The spatial distribution of focal stacks within the inner enamel layer of mandibular mouse incisors

Focal stacks are an alternative spatial arrangement of enamel rods within the inner enamel of mandibular mouse incisors where short rows comprised of 2–45 enamel rods are nestled at the side of much longer rows, both sharing the same rod tilt directed mesially or laterally. The significance of focal stacks to enamel function is unknown, but their high frequency in transverse sections (30% of all rows) suggests that they serve some purpose beyond representing an oddity of enamel development. In this study, we characterized the spatial distribution of focal stacks in random transverse sections relative to different regions of the inner enamel and to different locations across enamel thickness. The curving dentinoenamel junction (DEJ) in transverse sections complicated spatial distribution analyses, and a technique was developed to “unbend” the curving DEJ allowing for more linear quantitative analyses to be carried out. The data indicated that on average there were 36 ± 7 focal stacks located variably within the inner enamel in any given transverse section. Consistent with area distributions, focal stacks were four times more frequent in the lateral region (53%) and twice as frequent in the mesial region (33%) compared to the central region (14%). Focal stacks were equally split by tilt (52% mesial vs. 48% lateral, not significant), but those having a mesial tilt were more frequently encountered in the lateral and central regions (2:1) and those having a lateral tilt were more numerous in the mesial region (1:3). Focal stacks having a mesial tilt were longer on average compared to those having a lateral tilt (7.5 ± 5.6 vs. 5.9 ± 4.0 rods per row, p < 0.01). There was no relationship between the length of a focal stack and its location within the inner enamel. All results were consistent with the notion that focal stacks travel from the DEJ to the outer enamel the same as the longer and decussating companion rows to which they are paired. The spatial distribution of focal stacks within the inner enamel was not spatially random but best fit a null model based on a heterogenous Poisson point process dependent on regional location within the transverse plane of the enamel layer.     

Correlation of the arrangement pattern of enamel rods and secretory ameloblasts in pig and monkey teeth: A possible role of the terminal webs in ameloblast movement during secretion

Enamel rod architecture and ameloblast arrangement were examined in pig and monkey teeth using light microscopy and scanning and transmission electron microscopy. Enamel rods in the pig teeth were arranged in longitudinal straight rows in the initial enamel layer, in longitudinal wavy rows in the inner enamel layer, and in a staggered pattern in the outer enamel layer. Rod decussation was seen only in the inner layer. Cross-sectined enamel rods in the pig were arcade-shaped in the initial and inner layers, and mostly round in shape with circular boundaries in the outer layer. Arrangement of secretory ameloblasts at the level of the distal terminal web and Tomes' processes, and shape of Tomes' processes, corresponded to those of the enamel rod in the enamel layers. Distal terminal webs were well developed between straight rows of the ameloblasts forming the initial layer and between wavy rows of the ameloblasts forming the inner layer, and less developed within a row. The filament bundles in the distal terminal webs were also oriented along the rows. However, in the ameloblasts forming the outer layer, which lost their row pattern, distal terminal web filaments were distributed uniformly at the cell periphery. A similar arrangement of wavy rows of ameloblasts at the level of distal terminal web and Tomes' processes was also seen in monkey teeth.     

Correlation of the arrangement pattern of enamel rods and secretory ameloblasts in pig and monkey teeth: a possible role of the terminal webs in ameloblast movement during secretion.

Enamel rod architecture and ameloblast arrangement were examined in pig and monkey teeth using light microscopy and scanning and transmission electron microscopy. Enamel rods in the pig teeth were arranged in longitudinal straight rows in the initial enamel layer, in longitudinal wavy rows in the inner enamel layer, and in a staggered pattern in the outer enamel layer. Rod decussation was seen only in the inner layer. Cross-sectioned enamel rods in the pig were arcade-shaped in the initial and inner layers, and mostly round in shape with circular boundaries in the outer layer. Arrangement of secretory ameloblasts at the level of the distal terminal web and Tomes' processes, and shape of Tomes' processes, corresponded to those of the enamel rod in the enamel layers. Distal terminal webs were well developed between straight rows of the ameloblasts forming the initial layer and between wavy rows of the ameloblasts forming the inner layer, and less developed within a row. The filament bundles in the distal terminal webs were also oriented along the rows. However, in the ameloblasts forming the outer layer, which lost their row pattern, distal terminal web filaments were distributed uniformly at the cell periphery. A similar arrangement of wavy rows of ameloblasts at the level of distal terminal web and Tomes' processes was also seen in monkey teeth.     

