Amelogenins: assembly, processing and control of crystal morphology.

The remarkable properties of enamel crystals and their arrangements in an extraordinary micro-architecture are clear indications that the processes of crystal nucleation and growth in the extracellular matrix are highly controlled. The major extracellular events involved in enamel formation are: (a) delineation of space by the secretory ameloblasts and the dentino-enamel junction; (b) self-assembly of amelogenin proteins to form the supramolecular structural framework; (c) transportation of calcium and phosphate ions by the ameloblasts resulting in a supersaturated solution; (d) nucleation of apatite crystallites; and (e) elongated growth of the crystallites. Finally, during the 'maturation' step, rapid growth and thickening of the crystallites take place, which is concomitant with progressive degradation and eventual removal of the enamel extracellular matrix components (mainly amelogenins). This latter stage during which physical hardening of enamel occurs is perhaps unique to dental enamel. We have focused our in vitro studies on three major extracellular events: matrix assembly, matrix processing and control of crystal growth. This paper summarizes current knowledge on the assembly, processing and effect on crystal morphology by amelogenin proteins. The correlation between these three events and putative functional roles for amelogenin protein are discussed.     

«Relative growth» from the dentino-enamel junction in primate maxillary molars

The dentino-enamel junction is critical throughout growth to mature crown configurations, being the interface between the papilla and the dental cap. Enamel deposition occurs relatively late and often causes changes from the pattern residing in the dentino-enamel junction. Primate teeth (mostly M¹) have been stripped of enamel after measurement and mapping of the original crown. Relative growth, a variant of static adult (allomorphic) allometries, is assessed by displacement of enamel basal crown component landmarks from dentine homologues relative to tooth size. The hypothesis that differential enamel growth reflects evolutionary history is supported by the positive allometry and shape differences in enamel versus dentine landmarks among phyletically enlarged and dentally-reduced primates.     

The Functional Width of the Dentino-Enamel Junction Determined by AFM-Based Nanoscratching

The dentino-enamel junction (DEJ) constitutes a structurally unique interphase uniting two mineralized tissues with very different matrix composition and physical properties. Its excellent biomechanical properties have drawn interest as a biomimetic model for joining dissimilar materials. In order to characterize the functional width of the DEJ, nanoscratching experiments were performed on human third molars. Friction coefficients of enamel, of dentin, and at the DEJ were obtained with a nanoscratch tester attached to an atomic force microscope (AFM). Normal loads in the range of 50 to 600 microN were applied to a spherical diamond indenter (r = 10 microm), which was driven 10 microm across the sample surface, recording the lateral force. Imaging with an AFM facilitated exact positioning of the scratches. The friction coefficient of intertubular dentin was 0.31 +/- 0.05, significantly above the coefficient of enamel of 0.14 +/- 0.02. The increased friction of dentin is attributed to the higher content of organic phases. Scratches performed across the interphase between enamel and dentin showed a sharp monotonic change in the friction coefficient. The average width of the slope between the friction coefficients of dentin and enamel was 2.0 +/- 1.1 microm and is assumed to represent the functional width of the dentino-enamel junction. The effect of the scalloped structure of the DEJ on its functional width as determined by mechanical testing is discussed.     

Immunofluorescent localization of amelogenins in developing bovine teeth.

Antiserum was prepared to the proteins (amelogenins) isolated from fetal bovine enamel matrix. This antiserum was used to localize the amelogenins in the developing bovine molar by immunofluorescent microscopy. Amelogenins could be identified in the preameloblasts before enamel matrix deposition had begun as well as in the secretory ameloblasts. The closely adherent layer of stratum intermedium cells also contained some immunoreactive material, suggesting that they may contribute protein to the enamel matrix. The newly deposited enamel matrix consisted of brightly fluorescent particles. Mature enamel matrix did not contain the immunoreactive protein except in a thin layer along the dentino-enamel junction and adjacent to the ameloblasts. No other portion of the tooth bud or other tissues reacted with the specific antiserum.     

