THE STRUCTURE AND ORGANIZATION OF, AND THE RELATIONSHIP BETWEEN THE ORGANIC MATRIX AND THE INORGANIC CRYSTALS OF EMBRYONIC BOVINE ENAMEL

Electron microscope and electron diffraction studies of developing embryonic bovine enamel have revealed the organization of the organic matrix and the inorganic crystals. The most recently deposited inorganic crystals located at the ameloblast-enamel junction are thin plates, approximately 1300 A long, 400 A wide, and 19 A thick. During maturation of the enamel, crystal growth occurs primarily by an increase in crystal thickness. Statistical analyses failed to show a significant change in either the width or the length of the crystals during the period of maturation studied. Even in the earliest stages of calcification, the crystals are organized within the prisms so that their long axes (c-axes) are oriented parallel to the long axes of the prisms but randomly distributed about their long axes. With maturation of the enamel, the crystals become more densely packed and more highly oriented within the prisms. The organic matrix in decalcified sections of enamel is strikingly similar in its over-all organization to that of the fully mineralized tissue. When viewed in longitudinal prism profiles, the intraprismatic organic matrix is composed of relatively thin dense lines, approximately 48 A wide, which are relatively parallel to each other and have their fiber axes parallel to the long axes of the prisms within which they are located. Many of these dense lines, which have the appearance of thin filaments, are organized into doublets, the individual 48 A wide filaments of the doublets being separated by approximately 120 A. When observed in oblique prism profiles, the intraprismatic organic matrix is likewise remarkably similar in general orientation and organization to that of the fully mineralized tissue. Moreover, the spaces between adjacent doublets or between single filaments have the appearance of compartments. These compartments, more clearly visualized in cross- or near cross-sectional prism profiles, are oval or near oval in shape. Therefore, the appearance of the intraprismatic organic matrix (in longitudinal, oblique, and cross-sectional prism profiles) indicates that it is organized into tubular sheaths which are oriented with their long axes parallel to the long axes of the prisms in which they are located, but randomly oriented about their own long axes, an orientation again remarkably "blue printing" that of the inorganic crystals. The predominant feature of the walls of the tubular sheaths, when viewed in cross- or near cross-section, is that of continuous sheets, although in many cases closely packed dot-like structures of approximately 48 A were also observed, suggesting that the wall of the sheaths consists of a series of closely packed filaments. The 48 A wide dense lines (filaments) representing the width of the sheath wall were resolved into two dense strands when viewed in longitudinal prism profiles. Each strand was 12 A wide and was separated by a less electron-dense space 17 A wide. The intraprismatic organic matrix is surrounded by a prism sheath which corresponds in mineralized sections to the electron-lucent uncalcified regions separating adjacent prisms. Structurally, the prism sheaths appear to consist of filaments arranged in basket-weave fashion.     

THE ULTRASTRUCTURE OF THE ENAMEL IN THE GIANT PANDA OF PLEISTOCENE

<正> Enamel ultrastructures in the molar teeth of the giant panda, including Ailuropoda microta of the Early Pleistocene, Ailuropoda melanoleuca bacont of the Middle and Late Pleistocene and a living form, Ailuropoda rnelanoleuca, have been investigated by scanning electron microscopy. Transverse and longitudinal sections of enamel were made in order co evaluate shape, size and arrangement of the prisms. The sections were etched then with 0.074 M H_3PO_4 for 30-60 sec. Our investigations have shown certain features of the enamel which allow us to recognize differences among Ailuropoda on the basis of examination of large areas of the enamel. The results are summarized below.     

On the nature of the opaque and translucent enamel regions of some macropodinae (Macropus giganteus,Wallabia bicolor and Peradorcas concinna)

Summary Teeth of three macropod species, M. giganteus, W. bicolor and P. concinna, have been studied using the techniques of light microscopy, scanning- and transmission-electron microscopy and hardness measurement. Light microscope observations showed that the teeth of these species had a translucent enamel region close to the dentine and an outer opaque enamel region at the tooth's surface. These regions were not related to the presence or absence of tubules which are a characteristic feature of marsupial enamel. Hardness tests showed that the opaque enamel was softer than the translucent enamel. Scanning electron microscope observations revealed that there was no correlation between any particular prism packing or orientation and the opaque and translucent enamel regions. Transmission electron microscope observations showed that the translucent enamel region consisted of well defined prisms and well packed, lath-like crystals, whereas the opaque enamel was disrupted by voids (which ranged in size from enlarged micropores to about 2 m in diameter in extreme cases) between crystals and some randomly oriented, loosely packed crystals. This disruption within the opaque enamel region was more common at prism boundaries but pockets of disrupted enamel were also found within prisms and interprismatic regions. The opacity of the enamel was caused by scattering of light from the voids. The ultrastructure of the opaque enamel region indicated that this region was hypomineralized; hardness tests and polarized light microscope observations were consistent with these results.     

