Outer Enamel Research Articles

The Role of Na:K:2Cl Cotransporter 1 (NKCC1/SLC12A2) in Dental Epithelium during Enamel Formation in Mice.

Na+:K+:2Cl− cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl− transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl−-dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl− ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.

Hey1 and Hey2 are differently expressed during mouse tooth development

The Hey family (also known as Chf, Herp, Hesr, and Hrt) is a set of Hairy/Enhancer of Split-related basic helix-loop-helix type transcription factors. Hey1, Hey2, and HeyL have been identified in mammals. Although Hey proteins are known to regulate cardiovascular development, muscle homeostasis, osteogenesis, neurogenesis, and oncogenesis, their roles in tooth development have been largely obscure. Therefore, this study aimed to clarify detailed spatiotemporal expression patterns of Hey1 and Hey2 in developing molars and incisors of mice by section in situ hybridization. Hey1 and Hey2 were not significantly expressed in tooth germs at epithelial thickening, bud, and cap stages during molar development. In the dental epithelium in molars at the bell stage and incisors, Hey2 transcripts were restricted to the undifferentiated inner enamel epithelium and down-regulated in preameloblasts and ameloblasts. On the other hand, Hey1 was mainly expressed in preameloblasts and down-regulated in differentiated ameloblasts. Both genes were not significantly expressed in other dental epithelial tissues, including the outer enamel epithelium, stellate reticulum, and stratum intermedium cells. In the dental mesenchyme, Hey1 was intensely transcribed in the subodontoblastic layer of the dental pulp in both molars and incisors, whereas Hey2 was barely detectable in mesenchymal components. Our data implied that Hey2 function is restricted to transient amplifying cells of the ameloblast cell lineage and that Hey1 plays a role in the composition of the subodontoblastic layer, in addition to ameloblast differentiation. These findings provide novel clues for the better understanding of tooth development.

Durability of adhesive bonds to tooth structure involving the DEJ.

The importance of the Dentin Enamel Junction (DEJ) to the durability of adhesive bonds to tooth structure is unclear. In fact, no investigation has been reported on contributions of the DEJ to the fatigue resistance of the bonded interface. In this study, the durability of adhesive bonds to tooth structure involving the DEJ was quantified and compared to that of adhesive bonds to enamel only, not including the DEJ. Two different configurations of enamel bonding were considered, including when tensile stress is focused on the outer enamel (occlusal configuration) or the inner decussated enamel (decussated configuration). The resistance to failure for all bonded interfaces was assessed under both static and cyclic loading to failure. Results showed that the durability of the bonded interfaces was primarily a function of their resistance to crack initiation and growth. The bonded interface strength involving the DEJ was significantly (p ≤ 0.05) greater than that of bonds to enamel only with occlusal configuration, under both static and cyclic loading. While the fatigue strength of bonds involving the DEJ was approximately 20% greater than that for enamel bonds with occlusal configuration (7.7MPa) it was lower than that of enamel with the decussated configuration. The DEJ deterred cracks from extending readily into the dentin but it did not prevent fatigue failure. These results suggest that the durability of bonds to enamel are most dependent on the enamel rod decussation and that the DEJ plays a minor role.

The role of β-catenin in tooth development

Background:The present experimental study was conducted on tooth development during embryogenesis in developing mouse. with main objective to identify the expression and action β -Catenin in development of teeth and mophogenesis. Materials and Methods:This study comprised a total of (21) sections were selected during different stages of tooth development (bud, cap and bell stage), (7) sections from each stage in developing mouse. The expression of Beta-Catenin was carried out on 4μm specimen sections using Immuno histochemical staining of β-Catenin antibody. The staining demonstrated the expression intensity.Results:Results shows intense nuclear and cytoplasmic staining in inner enamel epithelium, outer enamel epithelium and in adjacent dental papilla and ecto-mesenchymal cells surrounding the tooth germ, that will give rise to a definite dental sac,while the stellate reticulum, shows only moderate cytoplasmic and not nuclear staining.Conclusion: β-catenin is highly expressed during tooth development stages due to its role in cell signaling transmission between the enamel knot and the surrounding mesenchymal cells to induce tooth formation and its morphogenesis.