Dentin sialoprotein and dentin phosphoprotein overexpression during amelogenesis

The gene for dentin sialophosphoprotein produces a single protein that is post-translationally modified to generate two distinct extracellular proteins: dentin sialoprotein and dentin phosphoprotein. In teeth, dentin sialophosphoprotein is expressed primarily by odontoblast cells, but is also transiently expressed by presecretory ameloblasts. Because of this expression profile it appears that dentin sialophosphoprotein contributes to the early events of amelogenesis, and in particular to those events that result in the formation of the dentino-enamel junction and the adjacent "aprismatic" enamel. Using a transgenic animal approach we have extended dentin sialoprotein or dentin phosphoprotein expression throughout the developmental stages of amelogenesis. Overexpression of dentin sialoprotein results in an increased rate of enamel mineralization, however, the enamel morphology is not significantly altered. In wild-type animals, the inclusion of dentin sialoprotein in the forming aprismatic enamel may account for its increased hardness properties, when compared with bulk enamel. In contrast, the overexpression of dentin phosphoprotein creates "pitted" and "chalky" enamel of non-uniform thickness that is more prone to wear. Disruptions to the prismatic enamel structure are also a characteristic of the dentin phosphoprotein overexpressing animals. These data support the previous suggestion that dentin sialoprotein and dentin phosphoprotein have distinct functions related to tooth formation, and that the dentino-enamel junction should be viewed as a unique transition zone between enamel and the underlying dentin. These results support the notion that the dentin proteins expressed by presecretory ameloblasts contribute to the unique properties of the dentino-enamel junction.     

A simple,disposable, and improved organ culture system for maintaining three-dimensional development of mouse embryonic molars

Summary Mandibular first molars from 17-d-old mouse embryos were cultured in vitro for 2 to 4 d by a simple, disposable, improved floatation method. This method consisted of using a 24-well multidish and a plastic culture chamber with a membrane filter. The improved floatation method, as well as our previous method, was capable of the three-dimensional development of tooth germs. Cytodifferentiation of odontoblasts and ameloblasts and formation of extracellular matrices were accelerated by the present culture system, in comparison with our previous method. All the molars cultivated by this method were very similar in morphology to in vivo. On Day 2 of culture the terminal cytodifferentiation of odontoblasts and the formation of predentin were ascertained in the bucco-lingual sections of the cultured molars. A thick layer of predentin was formed at the tip of the cusp and gradually decreased toward the cervical loop and the fissure between the buccal and ligual cusps. On Day 4 in vitro, secretory ameloblasts produced enamel matrix, and the mineralized enamel showed prismatic structure very similar to that in vivo. Dentin and predentin also were normal in ultrastructure. The extracellular matrices (enamel, dentine, and predentin) were formed in line with the pattern of the cusp and the formation of matrices normally started at the tip of the cusp. We conclude that the three-dimensional development of whole tooth germs in vitro may be very important for normal expression of the developmental program intrinsic to mouse embryonic molars.     

The presence and possible functions of the matrix metalloproteinase collagenase activator protein in developing enamel matrix.

The developing enamel matrix contains mostly amelogenins, which are hydrophobic proline-rich proteins. During amelogenesis, the amelogenins are presumably hydrolysed and removed from the enamel. Recently a number of metalloproteinases that may be important in amelogenesis have been identified in zymograms of the developing enamel matrix. In the present study an antibody specific for the matrix metalloproteinase collagenase activator protein (CAP) was characterized and used to identify this metalloproteinase in enamel. Immunoblotting showed that the CAP proteinase was present in the enamel matrix. Immunohistochemistry confirmed that the proteinase is localized in the enamel matrix, most specifically along the dentino-enamel junction. Purified CAP was found to hydrolyse amelogenin protein. Possible functions of the proteinase in the enamel matrix are discussed.     

Histochemical observations of protein-bound amino groups in human developing deciduous teeth

Summary Human developing deciduous teeth were studied by histochemical methods for protein groups; dinitrofluorobenzene (DNFB) H-acid stain for tyrosine histidine and tryptophan, coupled tetrazonium reaction with pre-iodination for histidine, diazotization and Millon's reaction for tyrosine revealed strong staining in the granular precursor of ameloblastic cytoplasma, pre- and young enamel layers. But only alloxan-Schiff stain showed a more intense stainability in the dentin matrix and pre-enamel than the young enamel matrix. The reaction of sulfhydryl and disulfide groups was observed in the matrix-forming ameloblast and Tomes' process with some variation of staining in the enamel matrix. The present histochemical results of protein groups in the enamel matrix were similar to those of the cornified layer of skin.Ameloblasts moderately stained with DNFB H-acid, coupled tetrazonium and alloxanschiff reaction were similarly stained in both matrix-forming stage and reduced stage. Increasing stainability of odontoblasts was limited in the matrix-forming stage.Pronounced changes of staining intensity in the enamel matrix for protein groups were found especially in the intra-rod organic substance with progress of mineralization and growth of deposited mineral crystallites, starting from the region of apex of cusp, parallel to the dentino-enamel junction.The stainability of the dentin matrix for protein groups increases following decalcification, and non-decalcified dentin was weakly stained for the alloxan-schiff method, coupled tetrazonium reaction, sulfhydryl and disulfide method.     