Effect of prism orientation and loading direction on contact stresses in prismatic enamel of primates: implications for interpreting wear patterns

The ability of prisms to effectively dissipate contact stress at the surface will influence wear rates in teeth. The aim of this investigation was to begin to quantify the effect of prism orientation on surface stresses. Seven finite element models of enamel microstructure were created, each model differing in the angulation of prism orientation with regard to the wear surface. For validation purposes, the mechanical behavior of the model was compared with published experimental data. In order to test the enamel under lateral loads, a compressed food particle was dragged across the surface from the dentino-enamel junction (DEJ) towards the outer enamel surface (OES). Under these conditions, tensile stresses in the enamel model increased with increases in the coefficient of friction. More importantly, stresses were found to be lowest in models in which the prisms approach the surface at lower angles (i.e., more obliquely cut prisms), and highest when the prisms approached the surface at 60 degrees (i.e., less obliquely cut). Finally, the direction of travel of the simulated food particle was reversed, allowing comparison of the difference in behavior between trailing and leading edge enamels (i.e., when the food particle was dragged either towards or away from the DEJ). Stresses at the trailing edge were usually lower than stresses at the leading edge. Taken together with what is known about prism orientation in primate teeth, such findings imply greater wear resistance at the intercuspal region and less wear resistance at the lateral enamel at midcrown. Such findings appear to be supported by archeological evidence.     

Enamel microstructure in lemuridae (mammalia,primates): Assessment of variability

This study describes the molar enamel microstructure of seven lemurid primates: Hapalemur griseus, Varecia variegata, Lemur catta, Lemur macaco, Lemur fulvus rufus, Lemur fulvus fulvus, and Lemur fulvus albifrons. Contrary to earlier accounts, which reported little or no prism decussation in lemurid enamel, both Lemur and Varecia molars contain a prominent inner layer of decussating prisms (Hunter-Schreger bands), in addition to an outer radial prism layer, and a thin, nonprismatic enamel surface layer. In contrast, Hapalemur enamel consists entirely of radial and, near the surface, nonprismatic enamel. In addition, for all species, prism packing patterns differ according to depth from the tooth surface, and for all species but Varecia (which also has the thinnest enamel of any lemurid), average prism area increases from the enamel-dentine junction to the surface; this may be a developmental solution to the problem of accommodating a larger outer surface area with enamel deposited from a fixed number of cells. Finally, contradicting some previous reports, Pattern 1 prisms predominate only in the most superficial prismatic enamel. In the deeper enamel, prism cross-sections include both closed (Pattern 1) and arc-shaped (Pattern 2 or, most commonly, Pattern 3). This sequence of depth-related pattern change is repeated in all taxa. It should also be emphasized that all taxa can exhibit all three prism patterns in their mature enamel. The high degree of quantitative and qualitative variation in prism size, shape, and packing suggests that these features should be used cautiously in phylogenetic studies. Hapalemur is distinguished from the other lemurids by unique, medially constricted or rectangular prism cross-sections at an intermediate depth and the absence of prism decussation, but, without further assessment of character polarity, these differences do not clarify lemurid phylogenetic relations. Some characters of enamel microstructure may represent synapomorphies of Lemuridae, or of clades within Lemuridae, but homoplasy is likely to be common. Homoplasy of enamel characters may reflect functional constraints. © 1994 Wiley-Liss, Inc.     

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.     

Scanning electron microscopy and calcification in amelogenesis imperfecta in anterior and posterior human teeth

Teeth fragments from members of a family clinically and genetically diagnosed as having amelogenesis imperfecta were studied by scanning electron microscopy and X-ray microprobe analysis to establish the morphological patterns and the quantitative concentration of calcium in the enamel of anterior (canine, incisor) and posterior (premolar and molar) teeth. The prism patterns in the enamel of teeth from both regions were parallel or irregularly decussate, with occasional filamentous prisms accompanied by small, irregularly rounded formations. Prismless enamel showed the R- and P-type patterns. Calcium levels in enamel of amelogenesis imperfecta and control teeth differed significantly between anterior and posterior teeth, indicating that the factors that influence normal mineralization in different regions of the dental arch are not altered in the process of amelogenesis imperfecta.     