Irx1 regulates dental outer enamel epithelial and lung alveolar type II epithelial differentiation

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1-/- mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development.

Localization of RELM-β/FIZZ2 Is Associated with Cementum Formation.

Resistin-like molecule-β/found in inflammatory zone 2 (RELM-β/FIZZ2) is a cysteine-rich secretory protein that is localized in the epithelium of the gastrointestinal tract and lung alveoli. Previous reports have suggested that this protein regulates glucose metabolism and inflammation. In the present study, to analyze the involvement of RELM-β/FIZZ2 in tooth development, we immunohistochemically examined the localization of RELM-β/FIZZ2 in tooth germs of embryonic days (E) 15-20 and postnatal days (P) 7-42 rats. RELM-β/FIZZ2 was hardly detected in the tooth germ at the bud (E15) stage. However, at the cap (E17) and bell (E20) stages, this protein was detectable in the inner enamel epithelium; whereas cells in the other parts of the enamel organ including the outer enamel epithelium and stellate reticulum did not show the immunoreactivity. During the root formation stage (P14-28), cells in Hertwig's epithelial root sheath (HERS) localized RELM-β/FIZZ2. Intense immunoreactivity was also seen in the matrix of the root dentin facing the HERS and the dental follicle. This reactivity was not present on the more upwardly located dentin surface. In contrast, cementum matrix positive for osteopontin and bone sialoprotein was observed on the dentin instead of immunoreactivity for RELM-β/FIZZ2. Osterix-positive cells, indicating cementoblast progenitors, were also detected in the dental follicle near the matrix positive for RELM-β/FIZZ2. These results suggest that RELM-β/FIZZ2 secreted by the inner enamel epithelium was mainly localized in the matrix at the surface of the apical root dentin and might be involved in cementogenesis. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1865-1874, 2017. © 2017 Wiley Periodicals, Inc.

Homology, homoplasy and cusp variability at the enamel-dentine junction of hominoid molars.

Evolutionary studies of mammalian teeth have generally concentrated on the adaptive and functional significance of dental features, whereas the role of development on phenotypic generation and as a source of variation has received comparatively little attention. The present study combines an evolutionary biological framework with state-of-the-art imaging techniques to examine the developmental basis of variation of accessory cusps. Scholars have long used the position and relatedness of cusps to other crown structures as a criterion for differentiating between developmentally homologous and homoplastic features, which can be evaluated with greater accuracy at the enamel-dentine junction (EDJ). Following this approach, we collected digital models of the EDJ and outer enamel surface of more than 1000 hominoid teeth to examine whether cusp 5 of the upper molars (UM C5) and cusps 6 and 7 of the lower molars (LM C6 and LM C7) were associated each with a common developmental origin across species. Results revealed that each of these cusps can develop in a variety of ways, in association with different dental tissues (i.e. oral epithelium, enamel matrix) and dental structures (i.e. from different cusps, crests and cingula). Both within and between species variability in cusp origin was highest in UM C5, followed by LM C7, and finally LM C6. The lack of any species-specific patterns suggests that accessory cusps in hominoids are developmentally homoplastic and that they may not be useful for identifying phylogenetic homology. An important and unanticipated finding of this study was the identification of a new taxonomically informative feature at the EDJ of the upper molars, namely the post-paracone tubercle (PPT). We found that the PPT was nearly ubiquitous in H.neanderthalensis and the small sample of Middle Pleistocene African and European humans (MPAE) examined, differing significantly from the low frequencies observed in all other hominoids, including Pleistocene and recent H.sapiens. We emphasize the utility of the EDJ for human evolutionary studies and demonstrate how features that look similar at the external surface may be the product of different developmental patterns. This study also highlights the importance of incorporating both developmental and morphological data into evolutionary studies in order to gain a better understanding of the evolutionary significance of dental and skeletal features.

Micro-CT and FE-SEM enamel analyses of calcium-based agent application after bleaching.

