dentine, and a Ca:P ratio of 1.61. In addition, the glycosylaminoglycan content of periodontal ligament, cementum and alveolar bone was compared using cellulose acetate electrophoresis. Periodontal ligament contained predominantly dermatan sulfate while cementum and alveolar bone contained mostly chondroitin sulfate. A role for glycosylaminoglycans in maintaining the unmineralized state of the periodontal ligament is suggested. Control of expression of specific proteoglycan species on a spatially restricted basis is presumably central to this role.

Isolation and characterisation of an alternatively-spliced rat amelogenin cDNA: LRAP--a highly conserved, functional alternatively-spliced amelogenin?

A cDNA coding for a 59 amino acid polypeptide containing both the carboxy- and amino-termini, but lacking the central domain, of the rat tooth enamel matrix protein, amelogenin, was cloned and sequenced. The deduced polypeptide sequence indicates that this cDNA was derived from an amelogenin RNA molecule by using an alternative intra-exonic 3' splice acceptor site. This alternatively spliced product is almost identical to products previously identified in both cow and mouse enamel organs: the leucine-rich amelogenin peptide (LRAP). The conservation of this truncated polypeptide across the species suggests that it may have an important role in the formation of tooth enamel.

Immunohistochemical investigation of epidermal growth factor receptor expression during periods of accelerated rat incisor eruption.

The effect of accelerated odontogenesis of the rat mandibular incisor on the expression of receptors for epidermal growth factor (EGF) was examined using specific monoclonal antibodies to the receptor molecule. Acceleration of odontogenesis was achieved by regular trimming of the tooth crown. At normal eruption rates the major area of cross-reactivity was over the secretory ameloblasts. Some labelling of the papilla and preameloblasts was evident. When proliferation was increased the major area of effect was at the preodontoblast/odontoblast boundary where there was a marked increase in labelling, initially at the proximal end of the cell adjacent to the basal lamina. The ameloblasts did not show such a dramatic increase in receptor numbers. Increase in labelling was also evident in the remainder of the papilla. The results suggest that an increase in proliferation with normal morphogenesis is associated with an overall increase in the numbers of EGF receptors, particularly in a population of cells immediately before and after elongation and differentiation of odontoblasts.