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The revolution in genetics has rapidly increased our knowledge of human and mouse genes that are critical for the formation of dental enamel and helps us understand how enamel evolved. In this graphical review we focus on the roles of 41 genes that are essential for the secretory stage of amelogenesis when characteristic enamel mineral ribbons initiate on dentin and elongate to expand the enamel layer to the future surface of the tooth. Based upon ultrastructural analyses of genetically modified mice, we propose a molecular model explaining how a cell attachment apparatus including collagen 17, α6ß4 and αvß6 integrins, laminin 332, and secreted enamel proteins could attach to individual enamel mineral ribbons and mold their cross-sectional dimensions as they simultaneously elongate and orient them in the direction of the retrograde movement of the ameloblast membrane., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed "shedding", while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel "dahlite" crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dental enamel forms extracellularly as thin ribbons of amorphous calcium phosphate (ACP) that initiate on dentin mineral in close proximity to the ameloblast distal membrane. Secreted proteins are critical for this process. Enam -/- and Ambn -/- mice fail to form enamel. We characterize enamel ribbon formation in wild-type (WT), Amelx -/- and Mmp20 -/- mouse mandibular incisors using focused ion beam scanning electron microscopy (FIB-SEM) in inverted backscatter mode. In Amelx -/- mice, initial enamel mineral ribbons extending from dentin are similar in form to those of WT mice. As early enamel development progresses, the Amelx -/- mineral ribbons develop multiple branches, resembling the staves of a Japanese fan. These striking fan-shaped structures cease growing after attaining ~ 20 µm of enamel thickness (WT is ~ 120 µm). The initial enamel mineral ribbons in Mmp20 -/- mice, like those of the Amelx -/- and WT, extend from the dentin surface to the ameloblast membrane, but appear to be fewer in number and coated on their sides with organic material. Remarkably, Mmp20 -/- mineral ribbons also form fan-like structures that extend to ~ 20 µm from the dentin surface. However, these fans are subsequently capped with a hard, disorganized outer mineral layer. Amelogenin cleavage products are the only matrix components absent in both Amelx -/- and Mmp20 -/- mice. We conclude that MMP20 and amelogenin are not critical for enamel mineral ribbon initiation, orientation, or initial shape. The pathological fan-like plates in these mice may form from the lack of amelogenin cleavage products, which appear necessary to form ordered hydroxyapatite.

Background: Truncation FAM83H mutations cause human autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI), an inherited disorder characterized by severe hardness defects in dental enamel. No enamel defects were observed in Fam83h null mice suggesting that Fam83h truncation mice would better replicate human mutations., Methods: We generated and characterized a mouse model (Fam83h Tr/Tr ) expressing a truncated FAM83H protein (amino acids 1-296), which recapitulated the ADHCAI-causing human FAM83H p.Tyr297* mutation., Results: Day 14 and 7-week Fam83h Tr/Tr molars exhibited rough enamel surfaces and slender cusps resulting from hypoplastic enamel defects. The lateral third of the Fam83h Tr/Tr incisor enamel layer was thinner, with surface roughness and altered enamel rod orientation, suggesting disturbed enamel matrix secretion. Regular electron density in mandibular incisor enamel indicated normal enamel maturation. Only mildly increased posteruption attrition of Fam83h Tr/Tr molar enamel was observed at 7-weeks. Histologically, the Fam83h Tr/Tr enamel organ, including ameloblasts, and enamel matrices at sequential stages of amelogenesis exhibited comparable morphology without overt abnormalities, except irregular and less evident ameloblast Tomes' processes in specific areas., Conclusions: Considering Fam83h -/- mice showed no enamel phenotype, while Fam83h Tr/Tr (p.Tyr297*) mice displayed obvious enamel malformations, we conclude that FAM83H truncation mutations causing ADHCAI in humans disturb amelogenesis through a neomorphic mechanism, rather than haploinsufficiency., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Objective: Enamel structure reflects ameloblast function. By studying the structure of the superficial enamel, information about ameloblast function toward the end of the secretory stage may be obtained., Design: The superficial enamel in midcoronal areas of acid-etched facio-lingual sections from human third molars was studied in the scanning electron microscope (SEM)., Results: A great variation was observed in occurrence of prism-free enamel. Prism-free enamel dominated in 40% (mandibular) and 47% (maxillary) of observed areas and had a mean thickness of about 30μm. Striations in the prism-free enamel had an interstriae distance of about 3.3-3.8μm. The angle between prisms and enamel surface was about 60°, between prisms and Retzius lines about 45° and between Retzius lines and enamel surface about 15°. The distances between regularly occurring Retzius lines and between striations in the prism-free enamel both tended to decrease toward the enamel surface. Prisms could change direction as they approached the enamel surface, mostly in cervical direction. Where Retzius lines curved and converged occlusally, prisms tended to deviate in an occlusal direction., Conclusions: Judged from the incremental lines and occurrence of prism-free enamel, ameloblasts slow down and tend to lose their Tomes' process as they approach the end of secretion. The crystals of prism-free enamel belong to the same system as the interprism crystals of prismatic enamel. A method, based on the disposition of fine incremental lines, is suggested for evaluation of ameloblast dynamics in the last stage of enamel secretion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: High fluoride can induce stress-mediated apoptosis and degradation of ameloblasts. Fas ligand (FasL) has been regarded as a key regulator in intracellular responses for stress-induced apoptosis in reproductive or cancerous cell lineages. The objective of this study is to explore the role of FasL in the regulation of ameloblast ultrastructure damage., Design: Primary ameloblasts were isolated from the molar tooth germ of 4-day-old SD rats. The ameloblasts were incubated with 3.2mM NaF or nothing. After incubation for different time arranging from 12h to 72h, ELISA was used to detected the secretion levels of FasL in the medium. Then at 48h post treatment, the ameloblast ultrastructure was detected with Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), and expression of apoptotic proteins and peroxidative enzymes/products were examined. Finally, a specific FasL inhibitor was applied to co-treat the ameloblasts with NaF, and the ameloblast ultrastructure was detected with TEM and SEM., Results: The secretion of FasL was notably increased by 3.2mM NaF treatment, and the increase reached to the peak after incubation for 48h. High fluoride incubation damaged the ameloblast untrastructure manifesting a series of intracelluar stress responsing cell organelle destruction, and a marked increase in expression of apoptotic genes and oxidative stress. The FasL inhibitor treatment partially mitigated the untrastructure damage caused by high dose NaF., Conlusion: High-fluoride leads to damage of the ameloblast ultrastructure through paritially acitivating the FasL signalling pathway., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To investigate the possible biological mechanism of dental fluorosis at a molecular level., Material and Methods: Cultured LS8 were incubated with serum-free medium containing selected concentrations of NaF (0 ∼ 2 mM) for either 24 or 48 h. Subcellular microanatomy was characterized using TEM; meanwhile, selected biomolecules were analysed using various biochemistry techniques. Transient transfection was used to modulate a molecular pathway for apoptosis., Results: Apoptosis of LS8 was induced by NaF treatment that showed both time and concentration dependency. The activity of caspase-3, -8, -9 was found to be increased with NaF in a dose-dependent manner. Western blot revealed that the protein expression of p-ERK and p-JNK were decreased, while the expression of p-P38 was increased. Inhibition of the p-ERK and p-JNK pathways resulted in a similar decrease for caspase-3., Conclusion: During NaF-induced apoptosis of LS8, p-ERK and p-JNK were closely associated with induction of apoptosis, which might be a mechanism of dental fluorosis.

Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.

ClC-7 is a 2Cl(-) /1H(+) -exchanger expressed at late endosomes and lysosomes, as well as the ruffled border of osteoclasts. ClC-7 deficiencies in mice and humans lead to impaired osteoclast function and therefore osteopetrosis. Failure of tooth eruption is also apparent in ClC-7 mutant animals, and this has been attributed to the osteoclast dysfunction and the subsequent defect in alveolar bone resorptive activity surrounding tooth roots. Ameloblasts also express ClC-7, and this study aims to determine the significance of ClC-7 in enamel formation by examining the dentitions of ClC-7 mutant mice. Micro-CT analysis revealed that the molar teeth of 3-week old ClC-7 mutant mice had no roots, and the incisors were smaller than their age-matched controls. Despite these notable developmental differences, the enamel and dentin densities of the mutant mice were comparable to those of the wild-type littermates. Scanning electron microscopy showed normal enamel crystallite and prismatic organization in the ClC-7 mutant mice, although the enamel was thinner (hypoplastic) than in controls. These results suggested that ClC-7 was not critical to enamel and dentin formation, and the observed tooth defects may be related more to a resulting alveolar bone phenotype. Micro-CT analysis also revealed abnormal features in the calvarial bones of the mutant mice. The cranial sutures in ClC-7 mutant mice remained open compared to the closed sutures seen in the control mice at 3 weeks. These data demonstrate that ClC-7 deficiency impacts the development of the dentition and calvaria, but does not significantly disrupt amelogenesis., (© 2015 Wiley Periodicals, Inc.)

In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes., (© International & American Associations for Dental Research 2015.)

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation., (© International & American Associations for Dental Research 2014.)

Objective: The purpose of the present study was to investigate the occurrence and periodicity of enamel incremental lines in mouse molars in an attempt to draw attention to some key questions about the rhythm in the activity of the secreting ameloblasts during formation of mouse molar enamel., Methods: The mouse molars were ground, etched, and studied using scanning electron microscopy., Results: Lines interpreted as incremental lines generally appeared as grooves of variable distinctness, and were only observed cervically, in the region about 50-250μm from the enamel-cementum junction. The lines were most readily observable in the outer enamel and in the superficial prism-free layer, and were difficult to identify in the deeper parts of enamel, i.e. in the inner enamel with prism decussation. However, in areas where the enamel tended to be hypomineralized the incremental lines were observed as clearly continuous from outer into inner enamel. The incremental lines in mouse molar enamel exhibited an average periodicity of about 4μm, and the distance between the lines decreased towards the enamel surface., Conclusions: We conclude that incremental lines are to some extent visible in mouse molar enamel. Together with data from the literature and theoretical considerations, we suggest that they probably represent a daily rhythm in enamel formation. This study witnesses the layered apposition of mouse molar enamel and supports the theory that circadian clock probably regulates enamel development., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: Excess fluoride intake during tooth development is known to cause dental fluorosis. It has also been suggested that amoxicillin use in early childhood is associated with enamel hypomineralization. The aim was to investigate separate and combined effects of sodium fluoride (NaF) and amoxicillin on enamel formation in vitro., Design: Mandibular molar tooth germs of E18 mouse embryos were cultured for 10 days in a medium containing NaF (10, 12 or 15μM) and/or amoxicillin (0.5, 1, 2 or 3.6mg/mL) or sodium clavulanate (0.07mg/mL) alone or in combination with 0.5mg/mL of amoxicillin. Morphological changes were studied from the whole tooth photographs and histological tissue sections with light microscope., Results: Only with the highest concentrations of NaF or amoxicillin alone the extent of enamel in the first molars measured as the vertical enamel height/crown height ratio was reduced (p<0.01, p<0.001, respectively). At lower concentrations, combination of NaF (12μM) and amoxicillin (2mg/mL) significantly reduced enamel extent compared with the controls (p<0.001). Histologically, the ameloblasts were still columnar but poorly organized and the nascent enamel was often non-homogeneous. Enamel formation was not seen in any second molars exposed to 12μM NaF and 2mg/mL of amoxicillin (or higher concentrations) compared with the presence of enamel in half of the controls (p<0.001)., Conclusions: Amoxicillin and NaF dose dependently affect developing enamel of mouse molars in vitro and the effects are potentiative. The clinical significance of the results remains to be studied., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Rodent incisors exhibit pigmentation on their labial surfaces. Although previous studies have shown that this pigment is composed of iron, the existence of other elements has not been investigated. This study found that the lower incisors of CD61, also known as integrin β3, null mice (CD61(-/-)) lacked pigmentation. Although ameloblasts differentiated and formed enamel normally, no ferric ion accumulation was observed in maturation-stage ameloblasts in CD61(-/-) mice. Surface elements of control and CD61-/- lower incisors were compared by x-ray photoelectron spectroscopy (XPS). XPS analysis detected C, Ca, N, O, and P on the labial surfaces of lower incisors of both mice, whereas Fe was detected only in control samples. No peak of non-ferrous metal or other element was detected in either group. Quantitative RT-PCR analysis of 18 iron-transportation-related genes with mRNA from maturation-stage ameloblasts and ALC, a pre-ameloblastic cell line, was performed. The results suggested that CD61 regulates the expressions of Slc11a2 and Slc40a1, both of which are involved in iron transportation in epithelial tissues. These results suggested that the pigment on the labial surface of mouse incisors is composed of Fe and that both anemia and reduction of iron-transporting proteins may cause the loss of pigmentation in CD61(-/-) mice.

