Modulated regeneration of acid-etched human tooth enamel by a functionalized dendrimer that is an analog of amelogenin

In the bioinspired repair process of tooth enamel, it is important to simultaneously mimic the organic-matrix-induced biomineralization and increase the binding strength at the remineralization interface. In this work, a fourth-generation polyamidoamine dendrimer (PAMAM) is modified by dimethyl phosphate to obtain phosphate-terminated dendrimer (PAMAM-PO₃H₂) since it has a similar dimensional scale and peripheral functionalities to that of amelogenin, which plays important role in the natural development process of enamel. Its phosphate group has stronger affinity for calcium ion than carboxyl group and can simultaneously provide strong hydroxyapatite (HA)-binding capability. The MTT assay demonstrates the low cytotoxicity of PAMAM-PO₃H₂. Adsorption tests indicate that PAMAM-PO₃H₂ can be tightly adsorbed on the human tooth enamel. Scanning electron microscopy and X-ray diffraction are used to analyze the remineralization process. After being incubated in artificial saliva for 3 weeks, there is a newly generated HA layer of 11.23 μm thickness on the acid-etched tooth enamel treated by PAMAM-PO₃H₂, while the thickness for the carboxyl-terminated one (PAMAM-COOH) is only 6.02 μm. PAMAM-PO₃H₂ can regulate the remineralization process to form ordered new crystals oriented along the Z-axis and produce an enamel prism-like structure that is similar to that of natural tooth enamel. The animal experiment also demonstrates that PAMAM-PO₃H₂ can induce significant HA regeneration in the oral cavity of rats. Thus PAMAM-PO₃H₂ shows great potential as a biomimetic restorative material for human tooth enamel.     

Three-dimensional direction and interrelationship of prisms in cuspal and cervical enamel of dog tooth

The three-dimensional architecture of enamel prisms was examined in cuspal enamel and compared with that in cervical enamel by light and electron microscopy as well as computer-assisted reconstruction using the developing enamel of several dog teeth. Dog tooth enamel consists of two groups of alternately arranged enamel prisms oriented in opposite sideward directions basically forming thick horizontal rings, partly branching off from the stem. Along a 8–10 enamel prism-wide group, the enamel prisms emerge in parallel tilting uniformly to the same sideward direction. In cervices, groups of enamel prisms are arranged nearly in parallel displaying a regular arrangement of prisms. Approaching the cusp of tooth, the groups of enamel prisms fuse to a concentric cusp-centered arrangement and the prisms exhibit no periodic arrangement as shown in the cervical enamel. It is suggested that the three-dimensional structure of enamel becomes complicated close to the cusp, contributing to the chewing stress of tooth. Anat. Rec. 252:355–368, 1998. © 1998 Wiley-Liss, Inc.     

Evolutionary trajectories of tooth histology patterns in modern sharks (Chondrichthyes,Elasmobranchii)

During their evolutionary history, modern sharks developed different tooth mineralization patterns that resulted in very distinct histological patterns of the tooth crown (histotypes). To date, three different tooth histotypes have been distinguished: (i) orthodont teeth, which have a central hollow pulp cavity in the crown, encapsulated by a prominent layer of dentine (orthodentine); (ii) pseudoosteodont teeth, which have their pulp cavities secondarily replaced by a dentinal core of porous dentine (osteodentine), encased by orthodentine; and (iii) osteodont teeth, which lack orthodentine and the whole tooth crown of which consists of osteodentine. The aim of the present study was to trace evolutionary trends of tooth mineralization patterns in modern sharks and to find evidence for the presence of phylogenetic or functional signals. High resolution micro-computed tomography images were generated for the teeth of members of all nine extant shark orders and the putative stem group †Synechodontiformes, represented here by three taxa, to examine the tooth histology non-destructively. Pseudoosteodonty is the predominant state among modern sharks and represents unambiguously the plesiomorphic condition. Orthodonty evolved several times independently in modern sharks, while the osteodont tooth histotype is only developed in lamniform sharks. The two shark orders Heterodontiformes and Pristiophoriformes showed highly modified tooth histologies, with Pristiophorus exhibiting a histology only known from batomorphs (i.e. rays and skates), and Heterodontus showing a histological difference between anterior and posterior teeth, indicating a link between its tooth morphology, histology and durophagous lifestyle. The tooth histotype concept has proven to be a useful tool to reflect links between histology, function and its taxonomic value for distinct taxa; however, a high degree of variation, especially in the pseudoosteodont tooth histotype, demonstrates that the current histotype concept is too simplistic to fully resolve these relationships. The vascularization pattern of the dentine might offer new future research pathways for better understanding functional and phylogenetic signals in the tooth histology of modern sharks.     