The epithelial genotype controls the pattern of extracellular enamel prism formation

Abstract. Enamel formation in the developing tooth organ is the product of epithelial-mesenchymal interactions which result in the differentiation of ameloblasts, the secretion of enamel proteins, and the production of a highly organized extracellular matrix. The three-dimensional organization of enamel prisms is species-specific: irregular polygonshaped in rabbit and rectangular-shaped in mouse. We designed experiments to test the hypothesis that three-dimensional organization of enamel prism formation is genetically determined by epithelium; the prediction being that speciesspecific enamel prism pattern formation is expressed independent of mesenchymal instructions. Our strategy employs scanning electron microscopy to examine enamel prism patterns formed during rabbit and mouse tooth morphogenesis in situ and in vitro, and to then determine the specific tissue type required for regulating these patterns using heterotypic tissue recombinations. Morphometric analyses demonstrated that cap stage tooth organs cultured on the chick chorioallantoic membrane (CAM) formed enamel prisms equivalent to prism patterns observed for in situ controls. Heterotypic tissue recombinations, using cap stage molar organs, formed rabbit-like prisms with rabbit epithelium/mouse mesenchyme, and mouse-like prisms with mouse epithelium/rabbit mesenchyme. These results indicate that dental papilla mesenchyme has no apparent influence on enamel prism pattern formation. Enamel prism pattern appears to be genetically regulated by the inner enamel epithelium.     

Immunochemical and immunohistochemical studies,using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13–17 kDa nonamelogenins,on immature enamel of the pig and rat

Summary Enamel proteins were extracted from the newly formed layer of immature porcine enamel, and the 25 kDa amelogenin, 89 kDa enamelin and 13–17 kDa nonamelogenins were purified. Specific antisera were raised against these proteins. Antibodies specific to the C-terminal region (residues 149–173) of the 25 kDa amelogenin were generated by absorption of the anti-25 kDa amelogenin serum with 20 kDa amelogenin, which contains residues 1–148 of the antigen. Immunoelectrotransfer blotting of the extracted porcine enamel proteins showed that the anti-25 kDa amelogenin serum recognized the 25 kDa and other low and high molecular weight amelogenins. The C-terminal specific anti-25 kDa amelogenin serum reacted only with amelogenins having molecular weights over 23 kDa. The anti-89 kDa enamelin serum recognized the 89 kDa enamelin and lower molecular weight proteins, but neither the amelogenins nor the 13–17 kDa nonamelogenins. The antiserum against the 13–17 kDa nonamelogenins showed no cross reactivity to the 89 kDa enamelin, but recognized higher molecular weight nonamelogenins. In immunohistochemical preparations of the porcine tooth germs, the 25 kDa amelogenin-like immunoreactivity over immature enamel decreased in a gradient from the enamel surface to the middle layer. In the inner layer immunoreactivity was concentrated over the prism sheaths. The C-terminal specific 25 kDa amelogenin-like immunoreactivity was intense at the outer layer of immature enamel and decreased sharply toward the middle layer. Prism sheaths were intensely stained by the antiserum to the 13–17 kDa nonamelogenins. The 89 kDa enamelin-like immunoreactivity over enamel prisms was intense at the outer layer and decreased toward the middle layer. Staining by the anti-89 kDa enamelin serum of prism sheaths was faint. In immature rat incisor enamel, the C-terminal specific 25 kDa amelogenin antiserum demonstrated a staining pattern similar to that in the immature enamel of the pig. Distinct 13–17 kDa nonamelogenin-like and 89 kDa enamelin-like immunoreactivities were found especially in the layer adjacent to the Tomes' process. We conclude that some enamel proteins are degraded soon after their secretion from the secretory ameloblast in the rat and the pig. The specific enamel proteins which reacted with the antiserum to the 13–17 kDa nonamelogenins seem to be involved with the formation of prism sheaths in immature porcine enamel, but not in rat incisor enamel.     