Embryonic dental cells were used to check a series of criteria to be achieved for tooth engineering. Implantation of cultured cell-cell re-associations led to crown morphogenesis, epithelial histogenesis, organ vascularization, and root and periodontium development. The present work aimed to investigate the organization of predentin/dentin, enamel, and cementum which formed and mineralized after implantation. These implants were processed for histology, transmission electron microscopy, x-ray microanalysis, and electron diffraction. After two weeks of implantation, the re-associations showed gradients of differentiating odontoblasts. There were ciliated, polarized, and extended cell processes in predentin/dentin. Ameloblasts became functional. Enamel crystals showed a typical oriented arrangement in the inner and outer enamel. In the developing root, odontoblasts differentiated, cementogenesis occurred, and periodontal ligament fibroblasts interacted with the root surface and newly formed bone. The implantation of cultured dental cell re-associations allows for reproduction of complete functional differentiation at the cell, matrix, and mineral levels.

Nectins are immunoglobulin-like cell adhesion proteins and their interactions recruit various cell-cell junctions. Mutations in human NECTIN-1 cause an ectodermal dysplasia syndrome, but Nectin-1 null mice have only slight defects in teeth, suggesting compensation by other nectin(s). We observed overlapping expression of nectin-3 with nectin-1 and enamel abnormality in the nectin-3 mutant. We, therefore, generated nectin-1;nectin-3 compound mutants. However, all teeth developed and no significant dental abnormalities were observed before birth. At postnatal day 10, the upper molars of compound mutants exhibited conical crown shape and retarded enamel maturation. Nectin-1 was expressed in ameloblasts whereas nectin-3 was expressed in neighboring stratum intermedium cells at this stage. The immunohistochemical localization and electron microscopical observations indicated that the desmosomal junctions between stratum intermedium and ameloblasts were significantly reduced. These results suggest that heterophilic interaction between nectin-1 and nectin-3 recruits desmosomal junctions, and that these are required for proper enamel formation.

Teeth form as appendages of the ectoderm and their morphogenesis is regulated by tissue interactions mediated by networks of conserved signal pathways. Micro-RNA (miRNA) pathway has emerged as important regulator of various aspects of embryonic development, but its function in odontogenesis has not been elucidated. We show that the expression of RNAi pathway effectors is dynamic during tooth morphogenesis and differentiation of dental cells. Based on microarray profiling we selected 8 miRNAs expressed during morphogenesis and 7 miRNAs in the incisor cervical loop containing the stem cell niche. These miRNAs were mainly expressed in the dental epithelium. Conditional deletion of Dicer-1 in the epithelium (Dcr(K14)(-)(/)(-)) resulted in rather mild but significant aberrations in tooth shape and enamel formation. The cusp patterns of the Dcr(K14)(-)(/)(-) molar crowns resembled the patterns of both ancestral muroid rodents and mouse mutants with modulated signal pathways. In the Dcr(K14)(-)(/)(-) incisors, longitudinal grooves formed on the labial surface and these were shown to result from ectopic budding of the progenitor epithelium in the cervical loop. In addition, ameloblast differentiation was impaired and resulted in deficient enamel formation in molars and incisors. To help the identification of candidate target genes of the selected tooth enriched miRNAs, we constructed a new ectodermal organ oriented database, miRTooth. The predicted targets of the selected miRNAs included several components of the main morphogenetic signal pathways regulating tooth development. Based on our findings we suggest that miRNAs modulate tooth morphogenesis largely by fine tuning conserved signaling networks and that miRNAs may have played important roles during tooth evolution., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

The three-dimensional architecture of enamel prisms at early stages of enamel formation and its spatial relationship to the Hunter-Schreger bands were examined in canine tooth germs by light and electron microscopy. In serial semithin sections of demineralized tooth germs tangential to the enamel-dentin junction, a straight row of enamel prisms was depicted along the longitudinal tooth axis at the level of the enamel-dentin junction and then their three-dimensional arrangement was reconstructed using computer software. The spatial arrangement of the groups of enamel rods oriented in specific sideward directions was also reconstructed in deep layers of the enamel. Initially, all enamel prisms were parallel to perpendicular toward the enamel-dentin junction, but at 10µm from the enamel-dentin junction, some small specks, or groups of enamel prisms--tilting to the right or the left--emerged as small islands. In each speck of enamel prism, the inclined prisms were uniformly oriented in a sideward direction and gradually expanded their boundary until merging with the neighboring specks inclined in the same direction. Consequently, at 50µm from the enamel-dentin junction, the group of enamel prisms oriented either to the right or the left formed alternately arranged horizontal belt-like zones, corresponding to the parazone or the diazone of the Hunter-Schreger bands. Reversed images of scanning electron-micrographs of the exposed surfaces of the developing enamel revealed round and bulb-like profiles of Tomes' processes at early amelogenesis and its changes into a characteristic structure combined with flat secretory and enclosing nonsecretory faces that dictated the orientation of corresponding enamel prisms. The results suggest that the groups of enamel prisms oriented in sideward directions first appear as small island-like specks near the enamel-dentin junction, which later merge and form alternating horizontal belt-like zones as a consequence of morphological changes of the Tomes' processes. However, the mechanisms whereby the functional grouping of secretory ameloblasts with similarly oriented Tomes' processes is induced are yet to be determined.

The aim of this study was to examine the intrauterine hypoxia influence on dental hard tissue development. Pregnant rats were exposed in hypoxic environments between day 14 and 19 of pregnancy. The study was performed on 36 newborn albino rats. Analysis of nucleolar organizator parameters were performed in enameloblasts, odontoblasts and saliva gland epitheliocytes. Data obtained demonstrated that intrauterine hypoxia decreased nucleolar organizator quantity in enameloblasts of tooth germ.

Mouse incisor enamel can be divided into four layers: an inner prism-free layer; an inner enamel with prism decussation; outer enamel with parallel prisms; and a superficial prism-free layer. We wanted to study how this complex structural organization is established in the very first enamel formed in wild-type mice and also in Tabby mice where enamel coverage varies considerably. Unworn incisors from young female wild-type and Tabby mice were ground, etched, and analyzed using scanning electron microscopy. In both wild-type and Tabby mice, establishment of the enamel structural characteristics in the initially formed enamel proceeded as follows, going from the incisal tip in an apical direction: (i) a zone with prism-free enamel, (ii) a zone with occasional prisms most often inclined incisally, and (iii) a zone where prism decussation was gradually established in the inner enamel. The distribution of enamel in Tabby mice exhibited considerable variability. The sequence of initial enamel formation in mouse incisors mimics development from a primitive (prism-free) structure to an evolved structure. It is suggested that genes controlling enamel distribution are not associated with genes controlling enamel structure. The control of ameloblast configuration, life span, organization in transverse rows, and movement is important for establishing the characteristic mature pattern of mouse incisor enamel.