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.     

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.     

The expression of slow myosin during mammalian somitogenesis and limb bud differentiation

The developmental pattern of slow myosin expression has been studied in mouse embryos from the somitic stage to the period of secondary fiber formation and in myogenic cells, cultured from the same developmental stages. The results obtained, using a combination of different polyclonal and monoclonal antibodies, indicate that slow myosin is coexpressed in virtually all the cells that express embryonic (fast) myosin in somites and limb buds in vivo as well as in culture. On the contrary fetal or late myoblasts (from 15-d-old embryos) express in culture only embryonic (fast) myosin. At this stage, muscle cells in vivo, as already shown (Crow, M.T., and F.A. Stockdale. 1986. Dev. Biol. 113:238-254; Dhoot, G.K. 1986. Muscle & Nerve. 9:155-164; Draeger, A., A.G. Weeds, and R.B. Fitzsimons. 1987. J. Neurol. Sci. 81:19-43; Miller, J.B., and F.A. Stockdale. 1986. J. Cell Biol. 103:2197-2208), consist of primary myotubes, which express both myosins, and secondary myotubes, which express preferentially embryonic (fast) myosin. Under no circumstance neonatal or adult fast myosins were detected. Western blot analysis confirmed the immunocytochemical data. These results suggest that embryonic myoblasts in mammals are all committed to the mixed embryonic-(fast) slow lineage and, accordingly, all primary fibers express both myosins, whereas fetal myoblasts mostly belong to the embryonic (fast) lineage and likely generate fibers containing only embryonic (fast) myosin. The relationship with current models of avian myogenesis are discussed.     

Lipids in the developing enamel of the rat incisor. Parallel histochemical and biochemical investigations

Morphologic examination of the developing enamel of rat incisors showed the presence of cell processes and remnants. Histochemical investigation of rapid-frozen freeze-substituted samples, using p-phenylenediamine or a phosphotungstic acid chromic acid mixture, revealed osmiophilic components which were extractable in chloroform-methanol solution and were located inside the tubule-like structures of the extracellular matrix. The presecretory cell-rich and developing enamel zones underwent quantitative and qualitative lipid analysis. Comparison of the biochemical data as well as of the morphological observations, suggests a cellular origin for enamel lipids randomly adsorbed by extracellular matrix components during enamel processing. The nature of the material which appeared as an osmiophilic intra-tubular filling is still unresolved.     

Mucopolysaccharides in odontogenesis

Summary Localization of sulfomucopolysaccharides in developing teeth of Swiss albino mice was detected by S³⁵ autoradiography and histochemistry.A positive correlation was found to exist between autoradiographic and histochemical data with regard to the localization of sulfomucopolysaccharides. Autoradiography, however, revealed some sites of localization which were not detectable by histochemistry, namely, the odontoblasts and stratum intermedium.Fetuses which received the isotope via maternal injection at the cap stage of tooth development and were sacrificed after 2 hours of isotope action displayed rapid incorporation of the isotope in the components of the dental papilla. In the enamel organ, however, only moderate activity was recorded. When the time interval between injection and sacrifice of the experimental animals was increased to 20 hours, intense activity was observed in the enamel organ. With progressively longer intervals between injection and sacrifice, S³⁵ was demonstrable first in odontoblasts and later in the predentin. This occurred as a band or active zone which migrated toward the dentino-enamel junction. With the increasing intervals between injection and sacrifice, first the odontoblasts were active, then predentin was active while the odontoblasts became reduced in activity, after which the dentin matrix gained activity while the predentin decreased somewhat in activity. This pattern is consistent with appositional growth. A linear band of activity was not observed in the enamel matrix; rather, the activity was present as a diffuse stippling over a relatively large area of the matrix. The sulfomucopolysaccharide which existed in dentin matrix was postulated to have originated from the cells of both the odontoblastic layer and the dental papilla.Supported by PHS Grant No 2800-02, Tooth Germ Development, National Institute of Dental Research, National Institutes of Health.     