Human enamel rod presents anisotropic nanotribological properties

The AFM combined nanoindentation was performed to observe the ultrastructure of enamel rod from various section plans and positions while probing their mechanical and tribological properties of the area. The nanohardness and the elastic modulus of the head region of the enamel rods are significantly higher than that of the tail region and the axial-sectional plane. Both nanohardness and elastic modulus gradually decrease from enamel surface toward dentino-enamel junction. Such a variation correlates well with the decreasing trend of calcium composition from our element analysis. The friction coefficient and nanowear of the enamel showed an inversed trend to the hardness with respect to their relative topological position in the long axis of enamel rod toward DEJ. The relationship between the nanowear depth and the distance from the outer enamel surface to DEJ presented exponential function. The results presented clarify the basic nanomechanical and nanotribological properties of human enamel rods and provide a useful reference for the future development of dental restorative materials.     

A freeze-fracture study of the topographic relationship between inner enamel-secretory ameloblasts in the rat incisor

A correlated study using freeze-fracture replicas and routine thin sectioning was done on the ameloblasts which secrete inner enamel in the rat incisor. These ameloblasts are columnar cells aligned in rows parallel to the cross-sectional plane of the incisor. Each cell has a Tomes' process at its distal end which is contained within a cavity of interrod enamel. Tomes' processes of a particular row of cells are inclined towards the mesial side of the tooth, while those of the next row are inclined towards the lateral side, so that the processes cross each other at 90°. Freeze-fracture replicas were used to examine the surfaces of ameloblasts between adjacent rows and the surfaces of the cells within the row. Replicas of the surfaces between rows showed that ameloblasts are curved so that their proximal and distal ends (Tomes' processes) are both directed towards the same side of the tooth. The surface of each row bears the impression left upon it by the cells in the adjacent row which have been fractured away. This cell-impression is also curved, but in the opposite direction to the cells which it crosses. It was thus shown that not only the Tomes' processes of adjacent rows but the entire cell bodies cross one another. This crossing is slight at the proximal ends of the cells, but marked at the level of the distal junctional complex. At this level a cell from a particular row curves across several cells of the neighbouring row and forms large tight junctions with at least two cells in that row. The proximal and distal junctional complexes form zones of tight junctions around the cell. The distal complex is more extensive than the proximal and the tight junctions are better developed between adjacent rows. Gap junctions are, however, larger between cells within the rows. The study demonstrated the effectiveness of freeze-fracture replicas in obtaining 3-dimensional details about ameloblast shape, and this information was used to evaluate the supposed movement of ameloblasts during inner enamel formation.     

The Function and Structure of the Marsupial Enamel

The aims of this study are to clarify the structure of tubular enamel and the function of enamel tubules on the marsupial of opossum (Monodelphis domestica). Almost all enamel prisms, surrounded by interprismatic enamel, ran obliquely from the dentinoenamel junction (DEJ), and bent near the enamel surface. The enamel tubules are distributed in both enamel prisms and the interprismatic enamel near the DEJ. From the middle to the surface of the enamel, one enamel tubule ran within a single enamel prism. Most of enamel tubules continued from the DEJ to near the enamel surface. It is suggested that each enamel tubule developed in relation to one ameloblast. The fibers of odontoblastic process penetrated the DEJ from the dentinal tubules into the enamel tubules, and some branched across the enamel prisms. The odontoblastic process may be actively cross into the ameloblastic layer and may be involved in the formation of enamel tubules. After in vivo injection of tetracycline, tetracycline labeling showed that the odontoblastic tubules continued to enamel tubules. And strontium was detected in enamel tubules from the DEJ to the enamel surface, as was the dentinal tubules. In conclusion, there was active transport by the odontoblast and it's process through the enamel tubules.     