Although the nonamelogenin proteins, ameloblastin and enamelin, are both low-abundance and rapidly degrading components of forming enamel, they seem to serve essential developmental functions, as suggested by findings that an enamel layer fails to appear on teeth of mice genetically engineered to produce either a truncated form of ameloblastin (exons 5 and 6 deleted) or no enamelin at all (null). The purpose of this study was to characterize, by direct micro weighing, changes in enamel mineralization occurring on maxillary and mandibular incisors of mice bred for these alterations in nonamelogenin function (Ambn(+/+, +/-5,6, -5,6/-5,6), Enam(+/+, +/- ,-/-)). The results indicated similar changes to enamel-mineralization patterns within the altered genotypes, including significant decreases by as much as 50% in the mineral content of maturing enamel from heterozygous mice and the formation of a thin, crusty, and disorganized mineralized layer, rather than true enamel, on the labial (occlusal) surfaces of incisors and molars along with ectopic calcifications within enamel organ cells in Ambn(-5,6/-5,6) and Enam(-/-) homozygous mice. These findings confirm that both ameloblastin and enamelin are required by ameloblasts to create an enamel layer by appositional growth as well as to assist in achieving its unique high level of mineralization.

The organic matrix of calcified tissues comprises collagenous and/or noncollagenous matrix proteins (NCPs). Identification and precise mapping of these matrix components is essential for determining their function, formulating coherent hypotheses on their mechanism(s) of action, and developing novel therapeutic approaches based on biologics. Fibrillar collagen can be readily identified by its conspicuous structure, however, NCPs, in general, do not individually exhibit characteristic structural features that permit to identify them and morphologically determine their localization. To address this limitation, we have used immunocytochemistry, a form of "biochemistry on section", to correlate composition with structure. For cytochemical characterizations, including immunolabeling, our laboratory has opted for colloidal gold labelings and pioneered their application to calcified tissues because they yield high spatial resolution and are quantitative. Over the years, this approach has been applied to identify and map various NCPs in bone and teeth and, in this review of our work, we will emphasize some selected studies that highlight it application to also achieve functional information.

At a late stage of amelogenesis, a basement-membrane-like (BML) structure appears between mature ameloblasts and the enamel surface. Although this BML structure is known to contain certain basement membrane components, its detailed nature and role were not well defined. As such, this study examined the BML structure using high-resolution electron microscopy combined with immunohistochemical staining. Mandibular rat incisors were processed for the preparation of Epon sections for ultrastructural observations, and frozen sections were used for immunostaining laminin, heparan sulphate proteoglycan (HSPG) and type IV collagen. The BML structure was characterized by the presence of abundant ribbon-like 'double tracks', 4.5-5.0 nm wide; the form known to be taken by HSPG in basal laminae. The main ultrastructural component of basal laminae, known as 'cords', was replaced by fine filaments of type IV collagen. Immunohistochemical staining of the BML structure showed an intense reaction for HSPG, moderate staining for type IV collagen and negligible staining for laminin. These observations indicate that this structure is an atypical basement membrane in which the cord network is replaced by type IV collagen filaments. However, the BML structure was found to be unusually rich in HSPG, similar to kidney glomerular basement membrane. It is likely that this specialized basement membrane mediates firm attachment of mature ameloblasts to the enamel surface, and filters the influx and efflux of materials to and from enamel during maturation.

The development of analytical techniques for the measurement of trace elements in cellular compartments of developing teeth remains an important methodological issue in dental research. Recent advances in third generation synchrotron facilities have provided high brilliance X-ray sources that can be effectively used to study trace element distributions in small spatial regions with low detection limits. The present study describes for the first time the application of synchrotron radiation induced X-ray emission (SRIXE) in measuring the distribution of zinc and lead in the ameloblasts of developing Wistar rat teeth. Wistar rats were fed a standard rat diet, containing the normal dietary requirements of zinc, ad libitum and exposed to 100 ppm of lead in drinking water. Resin embedded sections of first mandibular molars were analysed using a 13.3 keV incident monochromatic X-ray beam focussed to a 0.2 microm spot. Characteristic X-rays arising from the entire thickness of the sample were measured using an energy dispersive detector for quantitative analysis of elemental concentrations. The results showed that intranuclear concentrations of zinc were greater than levels in the cytoplasm. Furthermore, nuclear and cytoplasmic concentrations of zinc in the maturation stage (742+/-27 and 424+/-25 ppm, respectively) were significantly higher than the zinc levels observed in the nucleus and cytoplasm of presecretory stage ameloblasts (132+/-10 and 109+/-10 ppm, respectively) (p<0.05). A clear lead signal above the background was not detected in the ameloblasts and lead concentrations could only be reliably measured in the developing enamel. Overall, SRIXE was an effective method of studying the spatial distribution of zinc in the cells of developing teeth and offered a unique combination of sub-micron spatial resolution and parts-per-million detection limits (0.8-1 and 0.6-1 ppm for zinc and lead, respectively).

High mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that can act as a transcription factor, a growth factor, or a cytokine. To elucidate a possible role for HMGB1 in tooth development, we have studied the expression of HMGB1 and its receptor RAGE (receptor for advanced glycation end-products) during the late fetal and early postnatal period of rat by using light- and electron-microscopic immunohistochemistry. Low HMGB1 protein expression was observed during fetal and newborn stages of tooth development. However, from postnatal day 5 (P5) onward, a marked increase occurred in the levels of the protein in most dental cell types. Expression was particularly high in ameloblasts and odontoblasts at regions of ongoing mineralization. Although most HMGB1 immunoreactivity was confined to cell nuclei, it was also present in odontoblast cytoplasm. At P5, ameloblasts and odontoblasts also showed RAGE immunoreactivity, and reverse transcription-polymerase chain reaction demonstrated both HMGB1 and RAGE mRNA in human dental pulp cells in vitro. Immunoblots performed on extracts from bovine dentin demonstrated a principal band at approximately 27 kDa, indicating that HMGB1 participates in tooth mineralization. The expression of both ligand and receptor suggests an autocrine/paracrine HMGB1 signalling axis in odontoblasts.