Aspects of cell proliferation kinetics of the inner dental epithelium during mouse molar and incisor morphogenesis: a reappraisal of the role of the enamel knot area.

First lower E-14 and E-16 mouse molars and E-13 lower incisors were cultured in vitro and either sequentially or continuously labelled with BrdU (5-bromo-2'-deoxyuridine). The behaviour of the non-cycling inner dental epithelial cells emerging from the enamel knot area of the molars was analysed by 3D (three dimensional) reconstructions of serial sections. These cells, as well as slow cycling cells underwent a coordinated temporo-spatial patterning leading to their patchy segregation at the tips of the forming cusps. In incisors (in vitro and in vivo), non-cycling cells were also present in the inner dental epithelium of the enamel knot area. However, these cells were not redistributed during incisor morphogenesis. These non-dividing inner dental epithelium cells of the enamel knot area which are either redistributed or not according to the tooth type specific morphogenesis might represent the organizers of morphogenetic units (OMU), the cusps.     

Enamel thickness of 45,X females' permanent teeth.

Enamel thicknesses in 45,X females', their male and female relatives', and population control males' and females' permanent tooth crowns were determined from radiographs. The results showed that the enamel layer in both maxillary first incisors and canines of 45,X females is definitely thinner than that of control males or females. Enamel in control males' and females' teeth was about equal in size. The distance between mesial and distal dentino-enamel junctions or the thickness of "dentin" was similar in 45,X females' and in control females' teeth, but definitely smaller than in control males' teeth. These findings show that in the presence of the second sex-chromosome in the chromosome complement, whether X or Y, there is a definite and equal increase in the amount of enamel. On the other hand, in the presence of the Y chromosome in the chromosome complement, relative to the second X chromosome, there is a definite increase in the thickness of the dentin. The results of earlier studies have indicated a direct growth-promoting effect of the sex chromosomes on tooth growth, and that the effect of X and Y chromosomes is different. The present results suggest that the influence of the X- and Y-chromosome gene(s) on amelogenesis is the same in quantitative terms but different in relation to the determination of the distance between dentino-enamel junctions; the Y chromosome is more effective than the X chromosome in that respect. It is postulated that this size-increasing effect of the Y-chromosome gene(s) might result from its profound effect on cell proliferations.     

Immunodetection of osteoadherin in murine tooth extracellular matrices

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.     

Cell-matrix interrelation and cell-to-cell connection in the secretory ameloblast layer of kitten teeth

To investigate the cell-matrix interrelation and the structure and permeability of the junctional complexes of secretory ameloblasts, molar tooth germs from kittens were examined by means of scanning electron microscopy, routine thin sections and freeze-fracture replication. Scanning electron microscopy showed remarkably dissolved growth fronts of enamel in materials that had been fixed with glutaraldehyde and then subjected to EDTA perfusion for 10 min. By the action of EDTA, intercrystallite spaces in rod and interrod enamel were prominently widened, and their longitudinal ends of crystallites displayed irregular and extremely sparse structures. In enamel rods surrounded entirely by interrod enamel, and in enamel rods of the typical key hole shape with successive interrod enamel participation, the most striking dissolution of crystallites occurred at the boundaries between rod and interrod enamel, where broad expanses of rod-sheath spaces were observed. In thin sections, the Tomes processes of secretory ameloblasts occupying the above rods were rectangular or variations of a rectangular shape, respectively; and interameloblast spaces opened to the enamel growth fronts, which corresponded to the junction between rod and interrod enamel. In enamel rods standing in regular rows and showing the typical arcade shape, the centers of the rods were drastically dissolved and exhibited single and deep slits, whereas the boundaries between rod and interrod enamel showed no wide furrows. The Tomes processes occupying such arcade-shaped rods were typically triangular, and the interameloblast space always joined the type-1 face of process, which is responsible for enamel rod formation. Secretory ameloblast possessed two sets of junctional complexes at the proximal and distal ends of the cell body. The distal one was situated proximally to the Tomes process. Freeze-fracture replication demonstrated the functional structures of these junctions: the proximal junction was fascia occludens, and the distal one incomplete zonula occludens with many free-ending tight junctional strands and interstrand spaces or a less developed irregular junction.     