The function and structure of the marsupial enamel

The aims of this study are to clarify the structure of tubular enamel and the function of enamel tubules on the marsupial of opossum (Monodelphis domestica). Almost all enamel prisms, surrounded by interprismatic enamel, ran obliquely from the dentinoenamel junction (DEJ), and bent near the enamel surface. The enamel tubules are distributed in both enamel prisms and the interprismatic enamel near the DEJ. From the middle to the surface of the enamel, one enamel tubule ran within a single enamel prism. Most of enamel tubules continued from the DEJ to near the enamel surface. It is suggested that each enamel tubule developed in relation to one ameloblast. The fibers of odontoblastic process penetrated the DEJ from the dentinal tubules into the enamel tubules, and some branched across the enamel prisms. The odontoblastic process may be actively cross into the ameloblastic layer and may be involved in the formation of enamel tubules. After in vivo injection of tetracycline, tetracycline labeling showed that the odontoblastic tubules continued to enamel tubules. And strontium was detected in enamel tubules from the DEJ to the enamel surface, as was the dentinal tubules. In conclusion, there was active transport by the odontoblast and it's process through the enamel tubules.     

Hidden contributions of the enamel rods on the fracture resistance of human teeth

The enamel of human teeth is generally regarded as a brittle material with low fracture toughness. Consequently, the contributions of this tissue in resisting tooth fracture and the importance of its complex microstructure have been largely overlooked. In this study an experimental evaluation of the crack growth resistance of human enamel was conducted to characterize the role of rod (i.e. prism) orientation and degree of decussation on the fracture behavior of this tissue. Incremental crack growth was achieved in-plane, with the rods in directions longitudinal or transverse to their axes. Results showed that the fracture resistance of enamel is both inhomogeneous and spatially anisotropic. Cracks extending transverse to the rods in the outer enamel undergo a lower rise in toughness with extension, and achieve significantly lower fracture resistance than in the longitudinal direction. Though cracks initiating at the surface of teeth may begin extension towards the dentin–enamel junction, they are deflected by the decussated rods and continue growth about the tooth’s periphery, transverse to the rods in the outer enamel. This process facilitates dissipation of fracture energy and averts cracks from extending towards the dentin and vital pulp.     

Three-dimensional changes in direction and interrelationships among enamel prisms in the dog tooth.

This study describes the three-dimensional features of enamel prisms and their arrangement in dog teeth. Tangential semithin sections of demineralized tooth germ were serially cut from the enamel surface to the enamel-dentin junction. Straight rows of enamel prisms parallel or perpendicular to the meridian were selected at the enamel-dentin junction; these prisms were reconstructed from micrographs with a personal computer. Near the enamel-dentin junction, the arrangement of enamel prisms appeared regular. Viewed from the enamel surface, the cut-ends of the enamel prisms that were parallel to the meridian at the enamel-dentin junction appeared as a sine curve, with 16 enamel prisms forming one period. The enamel prisms in a row perpendicular to the meridian were parallel to each other and deflected to the left or right from the enamel-dentin junction. Away from the enamel-dentin junction, the periodicity of the prisms gradually disappeared. The sine curve formed by the cut-ends of prisms in a row parallel to the meridian became irregular, and prisms in rows perpendicular to the meridian crossed each other. The semithin sections showed belt-like zones arranged perpendicular to the meridian. Each belt-like zone consisted of enamel prisms oriented in the same direction, those in neighboring zones being oriented in opposite directions. The disappearance of the regular arrangement of prisms was related to changes in their location in the belt-like zones.     

The development of enamel structure in rat incisors as compared to the teeth of monkey and man

The rat incisor is an excellent model system in which to study amelgenesis. However, the information obtained has not been extrapolated to the human because of alleged structural differences between the teeth. The obvious differences include continuous eruption in rat incisors and an enamel rod pattern in rats which seemingly differs from the keyhole pattern of human enamel. A comprehensive analysis was made of those features of enamel structure considered fundamental to the understanding of its formation. This was done by applying the knowledge of amelogenesis obtained in rat incisors to the teeth of monkey and man. The following points of basic similarity were established between these species: (1) Interrod enamel is secreted first. It forms the side walls of cavities which are initially occupied by Tomes' processes. (2) The formation of interrod cavities is followed by deposition of enamel rods within these spaces. (3) The rods conform to the shape of the cavities and are secreted from one surface of Tomes' process. (4) At the initial site of rod deposition its enamel is continuous with the interrod enamel wall. (5) Growth of the rod compresses the process to one side of the cavity resulting in an arcade-shaped "space" between the rod and the remaining interrod walls. This study demonstrates that it is no longer necessary to postulate a keyhole structure for primate enamel, and it has established that a fundamental similarity exists in the basic structure and in the mode of formation of enamel in all three species.     