The objective of the present study is to evaluate the effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on bleached enamel. A bleaching agent (35% hydrogen peroxide) was applied, 4×8min on premolar teeth (n=8). A CPP-ACP paste was applied for 7days. Prior and post-treatment, microtomography images were obtained and 3D regions of interest (ROIs) were selected, from outer enamel, extending to 110.2-μm depth. CT parameters of structure: thickness (St.Th), separation (St.Sp), and fragmentation index (Fr.I.) were calculated for each (ROI). Data was submitted to paired t tests at a 95% confidence level. The samples were evaluated at 3000 to 100,000 magnification. Quantitative analysis of enamel mineral content was also determined by SEM EDX. There was a significant increase in structure thickness and calcium content. The phosphorus content increased after bleaching. There was also a decreased separation and fragmentation index on the outer enamel to a depth of 56.2μm (p<0.05). There were no changes at 110.2-μm depth for the bleaching CPP-ACP association. A covering layer and decreased spaces between the hydroxyapatite crystals appeared around the enamel prisms, 7days after the CPP-ACP application. The application of a CPP-ACP provides a compact structure on the enamel's outer surface, for 7days, due to calcium deposition. CT parameters seem to be a useful tool for mineralizing and remineralizing future studies. CPP-ACP neutralizes any adverse effects on enamel surface when applied during a week after bleaching and minimizes any side effects of the bleaching treatment due to a more compact structure.

Spatial and temporal expression of Sox9 during murine incisor development.

Mouse incisors are capable of continuously growing due to the renewal of dental epithelium stem cells and mesenchymal stem cells residing at the proximal ends. The transcription factor Sox9 plays important roles in maintaining the stem cells of hair follicles, retinal progenitor cells and neural crest stem cells. Whether Sox9 is involved during mouse incisor development is not reported yet. In this study, we examined the expression pattern of Sox9 during mouse incisor development by in situ hybridization and immunohistochemistry. Sox9 mRNA and protein showed similar expression pattern from embryonic day (E) 13.5 to postnatal (PN) day10. At E13.5 and E14.5, Sox9 was strongly expressed in the dental epithelium. At E16.5, Sox9 started to be detected in the mesenchymal cells within the dental pulp, especially the dental pulp cells that adjacent to the labial cervical loop. Similarly with E14.5, Sox9 was strongly detected in the labial cervical loop, including the basal epithelium, the stellate reticulum and the outer enamel epithelium from E16.5 to PN10. The mesenchyme adjacent to the labial cervical loop also showed strong signal of Sox9. The spatiotemporal expression of Sox9 suggested its possible involvement during mouse incisor development.

Spatial distribution of trace element Ca-normalized ratios in primary and permanent human tooth enamel

The trace elements distribution embedded in tooth enamel offers a means to study exposure of toxic metals and allows the reconstruction of dietary behaviors. The quantification of most of the elements with a spatial high resolution (~50μm) is routinely achieved using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). However, the lack of a comprehensive framework of trace elements distribution in enamel jeopardizes any endorsed sampling strategy using LA-ICPMS. The present work is an effort to improve our knowledge on this issue. We studied a suite of 22 sectioned teeth with known dietary history, including 12 3rd molars from 12 living individuals and 10 primary teeth from 3 living individuals. Using LA-ICPMS, we measured Ca, Cu, Zn, Ni, Sr, Ba and Pb variations along 2 or 3 rasters from cervical to occlusal enamel. Calcium concentrations are lower in primary than in permanent teeth and do not vary spatially within a tooth suggesting that enamel matures homogeneously before eruption. The Pb/Ca ratio does not vary within tooth enamel and between primary and permanent tooth enamel. The Cu/Ca and Ni/Ca ratios do not vary within tooth enamel but discriminate primary from permanent tooth enamel. The Zn/Ca ratios are higher in permanent than in primary tooth enamel, and increase up to an order of magnitude in the last hundred of microns at the enamel surface. The Sr/Ca and Ba/Ca ratios are higher in permanent than in primary tooth enamel, and decrease from the enamel-dentine junction towards outer enamel in permanent but not in primary tooth enamel. Considering the Ca-normalized intra-tooth variations of Zn, Sr and Ba, we recommend to perform laser ablation rasters along the enamel-dentine junction because this area is likely to retain most of the original and complete chemical information related to individual's life.

