The 32kDa enamelin undergoes conformational transitions upon calcium binding

The 32 kDa hydrophilic and acidic enamelin, the most stable cleavage fragment of the enamel specific glycoprotein, is believed to play vital roles in controlling crystal nucleation or growth during enamel biomineralization. Circular dichroism and Fourier transform infrared spectra demonstrate that the secondary structure of the 32 kDa enamelin has a high content of α-helix (81.5%). Quantitative analysis on the circular dichroism data revealed that the 32 kDa enamelin undergoes conformational changes with a structural preference to β-sheet with increasing concentration of calcium ions. We suggest that the increase of β-sheet conformation in the presence of Ca²⁺ may allow preferable interaction of the 32 kDa enamelin with apatite crystal surfaces during enamel biomineralization. The calcium association constant (K_a = 1.55 (±0.13) × 10³ M⁻¹) of the 32 kDa enamelin calculated from the fitting curve of ellipticity at 222 nm indicated a relatively low affinity. Our current biophysical studies on the 32 kDa enamelin structure provide novel insights towards understanding the enamelin–mineral interaction and subsequently the functions of enamelin during enamel formation.     

Reprint of "Stereology meets electron tomography: towards quantitative 3D electron microscopy" [J. Struct. Biol. 159 (2007) 443-450

Self-assembly of the extracellular matrix protein amelogenin is believed to play an essential role in regulating the growth and organization of enamel crystals during enamel formation. The full-length amelogenin uniquely regulates the growth, shape, and arrangement of enamel crystals. Protein hydrolysis will ultimately facilitate a tissue with high mineral content. Protein processing is however highly specific suggesting a functional role of the cleaved amelogenins in enamel maturation. Here we hypothesize that the cooperative self-assembly of the recombinant full-length amelogenin 25 kDa and the 23 kDa proteolytic cleavage product is a function of pH, mixing ratio and incubation time and is associated with the isoelectric point of the protein. Self-assembly of amelogenin into nanospheres which increased in size with increasing pH was observed by atomic force microscopy. Elongated structures of about 100 nm length and 25 nm width formed over several days for amelogenin 25 and 23 kDa predominantly at pH-values of 6.5 and 7.5, respectively. When both proteins 25 and 23 kDa were mixed, self-assembled nanostrings of 200–300 nm length consisting of fused nanospheres were obtained at pH around 7.0 within 24 h. The protein nanostrings formed links over time and a continuous mesh was obtained after 7 days. Electrical conductivity data also showed gradual changes when both amelogenins were mixed in solutions supporting the idea that elongated structures form over extended periods of time. We propose that due to the difference in the isoelectric point, self-assembled nanospheres composed of 23 or 25 kDa amelogenin have opposite ionic charges at pH-values around 7.0 and thus experience ionic attraction that enables cooperative self-assembly.     

Variation in modern human enamel formation times

Most of what we know about the timing of human enamel formation comes from radiographic studies on children of known age. Here, we present new longitudinal data derived from a histological analysis of tooth enamel. Two samples, one from southern Africa and one from northern Europe, contained all anterior and molar tooth types. Two further samples contained only one tooth type: canines from a medieval Danish sample and third molars from a modern North American sample. Data were collected on 326 molars and 352 anterior teeth. Each tooth was sectioned and prepared for polarized light microscopy. We used daily enamel cross striations to determine cuspal enamel formation time, recorded the periodicity of long-period striae in the lateral enamel, and used this value to calculate enamel formation times for each decile of crown length. We present data that reveal some of the processes whereby differences in enamel formation times arise between our samples. Mean cuspal enamel formation times were similar in southern African and northern European anterior teeth, but differed in certain molar cusps. All the southern African anterior teeth completed enamel formation earlier. The greatest difference in mean chronological age at enamel completion was 5.2 vs. 6.2 years of age in lower canines. However, enamel completion times in the molar teeth showed few differences between the samples, with mean times for the longest forming cusps all falling between 3.0 years and 3.45 years. Our data suggest fewer differences between samples and smaller ranges of variation than in many radiographic studies and present a more realistic picture of worldwide variation in enamel formation times.     

Matrix metalloproteinase inhibition impairs the processing, formation and mineralization of dental tissues during mouse molar development

Organotypic cultures of embryonic mouse tooth germs were used to investigate the developmental expression and roles of MMPs in the formation and mineralization of dentin and enamel matrices. The spatially and temporally regulated expression of MMP-2, MMP-9 and MMP-20 in developing mouse tooth germs in vivo was maintained in culture. The inhibition of metalloproteinases activity by marimastat altered the morphogenesis and mineralization of the tooth germs associated with an inhibition of the activation of both MMP-20 and MMP-2. MMP inhibition deregulated the molecular processing of two major dental matrix proteins, amelogenin and dentin sialoprotein (DSP). This coincided with their accumulation and the loss of their normal distribution within the extracellular matrix, resulting in a defective mineralization of dentin and enamel matrices. These findings demonstrate the critical role of MMPs in the processing and maturation of the dental matrix.     