Dentin sialophosphpoprotein (Dspp) is transiently expressed in the early stage of secretory ameloblasts. The secretion of ameloblast-derived Dspp is short-lived, correlates to the establishment of the dentinoenamel junction (DEJ), and is consistent with Dspp having a role in producing the specialized first-formed harder enamel adjacent to the DEJ. Crack diffusion by branching and dissipation within this specialized first-formed enamel close to the DEJ prevents catastrophic interfacial damage and tooth failure. Once Dspp is secreted, it is subjected to proteolytic cleavage that results in two distinct proteins referred to as dentin sialoprotein (Dsp) and dentin phosphoprotein (Dpp). The purpose of this study was to investigate the biological and mechanical contribution of Dsp and Dpp to enamel formation. Transgenic mice were engineered to overexpress either Dsp or Dpp in their enamel organs. The mechanical properties (hardness and toughness) of the mature enamel of transgenic mice were compared with genetically matched and age-matched nontransgenic animals. Dsp and Dpp contributions to enamel formation greatly differed. The inclusion of Dsp in bulk enamel significantly and uniformly increased enamel hardness (20%), whereas the inclusion of Dpp weakened the bulk enamel. Thus, Dsp appears to make a unique contribution to the physical properties of the DEJ. Dsp transgenic animals have been engineered with superior enamel mechanical properties.

Since a considerable amount of the world population is exposed to high doses of fluoride, it is of special concern to investigate its action mechanisms during dental enamel development. In this study, the toxicity of fluoride in ameloblasts during enamel development was evaluated by means of ultrastructural morphometric analysis. A total of 18 male Wistar rats were distributed into three groups. In Group I, the animals received deionized drinking water ad libitum (negative control) and in Groups II and III, they received sodium fluorided (NaF) drinking water at doses of 7 and 100 ppm ad libitum, respectively, for 6 weeks. Morphometric data were expressed as volume density of the most significant organelles present in the secretory and maturation phases of amelogenesis such as RER, granules, lysosomes, phagic vacuoles, microfilaments and mitochondria. The results showed that the volume density of mitochondria in the 100 ppm experimental group was 29% (P < 0.05) higher than the control group in secretory ameloblasts. No remarkable differences were found in maturation ameloblasts for all organelles evaluated. Taken together, these data indicate that NaF at high doses is able to induce cellular damage in secretory ameloblasts, whereas no noxious effect was observed during maturation stage of amelogenesis as depicted by ultrastructural analysis.

Confocal laser scanning microscopy (CLSM) represents a recent acquisition in the study of biological samples stained for fluorescence observation. Particularly, this technique allows a bidimensional investigation of tissues and cells with the possibility to elaborate a three-dimensional model. The aim of this study is the use of this technique, as a complementary and not substitutive application of the histological examination, for the morphological and histopathological analysis in a case of mixed complex-composed odontoma. The analyzed specimen has been surgically removed in the superior frontal region in a 12 year-old boy and submitted to conventional histopathological analysis. The specimen, hematoxylin-eosin stained, has been subsequently submitted to confocal laser scanning microscopic analysis in autofluorescence by using a Nikons C1 system. This analysis has underlined not visible aspects in traditional optical microscopy, such as the mineralization of hard tissues and the morpho-structural organization of the cellular component. The presence of enamel and dentin may be observed in the different phases of odontogenesis with clear fluorescence gradients determined by the different mineralization degrees. Thus, the odontogenetic components appear strongly autofluorescent in the classical follicular configuration. Three-dimensional reconstruction is made possible by the acquisition of serial bidimensional images that are subsequently analysed by using a specific software device. This study shows the confocal laser scanning microscopy versatility in the analysis of odontogenic neoplasms with production of mineralized tissues.

Transforming growth factor-beta1 (TGF-beta1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF-beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF-beta1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF-beta1 in teeth impacts the adhesion process of ameloblasts to dentin.

Mutations in both the human amelogenin and human matrix metalloproteinase-20 (MMP20, enamelysin) genes cause amelogenesis imperfecta. Both genes have also been individually deleted from the mouse and each deletion results in defective dental enamel. Here, we compare the stage-specific progression of enamel development in continuously erupting mouse incisors from amelogenin null and MMP-20 null mice. Our goal was to closely examine differences in enamel and enamel organ structure between these mice that would allow a better understanding of each protein's function. The predominant feature of the amelogenin null incisors was the late onset of mineral deposition, with little or no protein present within the forming mineral. Conversely, the developing MMP-20 null incisors had a layer of protein between the apical surface of the ameloblasts and the forming enamel. Furthermore, the protein present within the enamel matrix was disorganized. An analysis of crystal structure demonstrated that the thin amelogenin null enamel was plate-like, while the MMP-20 null enamel had a disrupted prism pattern. These results suggest that amelogenin is essential for appositional crystal growth during the early to mid-secretory stage and for the maintenance of the crystal ribbon structure. They also suggest that MMP-20 is responsible for enamel matrix organization and for subsequent efficient reabsorption of enamel matrix proteins. Both genes are essential for the generation of full-thickness enamel containing the characteristic decussating prism pattern.

It was the purpose of this article to analyze the (micro) morphological structure of enamel at different stages of development in order to deduce movement patterns of ameloblasts during formation of the human dental primordium. Developing enamel and overlying ameloblasts were dried and fractured for scanning electron microscopy (SEM) and sectioned for transmission electron microscopy (TEM). Specimens of human permanent enamel were either fractured and/or ground and etched to visualize the enamel rods. All specimens were viewed by SEM. Moreover, three-dimensional reconstructions were made from serial ground sections of enamel blocks to follow the enamel rods for a longer distance. In addition, the outline of the dentino-enamel junction was analyzed under the SEM after removal (using nitric acid) of the enamel cap, and in serial histological sections. Two basic movements of the inner enamel epithelium can be derived from the micromorphological features: (i) the scalloped dentino-enamel junction may be a consequence of a bulged inner enamel epithelium owing to initial spatial impediment; and (ii) the undulating path of the enamel rods may be a consequence of unequal growth of the cells in the cervical loop.

We have recently identified a protein, RP59, in bone marrow cells and young osteoblasts, in cells involved in bone repair and in young erythroblasts and megakaryocytes. Here, we report immunohistochemical data at the light- and electron-microscope level indicating that RP59 is also present in newly secreted tooth enamel of the rat and in ameloblasts, the formative cells. In enamel matrix, RP59 was located proximal to secretory ameloblasts only, i.e. in newly secreted material. Distal enamel and enamel in association with maturation stage ameloblasts were unlabelled. Secretory ameloblasts contained RP59 in the matrix-proximal region including Tomes' processes, post-secretory ameloblasts in the cell-matrix interface. Western blotting of proteins from tooth germs identified RP59 as a band at 90 kD, co-migrating with RP59 from bone marrow and spleen. Antisera versus a chemically synthesised RP59 peptide and versus a bacteria-synthesised protein fragment reacted in the same manner. In situ hybridisation of tooth tissue revealed RP59 RNA specifically in ameloblasts. The reverse transcription/polymerase chain reaction method identified tooth RNA coding for RP59. Sequence analysis indicated that RP59 RNA from tooth and marrow had the same sequence. An internal sequence motif was found in rat RP59 resembling a signal implicated in secretion of the chicken "engrailed" gene product. The findings indicate that RP59 is a genuine product of ameloblasts and that it is secreted in the course of enamel formation together with other matrix components.