Structure and composition of tooth enamel in quaternary soricines (Mammalia)

One of the main characteristics of the soricines (Eulipotyphla, Mammalia) is the red pigmentation of the outer part of the enamel in the cusps of the teeth. This gives the group its popular name: the red-toothed shrews. Several studies of extant species show that they have a complex enamel structure with three different parts and that the pigmentation is related to the presence of Fe oxides or hydroxides in the outer enamel. We have prepared transverse sections of the incisors of fossil shrews. These belong to the Early Pleistocene shrew Dolinasorex glyphodon and to an indeterminate species of Sorex. FESEM and EDS analyses of the sections show that Fe percentages reach their highest values in the outer part of the intermediate layer of the enamel and in the internal part of the outermost layer. The variations in Fe content are related to the change in direction of the enamel prisms in the intermediate parts of the enamel, instead of being at the surface as previous studies suggested. The location of the Fe seems to be related to the strength of the tooth.

A Fourth KLK4 Mutation Is Associated with Enamel Hypomineralisation and Structural Abnormalities.

“Amelogenesis imperfecta” (AI) describes a group of genetic conditions that result in defects in tooth enamel formation. Mutations in many genes are known to cause AI, including the gene encoding the serine protease, kallikrein related peptidase 4 (KLK4), expressed during the maturation stage of amelogenesis. In this study we report the fourth KLK4 mutation to be identified in autosomal recessively-inherited hypomaturation type AI, c.632delT, p.(L211Rfs*37) (NM_004917.4, NP_004908.4). This homozygous variant was identified in five Pakistani AI families and is predicted to result in a transcript with a premature stop codon that escapes nonsense mediated decay. However, the protein may misfold, as three of six disulphide bonds would be disrupted, and may be degraded or non-functional as a result. Primary teeth were obtained from one affected individual. The enamel phenotype was characterized using high-resolution computerized X-ray tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and microhardness testing (MH). Enamel from the affected individual (referred to as KLK4 enamel) was hypomineralised in comparison with matched control enamel. Furthermore, KLK4 inner enamel was hypomineralised compared with KLK4 outer enamel. SEM showed a clear structural demarcation between KLK4 inner and outer enamel, although enamel structure was similar to control tissue overall. EDX showed that KLK4 inner enamel contained less calcium and phosphorus and more nitrogen than control inner enamel and KLK4 outer enamel. MH testing showed that KLK4 inner enamel was significantly softer than KLK4 outer enamel (p < 0.001). However, the hardness of control inner enamel was not significantly different to that of control outer enamel. Overall, these findings suggest that the KLK4 c.632delT mutation may be a common cause of autosomal recessive AI in the Pakistani population. The phenotype data obtained mirror findings in the Klk4−/− mouse and suggest that KLK4 is required for the hardening and mineralization of the inner enamel layer but is less essential for hardening and mineralization of the outer enamel layer.

Quiescent adult stem cells in murine teeth are regulated by Shh signaling.

The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed inthe enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.

In vivo Experimental Study on the Influence of Sodium Fluoride and Amoxicillin/Clavulanic Acid on the Minerals Composition of Dental Enamel

The aim of this study was to assess the morphological and chemical composition changes induced by the chronic intake of sodium fluoride (NaF) and Amoxicillin in mice enamel. 35 C57BL/6 adult male mice, were randomly divided into a control and 4 treatment groups (n = 7). After acclimatization, the experimental groups were simultaneously treated with 25 ppm (group 2 and 3) and 50ppm (group 4 and 5) of NaF, and 50mg/kg BW (group 2 and 4) and 100 mg/kg BW (group 3 and 5) of Amoxicillin as Amoxicillin/clavulanic acid (AMC). NaF was supplied through drinking water without restricting access, and AMC administered through subcutaneous injection, once per day, for 60 days. After harvesting, lower incisors� enamel was subjected to a scanning electron microscope (SEM) and to an energy dispersive X-ray analysis (EDX). In the treatment groups, SEM and EDX analysis in treatment groups showed an increasing trend of weight percentage (wt%) for C, N, O, F, Na and C/O, F/Fe ratio, and also a decreasing trend of wt% for P, Cl, Ca, Fe and Ca/P ratio. Morphological changes ranged from fissures and short grooves with pits-like appearance, in group 2 sometimes associated with limited demineralized areas looking like irregular scratches, up to demineralized areas extended in the outer enamel, which in group 5 gives the enamel the corroded look. The severity of the morphological changes in the mice enamel varied with the supplied dose of NaF and AMC, and had a uniform pattern in each experimental group. SEM analysis revealed a hypoplasia on the outer enamel and EDX analysis showed a hypomineralisation at the level of the outer enamel.