Light microscopy and computer three-dimensional reconstruction of the blood capillaries of the enamel organ of rat molar tooth germs

We performed a light microscope and a computer three‐dimensional reconstruction study of serial sections of the molar enamel organ of 3‐ and 5‐day‐old rats perfused with Indian ink through the arterial system. The tooth germs were fixed in Bouin's solution, embedded in paraffin, sectioned and stained with haematoxylin and eosin. For the three‐dimensional reconstruction, light micrographs of the serial sections were digitized, and aligned using the serial EM Align software downloaded from http://synapses.bu.edu/tools/ . After alignment, the boundaries of the India‐ink‐filled blood vessels were manually traced with a mouse using the software IGL trace (version 1.26b), also downloaded from the above website. After tracing, a three‐dimensional representation of the blood vessel contours was generated in a VRML format and visualized with the help of the software Cortona Web3D viewer (version 4.0) downloaded from http://www.parallelgraphics.com/products/cortona/ . Our results showed that in regions where ameloblasts are polarized the capillaries are arranged in three distinct levels: (1) penetrating and leaving capillaries in relation to the outer enamel epithelium; (2) capillaries crossing and branching inside the stellate reticulum; and (3) capillaries branching and anastomosing profusely within the stratum intermedium, thereby forming an extensive capillary plexus intimately associated with the cells of the stratum intermedium. The existence of a conspicuous capillary plexus intermingled with cells of the stratum intermedium, as shown in our results, suggests that some molecules produced by cells of the stratum intermedium could be released into the capillary plexus and thereafter carried to the dental follicle.     

The Permian reptile Opisthodontosaurus carrolli: a model for acrodont tooth replacement and dental ontogeny

Continuous tooth replacement is common for tetrapods, but some groups of acrodont lepidosaurs have lost the ability to replace their dentition (monophyodonty). Acrodonty, where the tooth attaches to the apex of the jawbone, is an unusual form of tooth attachment that has been associated with the highly autapomorphic condition of monophyodonty. Beyond Lepidosauria, very little is known about the relationship between acrodonty and monophyodonty in other amniotes. We test for this association with a detailed study of the dentition of Opisthodontosaurus, an unusual Early Permian captorhinid eureptile with acrodont dentition. We provide clear evidence, both histological and morphological, that there were regular tooth replacement events in the lower jaw of Opisthodontosaurus, similar to its captorhinid relatives. Thus, our study of the oldest known amniote with an acrodont dentition shows that acrodonty does not inhibit tooth replacement, and that many of the characteristics assigned to lepidosaurian acrodonty are actually highly derived features of lepidosaurs that have resulted secondarily from a lack of tooth replacement. In the context of reptilian dental evolution, we propose the retention of the simple definition of acrodonty, which only pertains to the relative position of the tooth at the apex of the jaw, where the jaw possesses equal lingual and labial walls. This definition of implantation therefore focuses solely on the spatial relationship between the tooth and the jawbone, and separates this relationship from tooth development and replacement.     

Immunohistochemical examination for the distribution of podoplanin-expressing cells in developing mouse molar tooth germs

We recently reported the expression of podoplanin in the apical bud of adult mouse incisal tooth. This study was aimed to investigate the distribution of podoplanin-expressing cells in mouse tooth germs at several developing stages. At the bud stage podoplanin was expressed in oral mucous epithelia and in a tooth bud. At the cap stage podoplanin was expressed on inner and outer enamel epithelia but not in mesenchymal cells expressing the neural crest stem cell marker nestin. At the early bell stage nestin and podoplanin were expressed in cervical loop and odontoblasts. At the root formation stage both nestin and podoplanin were weakly expressed in odontoblasts generating radicular dentin. Podoplanin expression was also found in the Hertwig epithelial sheath. These results suggest that epithelial cells of developing tooth germ acquire the ability to express nestin, and that tooth germ epithelial cells maintain the ability to express podoplanin in oral mucous epithelia. The expression of podoplanin in odontoblasts was induced as tooth germ development advanced, but was suppressed with the completion of the primary dentin, suggesting that podoplanin may be involved in the cell growth of odontoblasts. Nestin may function as an intermediate filament that binds podoplanin in odontoblasts.     

Loss of teeth and enamel in tetrapods: fossil record, genetic data and morphological adaptations