Mineralized tissues are unique in that they use proteins to attract and organize calcium and phosphate ions into a structured mineral phase, thus precise knowledge of the expression and extracellular distribution of matrix proteins is very important to understand their function. Tooth development is regulated by sequential and reciprocal interactions between neural crest-derived mesenchymal cells and the oral environment. However, the precise molecular mechanisms that mediate interactions between epithelium and mesenchymal cells are not clear, although basement membrane (BM) components have been shown to play important roles in these regulatory events. In addition, the extracellular matrix layer, whose main components are laminin, collagen IV, nidogen, and sulfated proteoglycan, and the BM layer are both considered to be involved with cell proliferation and differentiation. During tooth morphogenesis, extracellular matrices are dramatically changed. Further, the BM components, laminin and collagen IV support dental epithelium; however, in the late stage, they begin the processes of enamel matrix secretion and calcification, after which the BM structure between the dental epithelium and mesenchyme disappears. In addition, tooth abnormalities associated with several kinds of human diseases that cause mutations in the extracellular matrix, as well as the molecular mechanisms of the basement membrane and enamel matrix during tooth morphogenesis, are not clearly understood. In our review, we discuss the role of the extracellular matrix, with focus on the BM and enamel matrix during tooth morphogenesis.

Objectives: The aim of this study was to analyze the association between Hertwig's epithelial root sheath (HERS) cells, enamel matrix proteins (EMPs), and cementogenesis., Material and Methods: Porcine teeth were examined at the beginning of root formation by light and transmission electron microscopy. Colloidal gold immunocytochemistry was used to analyze the protein expression of amelogenin and ameloblastin., Results: Before and during disintegration of HERS, its cells displayed the cytologic features of protein synthesis and secretion. While some cells assumed an ameloblast-like phenotype, others extended their territory away from the root surface. A collagenous matrix filled the widening intercellular spaces, and tonofilaments and desmosomes were still present in cells featuring the morphologic characteristics of cementoblasts. Labeling for amelogenin was observed but ameloblastin was not immunodetected. Labeling was associated with organic matrix deposits that were sporadically and randomly distributed both along the root surface and away from it among the dissipated epithelial cells., Conclusions: These findings suggest that HERS' cells occasionally assume a lingering ameloblastic activity at the beginning of root formation in the pig. While the results do not support the hypothesis of a causal relationship between EMPs and cementogenesis, they lend support to the concept of an epithelial origin of cementoblasts.

Western blots analyses and gelatin zymography established the presence of matrix metalloproteinase (MMP)-2 and -9 in the forming zone of rat incisor. Light microscope immunohistochemistry carried out on undemineralized material provided evidence for strong MMP-2 staining in the secretory ameloblasts, odontoblasts, in the enamel organ, and in the pulp. A weaker staining was observed in predentin and in the outer part of the forming enamel. Using MMP-9 antibodies, the staining was generally weak, except for the secretory ameloblasts that were positively stained. Electron microscopic immunohistochemistry of undemineralized sections revealed a close association between gold-antibodies complexes and cytoskeletal microfilaments in the cytosol of secretory ameloblasts and odontoblasts, within the rough endoplasmic reticulum and along the plasma membrane. The striking feature of MMP-2 and -9 electron immunostaining was the particularly high labeling in the mantle dentin. By contrast, staining of tissue inhibitors of metalloproteinases (TIMP-1 and -2) was lowest in this region. We suggest that this uneven distribution may have some functional implications.

Apoptosis of ameloblasts were examined by the TdT-mediated dUTP-biotin nick end-labelling method and electron microscopy 8 h after injection of colchicine. The results showed that extensive apoptosis occurred in ameloblasts of secretion to maturation zones. To determine the possible involvement of stimulators in ameloblast apoptosis, Fas, Fas ligand, tumor-necrosis-factor alpha, and tumor-necrosis-factor receptor 1 were examined utilizing immunohistochemistry and Western blotting analysis. Only Fas was consistently detected in the secretion, transition and maturation ameloblasts and overlying enamel organ epithelia. These results suggest that ameloblasts could undergo apoptosis by colchicine and that one of the ameloblast apoptosis mediators would be the Fas receptor.

Odontogenesis of early larval non-pedicellate teeth, late larval teeth with a more or less distinct dividing zone and fully transformed pedicellate teeth in Ambystoma mexicanum (Urodela) was studied to obtain insights into the development of differently structured teeth in lower vertebrates. Using transmission electron microscopy we investigated five developmental stages: (1) papilla; (2) bell stage (secretion of the matrix begins); (3) primordium (mineralization and activity of ameloblasts starts); (4) replacement tooth (young, old); and (5) established, functional tooth. Development of the differently structured teeth is largely identical in the first three stages. Mineralization takes place in apico-basal direction up to the (prospective) pedicel (early and some late larvae) or up to the zone that divides the late larval and transformed tooth in pedicel and dentine shaft (pedicellate condition). Mineralization starts directly at the collagen and by means of matrix vesicles. First odontoblasts develop small processes that extend to the basal lamina of the inner epithelial layer of the enamel organ. The processes are small and lack organelles in early larval teeth, but become larger, arborescent, and contain some organelles in late larval and transformed teeth. The processes are surrounded by unmineralized matrix (predentine). Odontoblasts at the basis of the teeth, at the pedicel, and in the zone of division do not develop significant cytoplasmic processes that extend into the matrix. Cells of the inner enamel epithelium differentiate to ameloblasts that secrete the enamel. In the early larval tooth they show an extensive basal labyrinth that becomes regressive when the enamel layer is completed. In late larval and transformed teeth, however, a large cavity arises between the basal ruffled border of ameloblasts and their basal lamina. This cavity appears to mediate amelogenesis. A small apical zone in early, but not in late larval teeth directly below the thin enamel layer consists of enameloid and is free of dentine channels.