Structural Analysis of Enamel in Teeth from Head-and-Neck Cancer Patients Who Underwent Radiotherapy

Objective: To analyze macroscopic, microscopic, and ultrastructural aspects of enamel from head-and-neck cancer patients submitted to radiotherapy. Materials and Methods: Twenty sound extracted permanent molars were used and divided into 2 groups. The experimental group consisted of 10 molars from head-and-neck cancer patients submitted to radiotherapy with total doses that ranged from 50 to 70 Gy. Ten molars from patients who did not receive radiotherapy were matched with experimental-group samples by anatomic tooth group and comprised the control group. To perform a macroscopic analysis, standardized photos of different enamel faces were taken with a camera. Teeth were subjected to longitudinal cuts and hand polished to a final thickness of 0.1 mm. Enamel was analyzed under polarized light microscopy, and optical retardation values of birefringence were calculated in cervical, cusp, and occlusal pit areas. Subsequently, the same enamel areas were analyzed by scanning electron microscopy. Data from optical retardation values were statistically analyzed by 2-way ANOVA and Fisher's test (α < 0.05). Results: No macroscopic differences were observed between the irradiated and control groups. Polarized light microscopy analysis revealed that cervical enamel exhibited darker areas characterized by discrete birefringence patterns compared to the control enamel. Optical retardation values were only significantly different in the cervical enamel of the irradiated and control groups (p < 0.0001). Scanning electron microscopy analysis revealed more evident interprismatic spaces in the cervical and outer cusp enamel of irradiated samples. Conclusions: Head-and-neck radiotherapy reduced optical retardation values of birefringence in cervical enamel, and the interprismatic spaces became more evident.

Caries Progression Rate in Primary Teeth: A Retrospective Study.

The rate of caries progression in primary teeth has rarely been studied, with most studies on early childhood caries reporting the percentage of lesions that remain at a certain stage of disease over a period of time. The aim of the present study was to examine the prevalence and behavior of proximal and occlusal carious lesions in primary teeth among children from low socio-economic status. This retrospective study was based on bite-wing radiographs of 95 children aged 5-12 taken at 6-12 months intervals, with a follow-up period of at least three years. One hundred thirty-five teeth and 141 tooth surfaces were examined. The degree of proximal surfaces and occlusal caries advancement was scored and statistical analyses (Mann-Whitney, Kruskal-Wallis and Kaplan-Meier analysis) were performed to evaluate caries progression rate. The results revealed that approximately 0.8 years were required for a carious lesion to progress from the outer enamel to the dentino-enamel junction, and an additional 1.4 years for it to reach the inner part of the dentin. The caries progression rate found in the present study is more rapid than previously found and affects more the lower socio economic population.

Clinical performance of a combined approach for the esthetic management of fluorosed teeth: Three-year results.

The long-term clinical performance of combined therapy including enamel microabrasion and vital bleaching is debatable due to the abrasion of outer enamel surface. The aim of this longitudinal cohort study was to evaluate the 3-year clinical performance of enamel microabrasion in combination with vital tooth bleaching for the esthetic management of fluorosed teeth. Fifteen patients with 176 fluorosed incisors and canines were included in this study. All the teeth were treated with enamel microabrasion (Opalustre, Ultradent), and at-home bleaching technique (10% Opalescence PF, Ultradent). Fluorosed teeth were evaluated at baseline, after microabrasion, after combined therapy, and at 3-year follow-up in terms of esthetic criteria (esthetic appearance, brown stains, and opaque white areas), side effects, and patient satisfaction using visual analog scales. The data were statistically analyzed (α = 0.05). Fluorosed teeth revealed significantly better esthetic appearance after microabrasion and combined therapy and at 3-year follow-up compared to those at baseline (P P Conclusions: The combined therapy was effective in the esthetic management of fluorosed teeth. However, a significant relapse was observed in the esthetic appearance and brown stains of these patients after 3 years, which also reduced patient satisfaction.