Presence of enamel on the incisors of the llama (Lama glama) and alpaca (Lama pacos)

Attempts have been made to define the relationships among the South American camelids, the guanaco, llama, alpaca, and vicuna, by comparing the morphology of their incisors. The alpaca has been reported to have an incisor morphology similar to the vicuna, lacking enamel on the lingual surface. The llama and guanaco are said to have enamel on both the labial and lingual surface of their incisor teeth. These comparisons have been based on gross morphological observations and not on histologic analysis. This study was undertaken to determine whether or not alpaca teeth have enamel on the lingual surface. The cross-sectional histologic anatomy of the incisor teeth was compared in two closely related South American camelid species, the llama (Lama glama), and the alpaca (Lama pacos). Thick sections (300 μm) and scanning electron microscopy were the techniques utilized. The mandibular first, second, and third incisors were examined in four llamas and five alpacas. A substantial layer of enamel was present on all surfaces of all llama incisors. The enamel layer on the labial surface of the alpaca incisors closely resembled that found in the llama. The enamel layer on the lingual surface of the alpaca incisors, although greatly reduced, was distinctly present. Alpacas may be more closely related to guanacos and llamas than to vicunas. A histologic study of vicuna incisors would help to better define the relationships of the four camelids. Anat Rec. 249:441–449, 1997. © 1997 Wiley-Liss, Inc.     

Cell architecture in the papillary layer of rat incisor enamel organ at the stage of enamel maturation

Shape and arrangement of cells and extent of intercellular spaces were studied in sections of enamel organ cut in three planes: at right angles to the axis of the incisor tooth, at right angles to the axis of the ameloblasts, and parallel to the axes of the incisor and ameloblasts. The cells in contact with the base of the ameloblasts make up the proximal part of the papillary layer. They have a polygonal cross section close to the ameloblasts and point several sheet-like cell processes towards the blood vessels which invaginate in the papillary layer. Intercellular spaces of constant width pervade the proximal part and provide a direct and straight communication between blood vessels and ameloblasts. The cells of the ridges make up the distal part of the papillary layer, and are flattened in the direction of the ridges. Intercellular spaces in the ridges are narrower than in the proximal part and visible with the light microscope only during a fraction of the enamel maturation period. No distinct cell layers are visible within the papillary layer during enamel maturation. All cells in the papillary layer may be in contact with the basement membrane investing the enamel organ. The structure of the papillary layer changes with the different phases of enamel maturation. Functional aspects of the papillary layer are briefly discussed.     

Comparison between deciduous and permanent incisor teeth in morphology of bovine enamel.

Functional incisor teeth (deciduous and permanent teeth) from Bovidae (14 species) were prepared for scanning electron microscopic observation. Ultrastructural patterns of the enamel layer of deciduous and permanent incisor teeth varied (ex. prisms, arrangement pattern of matrices, and in thickness of enamel layer) in each species. The ultrastructures of prisms in longitudinal sections were classified into three types; A, radial, B, tangential, and C, mix of A and B arrangement enamel; modified Koenigswald's method (1982) in examined species. Type A was found in a large part of permanent and a small part of deciduous incisor teeth, while types B and C were mainly found in the deciduous teeth. These morphological features show the remarkable correlation between permanent and deciduous teeth.     

Mutations in RELT cause autosomal recessive amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt^−/− mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt^−/− enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations.     

Knife chatter during thin sectioning of rat incisor enamel can cause periodicities resembling cross-striations

Cross-striations are traditionally associated with the enamel rods in many species including man. Although these striations are obvious with light microscopy, their exact nature has been difficult to determine with the transmission electron microscope on thin sections of enamel. Thin section microscopy either reveals no structures that can be called cross-striations, or shows periodic light and dark bands across the rods. Superficially, these bands resemble chatter artifact. To test this possibility, rat incisor enamel was used because cross-striations have not been demonstrated on these enamel rods. Thin sections were prepared of enamel blocks oriented in various ways with respect to the cutting edge of the diamond knife. The sections showed either uniform enamel or light and dark bands over rod profiles or interrod enamel. Since these bands could be produced artifactually it is concluded that similar bands seen on enamel rods of other species may also be artifacts.     