Phospholipids have been identified in enamel and dentin. Before demineralization, a group of phospholipids extracted by lipid solvents was associated with cell membranes and is therefore closely related to cell growth and intracellular regulations. After demineralization, a second group of phospholipids, associated with the extracellular matrix, was extracted; this group is probably linked to the mineralized phase. Using imidazole-osmium tetroxide fixation of rat incisors, we stained cellular unsaturated fatty acids, so that we could visualize the membrane domains, coated pits, and endocytic inclusions. Filipin, a probe for cholesterol, varied in density along the plasma membrane of secretory ameloblasts, and allowed us to visualize membrane remnants inside the forming enamel. With respect to phospholipids located in the extracellular matrix, the malachite-green-glutaraldehyde (MGA) method or iodoplatinate (IP) reaction retains and visualizes enamel and dentin phospholipids. In predentin, aggregates appearing as granules and filaments, or liposome-like structures, were located in the spaces between collagen fibrils. In dentin, organic envelopes coating the crystals, also named "crystal-ghost" structures, outlined groups of collagen fibrils. Histochemical data provided evidence that phospholipids are co-distributed or interact with proteoglycans. Radioautography after IP reaction established that [3H] choline was detected in dentin as early as 30 min after the intravenous injection of the labeled precursor, before any labeling was seen in odontoblasts and predentin. This suggests that blood-serum-labeled phospholipids pass between odontoblasts, cross the distal permeable junctional complex, and diffuse in dentin prior to any cellular uptake and phospholipid synthesis. Pharmacologically and genetically induced pathology also supports the suggestion that phospholipids play an important role in the formation and mineralization of dental tissues.

The structure of rat incisor enamel is established at the topographically complex interface between secretory ameloblasts and forming enamel. The aim of this study was to gain additional information on this interface by sectioning parallel with the rows and the long axis of Tomes' processes and prisms. Rats were sacrificed and fixed by glutaraldehyde/paraformaldehyde perfusion. After dissection, demineralization and embedding transverse jaw/incisor segments were cut, reembedded, and reoriented. Sections were prepared for and observed in the transmission electron microscopy (TEM). The intraenamel part of Tomes' process was about 18 microns long. The forming prism occupied a longitudinally grooved invagination on its apical aspect. The parts of Tomes' process forming the side walls of the groove were attenuated and showed variation in extent and outline. Prism growth occurred over the whole grooved area. An estimation of Tomes' process secretory area in rat compared with data from humans suggests that there may be a relationship between secretory area and rate of prism formation. Prism crystals were oriented obliquely or parallel to the secretory surface of Tomes' process. At interprism growth sites matrix deposition was irregular and required some redistribution to conform to the pattern of interprism sheets.

Immunohistochemical studies using a polyclonal antibody, raised against the recombinant form of dentin matrix protein 1 (DMP1), show that DMP1 was detected mainly in odontoblasts in cultured mouse embryonic tooth germs. However, in restricted areas, DMP1 staining was also observed in secretory ameloblasts, in the stratum intermedium and stellate reticulum, but only when the odontoblasts located in front of them were unstained. When the embryonic tooth germs were cultured in the presence of inositol hexasulfate, a casein kinase I and II inhibitor, staining of odontoblasts was weak or nil, whereas, in contrast, ameloblasts and enamel organ were strongly immunolabelled, suggesting an enhanced translocation of DMP1 after secretion to the secretory ameloblasts and/or stratum intermedium and stellate reticulum. Moreover, DMP1--was shown to be a good substrate for gelatinase A (MMP-2), but not to gelatinase B (MMP- 9). We hypothesized that DMP1--or the sub-fractions cleaved by the MMP--could behave as diffusible signaling molecule (s) rather than as a true dentin extracellular matrix component.

At the beginning of the maturation stage of amelogenesis, ameloblasts deposit a basal lamina (BL) at the interface between their apical surface and maturing enamel. This structure is rich in glycoconjugates and is proposed to exhibit adhesive and/or filtering functions. To clarify its role, we have applied a recently developed surgical window model to locally administer tunicamycin (TM), an antibiotic that interferes with N-glycosylation, in the rat hemimandible using an osmotic minipump. Male Wistar rats were infused with either TM or saline as a control. Lectin-gold cytochemistry was performed to reveal glycoconjugates in the BL. Immunogold labeling of enamel proteins and albumin was carried out to verify whether depletion of N-linked sugars in the BL affects the content and distribution of endogenous and exogenous proteins in the enamel layer. Under the influence of the drug, the BL became irregular and exhibited alterations in structural organization and composition. The number of Helix pomatia agglutinin binding sites was not significantly affected but their distribution was altered. The labeling density of wheat germ agglutinin over the BL was slightly reduced. Immunoreactivity for enamel proteins showed only a small decrease, but that of albumin, both between ameloblasts and within the enamel layer, increased significantly. No structural alterations were observed in the contralateral incisor and in other sampled tissues and organs. These results demonstrate that it is possible to achieve a localized administration of TM without systemic side effects and lend support to the proposal that the BL represents a specialized structure with filtering functions.(J Histochem Cytochem 49:165-176, 2001)

Enamel prism-packing patterns reflect the past history of ameloblasts, providing information about growth patterns in tooth development. Here, the area and density of enamel prisms on the cuspal surface of molar teeth were measured to examine if the onset and rate of enamel apposition differ according to stage of development and/or cusp type. Scanning electron-microscopic images were taken from the mesiobuccal and distal cusp tips of 30 mandibular first permanent molars at different stages of development recovered from archaeological sites in Israel dating to the past 10000 years. Selected enamel microstructural characters were measured for each cusp. The mean area of prisms on the mesiobuccal (MB) cusp was significantly larger than that of the distal (D) cusp at all stages of development and the differences in prism area between cusps were significant for each stage of development. Prism density was significantly smaller on the MB cusp than the D cusp at all stages of development but no significant differences were found between early and later stages in each cusp. This was interpreted as indicating that enamel formation in the MB cusp was almost complete, even in the earliest tooth germs studied, whereas in the D cusp it was less advanced. The differences between MB and D cusps are proposed to result from asynchrony of enamel formation between the different cusps of molar teeth in recent populations. The method provides a non-destructive approach to the study of growth patterns in teeth and provides baseline data for comparison with fossil teeth.