Tooth enamel maturation reequilibrates oxygen isotope compositions and supports simple sampling methods

Oxygen isotope and major element zoning patterns of several disparate ungulate teeth were collected to evaluate the timing and geometry of enamel formation, records of isotope zoning, and tooth enamel sampling strategies. Isotopic zoning in mammalian tooth enamel encodes a sub-annual time series of isotopic variation of an animal’s body water composition, with a damping factor that depends on the specifics of how enamel mineralizes. Enamel formation comprises two stages: precipitation of appositional enamel with a high CO3:PO4 ratio, followed by precipitation of maturational enamel with a lower CO3:PO4. If appositional and maturational enamel both contribute to isotope compositions (but with different CO3:PO4), and if isotope compositions vary seasonally, paired δ18O values from CO3 and PO4 profiles should show a spatial separation. CO3 isotope patterns should be shifted earlier seasonally than PO4 isotope patterns. Such paired profiles for new and published data show no resolvable shifts, i.e. CO3 and PO4 δ18O profiles show coincident maxima and minima. This coincidence suggests that enamel maturation reequilibrates appositional isotope compositions. If enamel maturation establishes enamel isotope compositions, the geometry of maturation, not apposition, should be considered when devising sampling protocols. X-ray maps of Ca zoning show that the majority of enamel (inner and middle layers) mineralizes heavily at a high angle to the external tooth surface and the enamel-dentine junction over length scales of 2–4mm, while the outer enamel surface mineralizes more slowly. These data suggest that isotopic sampling strategies should parallel maturational geometry and focus on interior enamel to improve data fidelity. The magnitude of isotopic damping is also smaller than implied in previous studies, so tooth enamel zoning more closely reflects original body water isotopic variations than previously assumed.

Development of the egg tooth – The tool facilitating hatching of squamates: Lessons from the grass snake Natrix natrix

Most embryos of squamates use their egg tooth to facilitate hatching when their development is completed. After they are out of the shell, this tooth is shed and, in the case, of the grass snake (Natrix natrix), not replaced by a successor teeth. The structure of this transient tooth resembles the development and histology of the regular teeth of vertebrates. Morphological, histological and scanning electron microscopic observations indicated that the egg tooth of the grass snake has four developmental phases. Like the teeth of other vertebrate species, it undergoes oral epithelium thickening as well as the bud, cap and bell phases. However, due to the specialised function it performs, the egg tooth differs significantly from the other teeth both in its morphology and development. The egg tooth of Natrix natrix embryos is an unpaired true tooth, as in most squamates. Our study indicated that the egg tooth started its development in the rostral part of the snout by the thickening of the oral epithelium and there was a condensation of mesenchyme underneath it. It formed very early, around developmental stage III, at approximately the same time as the null-generation teeth. After the thickening of the oral epithelium, only one tooth germ is formed, in contrast to lizards in which two germs can be observed during their embryonic life; however, in the course of development, one regressed and the other shifted into the midline position and developed into the functional egg tooth. The next step in the egg tooth development was the differentiation of the enamel organ and the dental papilla. Three layers of the enamel organ developed – the inner enamel epithelium, the stellate reticulum and the outer enamel epithelium, while a superficial layer of the dental papilla differentiated into the odontoblasts. The egg tooth was ready to erupt when its development ended at developmental stage XII, after the hard tissues developed.

Histo-anatomic 3D printing of dental structures.

The creation of dental restorations with natural appearance and biomechanics represents a major challenge for the restorative team. The manufacturing-process of high-aesthetic restorations from tooth-coloured restorative materials is currently dominated by manual manufacturing procedures and the outcome is highly dependent on the knowledge and skills of the performing dental technician. On the other hand, due to the simplicity of the manufacturing process, CAD/CAM restorations from different material classes gain more and more acceptance in the daily routine. Multi-layered restorations show significant aesthetic advantages versus monolithic ones, but are difficult to fabricate using digital technologies. The key element for the successful automated digital fabrication of aesthetic anterior restorations seems to be the form of the individual dentine core as defined by dentine enamel junction (DEJ) covered by a more transparent layer of material imitating the enamel layer to create the outer enamel surface (OES). This article describes the possibilities and technologies available for so-called '4D-printing'. It introduces the digital manufacturing process of multilayered anterior teeth using 3D multipart printing, taking the example of manufacturing replicas of extracted intact natural teeth.