Enamel hypoplasia in the Middle Pleistocene hominids from Atapuerca (Spain)

The prevalence and chronology of enamel hypoplasias were studied in a hominid dental sample from the Sima de los Huesos (SH) Middle Pleistocene site at the Sierra de Atapuerca (Burgos, northern Spain). A total of 89 permanent maxillary teeth, 143 permanent mandibular teeth, and one deciduous lower canine, belonging to a minimum of 29 individuals, were examined. Excluding the antimeres (16 maxillary and 37 mandibular cases) from the sample, the prevalence of hypoplasias in the permanent dentition is 12.8% (23/179), whereas the deciduous tooth also showed an enamel defect. No statistically significant differences were found between both arcades and between the anterior and postcanine teeth for the prevalence of hypoplasias. In both the maxilla and the mandible the highest frequency of enamel hypoplasias was recorded in the canines. Only one tooth (a permanent upper canine) showed two different enamel defects, and most of the hypoplasias were expressed as faint linear horizontal defects. Taking into account the limitations that the incompleteness of virtually all permanent dentitions imposes, we have estimated that the frequency by individual in the SH hominid sample was not greater than 40%. Most of the hypoplasias occurred between birth and 7 years (N = 18, X = 3.5, SD = 1.3). Both the prevalence and severity of the hypoplasias of the SH hominid sample are significantly less than those of a large Neandertal sample. Furthermore, prehistoric hunter-gatherers and historic agricultural and industrial populations exhibit a prevalence of hypoplasias generally higher than that of the SH hominids. Implications for the survival strategies and life quality of the SH hominids are also discussed. © 1995 Wiley-Liss, Inc.     

True enamel matrix of the newt,Triturus pyrrhogaster,contains no sulfated glycoconjugates

Summary The ultrastructural distibution and histochemical properties of sulfated glycoconjugates were investigated in the developing enamel of the adult newt, Triturus pyrrhogaster, by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. Development and ultrastructure of the enamel were also studied. After deposition of the mantle dentin matrix to a certain thickness, the first enamel matrix, globular in shape, appeared in juxtaposition to the dental basement membrane and tended to be intermixed with the previously deposited dentin matrix. Subsequently, enamel matrix was deposited outside (ameloblastic side) of the dental basal lamina and formed a true enamel layer. Thus, developing enamel of the newt consists of two layers: (1) an inner layer made up of a dentin-enamel mixed matrix and (2) an outer layer composed of only true enamel matrix. HID-TCH-SP precipitates resulting from the abovementioned studies were found in the mixed matrix and were identified as chondroitin sulfates; in contrast, the true enamel matrix contained no sulfated glycoconjugates.     

Proliferative and functional stages of rat ameloblast differentiation as revealed by combined immunocytochemistry against enamel matrix proteins and bromodeoxyuridine

Summary A double-staining immunocytochemical technique was used for the simultaneous detection, at the light- and electron-microscopical level, of proliferating bromodeoxyuridine (BrdU)-labelled cells and enamel protein (EP)-producing cells in the inner enamel epithelium (IEE) of rat tooth germ. BrdU-positive cells were found in the region of the IEE close to the cervical loop and never displayed EP-like immunoreactivity. BrdU-immunoreactivity was confined to the nucleus of replicating cells. In contrast, epithelial cells displaying EP-like immunoreactivity were found in the region of the forming dental cusp and were consistently BrdU-negative. EP-like immunoreactivity was detectable in the cytoplasmic compartments involved in the exocrine secretion pathway and in the extra-cellular matrix close to EP-immunoreactive cells. These data support the view that withdrawal from the cell cycle in the IEE is a temporal prerequisite for acquiring the functional competence of secreting EP. Moreover, cycling cells and secretory cells in the IEE constitute two separate compartments that are spatially defined, and that exhibit clear-cut staining patterns with respect to BrdU- and EP-immunoreactivity, respectively. We thus propose that BrdU-incorporation and EP-production may be used as specific markers of the differentiation of the IIE cells in studies of the possible role of growth factors, their receptors and oncoproteins in this tissue.     

Enamel structure and microwear: an experimental study of the response of enamel to shearing force.

The anisotropic fracturing and differential wear properties of enamel microstructure represent factors that can obscure the predictive relationship between dental microwear and diet. To assess the impact of enamel structure on microwear, this in vitro experimental study examines the relative contributions to wear of three factors: 1) species differences in microstructure, 2) direction of shearing force relative to enamel prisms and crystallites, and 3) size of abrasive particles. Teeth of Lemur, Ovis, Homo, and Crocodylus, representing, respectively, the structural categories of prismatic patterns 1, 2, and 3 and nonprismatic enamel, were abraded by shearing forces (forces having a component directed parallel to abraded surfaces) and examined by scanning electron microscopy. Striation width increased with particle size for nonprismatic, but not for prismatic, specimens. Direction of shear relative to prism and crystallite orientation had a significant influence on striation width in only some prismatic enamels. The different responses of prismatic and nonprismatic enamels to abrasion reflect the influence of structure, but at the level of organization of crystallites rather than prisms per se. Such interactions explain in part the inability of striation width to discriminate among animals with different dietary habits. Heteroscedasticity and deviations from normality also may confound parametric analyses of microwear variables. Variation in crystallite orientation in prismatic enamels may contribute to optimal dental function through the property of differential wear in functionally distinct regions of teeth.