We examined by immunocytochemistry the localization of the AP-1 family proteins c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, and Fra-2 in rat incisor ameloblasts. Most of the antibodies against AP-1 family proteins, except for c-Fos-specific antibody, labeled ameloblast nuclei. The labeling intensity of the c-Jun, JunD, and Fra-2 antibodies was stronger than that of JunB, FosB, and Fra-1. Antibody reactivities of c-Jun, JunD, and Fra-2 were greatly enhanced during or after the transition zone. Furthermore, c-Jun antibodies labeled maturation ameloblasts in a cyclic pattern, which was correlated with ameloblast modulation. Disruption of ameloblast modulation by colchicine injection resulted in greatly decreased reactivity of the c-Jun antibody in the ameloblast nuclei of the maturation zone. Phospho-specific antibodies to c-Jun labeled ameloblast nuclei only weakly throughout the secretion, transition, and maturation zones. These results suggest that the stage-specific localization of AP-1 in ameloblasts is closely related to tooth enamel formation.

Post-translational modification of enamel proteins is regulated by casein kinases (CK) and results in binding sites for calcium ions that subsequently play a key role during the initial stages of mineralization. Phosphorylation may also influence the secretion and extracellular organization of enamel proteins. Previous studies indicated that inositol hexasulphate inhibited the activity of CK-I and/or CK-II in mouse tooth germs (Torres-Quintana et al., 1998). We hypothesized that inositol hexasulphate would also inhibit the activity of the specific casein kinase(s) identified in secretory ameloblasts, and would prove useful for determination of the extent to which phosphorylation might influence the organization of enamel proteins at early stages of enamel formation. To test this hypothesis, we dissected mandibular first molars from 18-day-old mouse embryos and cultured them for 11 days in the presence of 0-0.1 mM inositol hexasulphate. Ultastructural analysis revealed that the formation of enamel was largely impaired at an inhibitor concentration > or = 0.08 mM. Quantitative radioautographic analysis of [33P]phosphate incorporation indicated that radiolabeled phosphate normally secreted into forming enamel was retained within ameloblasts. In contrast, no significant difference was observed between control and inositol-hexasulphate-treated tooth germs when cultures were labeled with [3H]serine and [3H]proline. SDS-PAGE and Western blot analysis confirmed that while inositol hexasulphate inhibited CK-mediated phosphorylation, it did not significantly alter protein synthesis. We conclude that impairment of phosphorylation leads to intracellular accumulation of [3H]phosphate-containing material by ameloblasts. We also conclude that when non-phosphorylated enamel matrix proteins are secreted, they are either unable to form an enamel matrix that supports mineralization, or they diffuse throughout a poorly mineralized dentin.

The expression of heat shock protein (Hsp) 25 during odontogenesis in the dental pulp and enamel organ of rat incisors was investigated by immunocytochemistry and confocal microscopy. In the process of dentin formation, immature odontoblasts first exhibited Hsp 25-immunoreactivity, and increased in immunointensity with the advance of their differentiation. In the dental pulp, in contrast, intense immunoreaction in the mesenchymal cells became weak or negative in parallel with the progress of cell differentiation. The immunoreaction for Hsp 25 in the enamel organ revealed a characteristic stage-related alteration during amelogenesis. In secretory ameloblasts, the immunoreaction for Hsp 25 was found throughout their cell bodies, intense reactivity being located near the proximal and distal terminal webs. At the maturation stage, ruffle-ended ameloblasts (RA) consistently showed Hsp 25-immunoreactivity throughout the cell bodies, whereas smooth-ended ameloblasts (SA) lacking a ruffled border were weak in immunoreaction at the distal cytoplasm. Other cellular elements of the enamel organ were negative. The subcellular localization of Hsp 25-immunoreactivity in this study appeared essentially identical to that of actin filaments as demonstrated by confocal microscopy using rhodamine-labeled phalloidin. These immunocytochemical data suggest that the Hsp 25 molecule is involved in reinforcement of the cell layer following cell movement during odontogenesis and in the formation and maintenance of the ruffled border of RA.

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.

The internal epithelium of enamel organ and the below enamel surface during growth of the lower incisor, were examinated in ten Wistar rat 12-27 weeks old and weighing between 150/200 gr, by means of immuno histochemical, light and scanning electron microscopy techniques. Our specimens indicate that during the outer enamel secretion the anti-actin positivity goes from distal terminal web to infra nuclear region of cell body. The results of the present study do not support the active movement hypothesis, conversely they support the Warshawsky (1992) hypothesis, i.e. the distal terminal web permits the maintenance and the assembling of ameloblasts during enamel growth. Hence we do agree with Osborn (1970) who reported that, during secretion, ameloblasts move passively in response to secretory forces.

A basement membrane-like structure associated with the maturation stage ameloblasts of the monkey (Macaca fuscata) tooth germ was examined with high resolution electron microscopy. The tissue was prepared either with or without demineralization. This structure was composed of a lamina lucida-like (lamina lucida) and lamina densa-like (lamina densa) structure. The latter was made up of a fine "cord" network, the major constituent of the basement membrane. It was closely associated with the third layer of a 200 nm wide looser cord network. In specimens without demineralization the third layer and a part of the lamina densa were calcified, and it formed the edge of the enamel. This particular area had a higher electron density, and the size, shape, and arrangement of mineral crystals were different from those of the rest of the enamel. Also, mineralization appeared to be proceeding along the cords. These observations indicate that this dense layer is a highly specialized basement membrane which mediates the firm association of maturation stage ameloblasts with the enamel by means of the mineralization of a part of this basement membrane itself which becomes integrated as a part of the enamel. Also, this highly specialized manner of association is favorable with the reported control of the loss of organic substances in the maturing enamel by maturation stage ameloblasts.

Adequate preservation of the cells and matrix of mineralising tissues remains difficult, as organic components and initial mineral deposits may be lost during conventional processing for electron microscopy. In this study, we have reduced significantly the processing time using microwave irradiation. Rat molar tooth germs were fixed in 4% glutaraldehyde + 4% formaldehyde with 0.1 M sodium cacodylate in a laboratory microwave oven for two periods of 20 s with a maximal temperature of 37 degrees C. After conventional washing and post-fixation, specimens were dehydrated in graded ethanols under microwave irradiation for a total of 7 min 20 s. For comparison, some specimens were processed by conventional methods. After embedding, ultrathin sections were examined by electron microscopy. In differentiating ameloblasts and odontoblasts, plasma membranes, mitochondria, rough endoplasmic reticulum, the Golgi complex, together with all other cytoplasmic organelles exhibited excellent preservation. Microtubules, microfilaments and coated vesicles were particularly evident. Crystal-like mineral deposits were conspicuously present in relation to dentine matrix vesicles and collagen fibrils as well as in enamel matrix. The matrix of forming enamel had a globular electron-lucent appearance. It is concluded that this is a rapid method which provides a preserved or even improved morphology.