Since their recruitment in the oral cavity, approximately 450 million years ago, teeth have been subjected to strong selective constraints due to the crucial role that they play in species survival. It is therefore quite surprising that the ability to develop functional teeth has subsequently been lost several times, independently, in various lineages. In this review, we concentrate our attention on tetrapods, the only vertebrate lineage in which several clades lack functional teeth from birth to adulthood. Indeed, in other lineages, teeth can be absent in adults but be functionally present in larvae and juveniles, can be absent in the oral cavity but exist in the pharyngeal region, or can develop on the upper jaw but be absent on the lower jaw. Here, we analyse the current data on toothless (edentate) tetrapod taxa, including information available on enamel-less species. Firstly, we provide an analysis of the dispersed and fragmentary morphological data published on the various living taxa concerned (and their extinct relatives) with the aim of tracing the origin of tooth or enamel loss, i.e. toads in Lissamphibia, turtles and birds in Sauropsida, and baleen whales, pangolins, anteaters, sloths, armadillos and aardvark in Mammalia. Secondly, we present current hypotheses on the genetic basis of tooth loss in the chicken and thirdly, we try to answer the question of how these taxa have survived tooth loss given the crucial importance of this tool. The loss of teeth (or only enamel) in all of these taxa was not lethal because it was always preceded in evolution by the pre-adaptation of a secondary tool (beak, baleens, elongated adhesive tongues or hypselodonty) useful for improving efficiency in food uptake. The positive selection of such secondary tools would have led to relaxed functional constraints on teeth and would have later compensated for the loss of teeth. These hypotheses raise numerous questions that will hopefully be answered in the near future.     

Complex patterns of tooth replacement revealed in the fruit bat (Eidolon helvum)

How teeth are replaced during normal growth and development has long been an important question for comparative and developmental anatomy. Non‐standard model animals have become increasingly popular in this field due to the fact that the canonical model laboratory mammal, the mouse, develops only one generation of teeth (monophyodonty), whereas the majority of mammals possess two generations of teeth (diphyodonty). Here we used the straw‐coloured fruit bat (Eidolon helvum), an Old World megabat, which has two generations of teeth, in order to observe the development and replacement of tooth germs from initiation up to mineralization stages. Our morphological study uses 3D reconstruction of histological sections to uncover differing arrangements of the first and second‐generation tooth germs during the process of tooth replacement. We show that both tooth germ generations develop as part of the dental lamina, with the first generation detaching from the lamina, leaving the free edge to give rise to a second generation. This separation was particularly marked at the third premolar locus, where the primary and replacement teeth become positioned side by side, unconnected by a lamina. The position of the replacement tooth, with respect to the primary tooth, varied within the mouth, with replacements forming posterior to or directly lingual to the primary tooth. Development of replacement teeth was arrested at some tooth positions and this appeared to be linked to the timing of tooth initiation and the subsequent rate of development. This study adds an additional species to the growing body of non‐model species used in the study of tooth replacement, and offers a new insight into the development of the diphyodont condition.     

Megadontia, striae periodicity and patterns of enamel secretion in Plio-Pleistocene fossil hominins

Early hominins formed large and thick-enamelled cheek-teeth within relatively short growth periods as compared with modern humans. To understand better the developmental basis of this process, we measured daily enamel increments, or cross striations, in 17 molars of Plio-Pleistocene hominins representing seven different species, including specimens attributed to early Homo. Our results show considerable variation across species, although all specimens conformed to the known pattern characterised by greater values in outer than inner enamel, and greater cuspal than cervical values. We then compared our results with the megadontia index, which represents tooth size in relation to body mass, for each species to assess the effect of daily growth rates on tooth size. Our results indicate that larger toothed (megadont) taxa display higher rates or faster forming enamel than smaller toothed hominins. By forming enamel quickly, large tooth crowns were able to develop within the constraints of shorter growth periods. Besides daily increments, many animals express long-period markings (striae of Retzius) in their enamel. We report periodicity values (number of cross striations between adjacent striae) in 14 new specimens of Australopithecus afarensis, Paranthropus aethiopicus, Paranthropus boisei, Homo habilis, Homo rudolfensis and Homo erectus, and show that long-period striae express a strong association with male and average male–female body mass. Our results for Plio-Pleistocene hominins show that the biological rhythms that give rise to long-period striae are encompassed within the range of variation known for modern humans, but show a lower mean and modal value of 7 days in australopithecines. In our sample of early Homo, mean and modal periodicity values were 8 days, and therefore similar to modern humans. These new data on daily rates of enamel formation and periodicity provide a better framework to interpret surface manifestations of internal growth markings on fossil hominin tooth crowns. Importantly, our data on early hominin cross striation variation may now contribute towards solving difficult taxonomic diagnoses where much may depend on fragmentary molar remains and enamel structure.     

Mutational spectrum of FAM83H: the C-terminal portion is required for tooth enamel calcification

Dental enamel forms through the concerted activities of specialized extracellular matrix proteins, including amelogenin, enamelin, MMP20, and KLK4. Defects in the genes encoding these proteins cause non-syndromic inherited enamel malformations collectively designated as amelogenesis imperfecta (AI). These genes, however, account for only about a quarter of all AI cases. Recently we identified mutations in FAM83H that caused autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI). Unlike other genes that cause AI, FAM83 H does not encode an extracellular matrix protein. Its location inside the cell is completely unknown, as is its function. We here report novel FAM83H mutations in four kindreds with ADHCAI. All are nonsense mutations in the last exon (c.1243G>T, p.E415X; c.891T>A, p.Y297X; c.1380G>A, p.W460X; and c.2029C>T, p.Q677X). These mutations delete between 503 and 883 amino acids from the C-terminus of a protein normally comprised of 1179 residues. The reason these mutations cause such extreme defects in the enamel layer without affecting other parts of the body is not known yet. However it seems evident that the large C-terminal part of the protein is essential for proper enamel calcification.     