On thick and thin enamel in hominoids

Martin (1983, 1985) reviewed the significance of enamel thickness in hominoid evolution. He studied cut faces of hominoid teeth using the scanning electron microscope and related enamel prism packing patterns to both enamel formation rates and enamel thickness, although he did not present primary data on formation rates, which he summarised as being either “fast” or “slow.” Martin concluded that thick enamel formed at a fast rate represented the ancestral condition in the human and great ape clade. Thin enamel in African apes reflected a secondary reduction in secretion rates, with outer enamel being formed at a slow rate. The present study on ground sections of great ape and human teeth, using polarised light microscopy, was designed to measure the spacing between incremental growth lines in enamel, including striae of Retzius and prism cross striations, to determine rates of enamel formation in hominoids. Measurements on stria spacing showed that striae generally diverged as they passed outwards through enamel in all taxa. Cross-striation spacings also increased from inner to outer enamel. Secretion rates did not fall into two exclusive categories but varied, giving a spectrum of values generally increasing from within outwards at any one crown level and reducing in cervical enamel. There was no evidence for a reduction in enamel formation rates in outer enamel among African apes. These findings cast doubt on the proposition that the common ancestor of great apes and man had thick enamel formed at a fast rate. It is possible that thin enamel was the primitive condition, in which case thick enamel in humans and in Sivapithecus is derived, suggesting that thick enamel on low cusped teeth evolved on more than one occasion.     

Ultrastructure of enamel and dentine in extant dolphins (Cetacea: Delphinoidea and Inioidea)

Longitudinal and cross sections of teeth from 17 species of the Recent dolphins (Delphinoidea and Inioidea) were examined under scanning electron microscope to study the arrangement and ultrastructure of dental tissues with reference to phylogenetic and functional constraints. For most species, enamel had a simple bi-layered structure of radial enamel and an outer layer of prismless enamel. The outer prismless layer varied from 5 to 30 % of enamel thickness. The enamel of Burmeister’s porpoise (Phocoena spinipinnis) was entirely prismless. The prisms had an open sheath; tubules and tuft-like structures were common at the enamel-dentine junction. Cetacean dentine was characterized by irregularly distributed dentinal tubules in a relatively homogenous dentinal matrix. Radial enamel was observed in all Delphinoidea and in the franciscana (Pontoporia blainvillei), whereas the Amazon river dolphin (Inia geoffrensis) had prisms organized in Hunter–Schreger bands. HSB in enamel are regarded as a device for resisting propagation of cracks. These may occur due to increased functional demands, possibly related to the hardness of the species diet. Simplification in tooth shape and reduced biomechanical demands plausibly explain the primitive radial organization among delphinoids and Pontoporia. The HSB structure in the Amazon river dolphin, similar to those of extinct archaeocetes, seems to have secondary functional implications. However, the distribution of HSB in more-basal odontocetes is too poorly known to judge whether the HSB of Inia are a retained plesiomorphic feature or convergence.     

The prism pattern of rat molar enamel: a scanning electron microscope study.

Rat molar enamel has been studied by sectioning the enamel along various planes, and observing the etched surfaces in the SEM. It was found that the prism pattern was much more variable than in rat incisor enamel. Regions without prism decussation seemed to dominate in the occlusal half of the molars. Where present, prism decussation was of the uniserial lamellar type, but it varied considerably in distribution, extent, and distinctness. Prism decussation seemed to have a predilection for the cervical enamel, and was almost absent in the enamel on the occlusal surface. The interprismatic substance showed a characteristic configuration: In the inner enamel it appeared in the form of radially oriented sheets, which tended to delimit radially directed, single lines of prisms. In regions with prism decussation these single lines of prisms encompassed prisms belonging to different prism lamellae. In the outer part of the enamel the interprismatic substance exhibited a honeycomb appearance. The similarities and differences between the prism patterns of rat incisor and molar enamel may be of importance for understanding the mechanisms of amelogenesis, especially for the recognition of factors controlling the movement of ameloblasts.     

Variation in the prismatic arrangement of dental enamel surfaces

Slightly etched prisms of human dental enamel surfaces were examined in the scanning electron microscope. The crystals in the central region of prisms showed a denser arrangement, similar to the crystals on the periphery, which determine their form here. A crevice-like space could be observed between the central and the peripheral region of a prism. The prisms on the enamel surface showed a wide variety in shape being either of fish-scale or key-hole form, in other places fully irregular. There was no uniform prism on a single tooth, and an interprismatic substance was never found. On the surface of a deciduous tooth a prismless enamel surface was observed consisting of edges of crystallites, which did not unite to prism formation.     