Relationships Between Enamel Prism Decussation and Organization of the Ameloblast Layer in Rodent Incisors

Rodent enamel microstructure has been extensively investigated, primarily on the basis of 2D electronic microscopy data. The nature and dynamics of the ameloblasts (the enamel-secreting cells) have also been well studied. However, critical issues still remain surrounding exactly how the ameloblasts produce the astonishing microstructural complexity of enamel, and how this subtle architecture evolved through time. In this article, we used a new methodology based on confocal laser microscopy to reconstruct the enamel microstructure of rodent incisors in three dimensions (3D) with the ameloblasts in situ. We proposed interpretations regarding the possible relationships between the workings of the ameloblasts and the resulting enamel prisms, especially how the phenomenon of decussation is generated. Finally, we were able to represent the two main types of modern rodent incisor microstructures (uniserial and multiserial decussations), as a set of parameters that have been entered into the 3D enamel simulation software Simulenam to generate 3D models that can be digitally manipulated. Associating 2D data of incisor enamel microstructure of fossil rodents and Simulenam, it was then possible to better understand how the various decussation parameters evolved through time and gave rise to the two modern microstructure types from the same ancestral type (pauciserial). This study also confirmed that rodent and artiodactyl enamel do not share the same mechanism of decussation formation. Anat Rec, 302:1195–1209, 2019. © 2018 Wiley Periodicals, Inc.     

Scanning electron microscopic observations of surface prismless enamel formed by minute crystals in some human permanent teeth

It might have been recognized that surface prismless enamel with no prism sheaths or boundaries is formed by needle-shaped crystals in parallel arrangements and shows almost the same crystal size as the underlying prismatic enamel. This study found that some island- and band-shaped prismless enamel in third molars and dome-shaped prismless areas in the region adjacent to the partial hypoplastic enamel of a premolar were formed by minute crystals compared with the underlying prismatic enamel when observing afibrillar cementum and cementicle-like structures by scanning electron microscopy. Their minute crystals became smaller in size towards the natural enamel surface. In the band- and dome-shaped prismless enamel, the minute crystals abruptly changed to the usual-sized crystals in the underlying prismatic enamel, although the minute crystals in the dome-shaped prismless areas tended to show random arrangements. The band-shaped prismless layers might be similar to afibrillar cementum, but shallow Tomes’ process pits were present in the natural surface and no appositional laminations were observed in the fractured surface. The minute crystal formation in such prismless regions might be caused by the remarkable decline of ameloblast activity immediately after the disappearance of Tomes’ processes producing prism structures surrounded by prism sheaths during the final stage of amelogenesis.     

Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors

In the continuously erupting rat incisor the ameloblasts progress through distinct stages associated with the secretion and maturation of enamel. We have examined the possibility that the so-called "postsecretory" ameloblasts of the maturation stage of amelogenesis remain biosynthetically active and are engaged in the synthesis, secretion, and degradation of enamel gene products. The ultrastructural distribution of antigenic sites for enamel proteins was studied within enamel organ cells during the early maturation stage of amelogenesis in rat incisors by using the protein A-gold immunocytochemical technique and rabbit polyclonal antibodies developed against mouse amelogenins. All regions of amelogenesis from late secretion through the first complete modulation from ruffle-ended to smooth-ended ameloblasts were examined. Specific immunolabelling was found within the rough endoplasmic reticulum, Golgi saccules, secretory granules, and lysosomes of ameloblasts throughout these regions. The heaviest intracellular immunolabelling was found within secretory granules and lysosomes (multivesicular type). Quantitative analyses showed that the Golgi saccules and the multivesicular lysosomes of modulating ameloblasts were generally less immunoreactive compared to similar organelles in ameloblasts secreting the inner enamel layer. Radioautographic studies confirmed that ameloblasts of the maturation stage incorporated 3H-leucine and 3H-methionine and secreted labelled proteins into the enamel layer. Grain counts indicated that ameloblasts from the first ruffle-ended band incorporated about two-fold less 3H-methionine and secreted about tenfold less labelled proteins into the enamel compared to ameloblasts secreting the inner enamel layer. The results of this study confirm that ameloblasts do not terminate biosynthesis and secretion of enamel proteins once the final layer has been deposited on the surface of the developing enamel. They continue to form and release new proteins during the maturation stage which intermix with older proteins laid down initially during the secretory stage of amelogenesis. Secretory activity for enamel proteins has been detected in ameloblasts up to at least the second ruffle-ended phase of maturation, at which point the enamel matrix is partially soluble in EDTA.