On the scaling relationship between enamel prism length and enamel thickness in primate molars: a comment

As part of a larger study we developed a computer programme which allows the recreation of the complex 3-dimensional arrangement of prisms. Data presented in these earlier publications are re-analyzed to assess the relationship between projected prism length (i.e., enamel thickness) and the true prism length. Across primates, proportional prism deviation increases as the enamel becomes thicker. This supports suggestions that prism decussation may be particularly marked in large-bodied and thick-enameled species. There are differences, however, in scaling relationships between species, which correspond to the species' dietary adaptations. Finally, the findings highlight the importance of employing species-specific correction factors for the calculation of prism length (i.e., life-span of the ameloblast) for life history enquiry.     

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.     

Immunolocalization of enamel matrix protein-like proteins in the tooth enameloid of spotted gar,Lepisosteus oculatus,an actinopterygian bony fish

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.     

Expression survey of genes critical for tooth development in the human embryonic tooth germ.

In the developing murine tooth, the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed. To unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of several regulatory genes, including BMP4, FGF8, MSX1, PAX9, PITX2, and SHOX2, and compared them with that found in mice. All of these genes are known to play critical roles in murine tooth development. Our results show that these genes exhibit basically similar expression patterns in the human tooth germ compared with that in the mouse. However, slightly different expression patterns were also observed for some of the genes at certain stages. For example, MSX1 expression was detected in the inner enamel epithelium in addition to the dental mesenchyme at the bell stage of the human tooth. Moreover, FGF8 expression remained in the dental epithelium at the cap stage, while PAX9 and SHOX2 expression was detected in both dental epithelium and mesenchyme of the human tooth germ. Our results indicate that, although slight differences exist in the gene expression patterns, the human and mouse teeth not only share considerable homology in odontogenesis but also use similar underlying molecular networks.     

R‐spondins/Lgrs expression in tooth development

Background: Tooth development is highly regulated in mammals and it is regulated by networks of signaling pathways (e. g. Tnf, Wnt, Shh, Fgf and Bmp) whose activities are controlled by the balance between ligands, activators, inhibitors and receptors. The members of the R‐spondin family are known as activators of Wnt signaling, and Lgr4, Lgr5, and Lgr6 have been identified as receptors for R‐spondins. The role of R‐spondin/Lgr signaling in tooth development, however, remains unclear. Results: We first carried out comparative in situ hybridization analysis of R‐spondins and Lgrs, and identified their dynamic spatio‐temporal expression in murine odontogenesis. R‐spondin2 expression was found both in tooth germs and the tooth‐less region, the diastema. We further examined tooth development in R‐spondin2 mutant mice, and although molars and incisors exhibited no significant abnormalities, supernumerary teeth were observed in the diastema. Conclusions: R‐spondin/Lgr signaling is thus involved in tooth development. Developmental Dynamics 243:844–851, 2014. © 2014 Wiley Periodicals, Inc.     

Immunocytochemical investigation of neurovascular relationships in human tooth pulp

This study sought to explore the anatomical relationships between peptidergic nerves and blood vessels within human primary and permanent teeth. Extracted primary and permanent molars (n = 120) were split longitudinally, placed in Zamboni's fixative and the coronal pulps were processed for indirect immunofluorescence. Ten-micrometre-thick serial frozen pulp sections were triple-labelled using combinations of the following antisera: (1) protein gene-product 9.5 (PGP 9.5), a general neuronal marker; (2) one of the neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP) or neuropeptide Y (NPY); and (iii) the lectin Ulex europeus, a label for vascular endothelium. The mid-coronal pulp region was examined, using fluorescence microscopy, to determine the proportion of blood vessels showing a positive innervation (recorded when PGP 9.5-labelled nerves appeared to intersect the vessel wall). In addition, the percentage of these vascular-related nerves expressing each of the above neuropeptides was recorded. Overall, 20% of pulpal blood vessels appeared to have a positive innervation. In the main these were thick-walled arterioles. Capillaries, venules and lymphatics were mostly devoid of an associated innervation. Ninety-two per cent of vascular-related nerves expressed CGRP, 87% expressed SP, 15% expressed VIP and 80% expressed NPY. There were no significant differences in overall innervation or peptide-related innervation between primary and permanent teeth (P < 0.05, ANOVA), indicating that pulpal blood flow is likely to be subject to similar neurological control mechanisms in both dentitions.