Comparative Ultrastructure and Organization of the Prismatic Region of Two Bivalves and its Possible Relation to the Chemical Mechanism of Boring

The prismatic region of two bivalve molluscs exemplifies, inits structure and organization, one of the types of differentiatedcalcareous substrates through which boring organisms must penetrate. The oriented inorganic crystals, separated from one anotherby intercrystalline "spaces", are structurally organized intowell defined prisms. The prisms of each bivalve vary in shapeand size and are delineated from one another by electron-lucent,non-calcified regions. The demineralized organic matrix is also structurally organizedinto prisms, delineated from one another by prism sheaths, andan intraprismatic matrix structurally organized into closelypacked sheet-like compartments and subcompartments in whichthe inorganic crystals are deposited. The non-mineralized intercrystalline "spaces" between the individualinorganic crystals of the same or adjacent rows in a mineralizedsection are occupied by the walls of the intraprismalic sheet-likecompartments. Similarly, the non-calcified electronlucent regionsdelineating one mineralized prism from the next are occupiedby the thick prism sheaths. These portions of the organic matrixwhich fail to mineralize completely undoubtedly provide readypathways for the passage of solutes and solvents through thesetissues of highly ordered, densely packed, inorganic crystals.Moreover, the framework of the organic matrix, which fails tomineralize in these heavily calcified, molluscan substrates,may provide the primary, not the secondary source of chemicalattack during boring, for once the sheaths and compartmentssurrounding the crystals are broken down or solubilized, thecrystals are themselves loosened and freed for mechanical removalby shell-penetrating organisms.     

A scanning electron microscope study of aberrations in the prism pattern of rat incisor inner enamel.

The prism pattern in the inner enamel of adult rat incisors was studied with the SEM in unfixed tissues that had been sectioned, ground, polished, and etched. Six different types of aberrations in the prism pattern were encountered: 1. Prism lamellae may be shorter than the mesio-lateral width of enamel. 2. Prism lamellae may deviate from a transverse orientation. 3. Prism lamellae may "fuse" or "bifurcate." 4. Prisms of two adjacent lamellae may pursue a common course. 5. Prisms may change direction. 6. Variations exist in the outline of transversely cut prism profiles. Aberrations were observed at any distance from the dentino-enamel junction. These observations were used as a basis for an analysis of the movement of ameloblasts during rat incisor amelogenesis. It was concluded that it is physically possible for the ameloblasts to create the observed aberrations as they move along the path of the prisms. However, the aberrations seem to make it more difficult to understand the factors controlling ameloblast movement. Occasionally crystallite bridges connecting adjacent prisms were observed. A configuration resembling a bifurcating prism is pesented.     

Dietary adaptations of South African australopiths: inference from enamel prism attitude

The angle at which enamel prisms approach the wear surface holds information with regard to the stiffness of the tissue, as well as its wear resistance. Hence, analyses of prism orientation may shed light on questions of whether the thick enamel in hominins has evolved to confer stiffness or wear resistance to the teeth and may thus inform about the diet and behavioural ecology of these species. This was explored for Paranthropus robustus and Australopithecus africanus, whereby a distinction was made between prisms at the Phase I and Phase II facets. The results were compared with those obtained for Theropithecus, Macaca, and Potamochoerus for whom behavioural and/or experimental data are available, and were interpreted against simple mechanical principles. The South African hominins differ significantly in their relationships between wear facets and prism angulations. Teeth of P. robustus are better adapted to more vertical loads during mastication (Phase I), whereas those of A. africanus are better adapted to cope with more laterally-directed loads (Phase II) commonly associated with roll-crush and mastication. Overall, teeth of P. robustus appear stiffer, while those of A. africanus seem more wear resistant.     

Prism Size in Tooth Enamel of Some Late Cretaceous Mammals and Its Value in Multituberculate Taxonomy

Teeth from specified members of the two suborders of Late Cretaceous Multituberculata and two Late Cretaceous therians were studied. The enamel was prismatic on all teeth. In the therian representatives and the representatives of the suborder Ptilodontoidea of the Multituberculata, the prism diameters and densities per unit area were similar to those of recent mammals. In the representatives of the suborder Taeniolabidoidea the prisms were very large and their density per unit area was 5 to 8 times lower than in recent mammals. It is suggested that gigantoprismatic enamel is a characteristic of Taeniolabidoidea and could be used as a taxonomic criterion in multituberculate systematics.