Immunochemical characterization of a chicken egg yolk antibody to secretory forms of rat incisor amelogenin.

Amelogenins represent the major component of the organic matrix of enamel, and consist of several intact and degraded forms. A precise knowledge of their respective distributions throughout the enamel layer could provide some insight into their functions. To date, no antibody exists that can selectively detect the secretory forms of amelogenin. In this study we used the chicken egg yolk system to generate an antibody to recombinant mouse amelogenin. Immunoblots of whole homogenates from rat incisor enamel organs and enamel showed that the resulting antibody (M179y) recognized proteins corresponding to the five known secretory forms of rat amelogenin. Immunogold cytochemistry demonstrated that reactivity was restricted to ameloblasts and enamel. Secretory forms of amelogenin persisted in significant amounts throughout the enamel layer. The density of labeling was highest over the surface portion of the enamel layer, but enamel growth sites in this region showed a localized paucity of gold particles. Immunoreactivity was lowest over the mid-portion of the layer and increased moderately near the dentino-enamel junction. These results indicate that intact forms of amelogenin probably have a more complex distribution in the enamel layer than was heretofore suspected.     

Incremental enamel development in modern human deciduous anterior teeth

This study reconstructs incremental enamel development for a sample of modern human deciduous mandibular (n = 42) and maxillary (n = 42) anterior (incisors and canines) teeth. Results are compared between anterior teeth, and with previous research for deciduous molars (Mahoney: Am J Phys Anthropol 144 (2011) 204-214) to identify developmental differences along the tooth row. Two hypotheses are tested: Retzius line periodicity will remain constant in teeth from the same jaw and range from 6 to 12 days among individuals, as in human permanent teeth; daily enamel secretion rates (DSRs) will not vary between deciduous teeth, as in some human permanent tooth types. A further aim is to search for links between deciduous incremental enamel development and the previously reported eruptionsequence. Retzius line periodicity in anterior teeth ranged between 5 and 6 days, but did not differ between an incisor and molar of one individual. Intradian line periodicity was 12 h. Mean cuspal DSRs varied slightly between equivalent regions along the tooth row. Mandibular incisors initiated enamel formation first, had the fastest mean DSRs, the greatest prenatal formation time, and based upon prior studies are the first deciduous tooth to erupt. Relatively rapid development in mandibular incisors in advance of early eruption may explain some of the variation in DSRs along the tooth row that cannot be explained by birth. Links between DSRs, enamel initiation times, and the deciduous eruption sequence are proposed. Anterior crown formation times presented here can contribute toward human infant age-at-death estimates. Regression equations for reconstructing formation time in worn incisors are given.     

Synchrotron microComputed Tomography of the mature bovine dentinoenamel junction

The mature dentinoenamel junction (DEJ) is viewed by some investigators and the current authors, not as a fossilized, sharp transition between enamel and dentin, but as a relatively broad structural transition zone including the mantle dentin and the inner aprismatic enamel. In this study, the DEJ structure in bovine incisors was studied with synchrotron microComputed Tomography (microCT) using small cubes cut parallel to the tooth surface. The reconstructions revealed a zone of highly variable punctate contrast between bulk dentin and enamel; the mean linear attenuation coefficients and their standard deviations demonstrated that this zone averaged less mineral than dentin or enamel but had more highly variable structure than either. The region with the punctuate contrast is, therefore, the mantle dentin. The thickness of the mantle dentin seen in a typical data set was about 30 microm, and the mantle dentin-enamel interface deviated +/-15 microm from the average plane over a distance of 520 microm. In the highest resolution data ( approximately 1.5 microm isotropic voxels, volume elements), tubules in the dentin could be discerned in the vicinity of the DEJ. Contrast sensitivity was high enough to detect differences in mineral content between near-surface and near-DEJ volumes of the enamel. Reconstructions before and after two cubes were compressed to failure revealed cracks formed only in the enamel and did not propagate across the mantle dentin, regardless of whether loading was parallel to or perpendicular to the DEJ.