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Developmental complexity stemming from the dynamic interplay between genetic and biomechanic factors canalizes the ways genotypes and phenotypes can change in evolution. As a paradigmatic system, we explore how changes in developmental factors generate typical tooth shape transitions. Since tooth development has mainly been researched in mammals, we contribute to a more general understanding by studying the development of tooth diversity in sharks. To this end, we build a general, but realistic, mathematical model of odontogenesis. We show that it reproduces key shark-specific features of tooth development as well as real tooth shape variation in small-spotted catsharks Scyliorhinus canicula. Wevalidate our model by comparison with experiments in vivo. Strikingly, we observe that developmental transitions between tooth shapes tend to be highly degenerate, even for complex phenotypes. We also discover that the sets of developmental parameters involved in tooth shape transitions tend to depend asymmetrically on the direction of that transition. Together, our findings provide a valuable base for furthering our understanding of how developmental changes can lead to both adaptive phenotypic change and trait convergence in complex, phenotypically highly diverse, structures. [ABSTRACT FROM AUTHOR]

Enamel is formed by the repetitive secretion of a tooth‐specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115‐deficient mice show partial enamel hypomineralization, suggesting that other pH‐responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111‐KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111‐deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast‐specific protease, kallikrein‐related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation. [ABSTRACT FROM AUTHOR]

Background: Dental age (DA) estimation using two convolutional neural networks (CNNs), VGG16 and ResNet101, remains unexplored. In this study, we aimed to investigate the possibility of using artificial intelligence-based methods in an eastern Chinese population. Methods: A total of 9586 orthopantomograms (OPGs) (4054 boys and 5532 girls) of the Chinese Han population aged from 6 to 20 years were collected. DAs were automatically calculated using the two CNN model strategies. Accuracy, recall, precision, and F1 score of the models were used to evaluate VGG16 and ResNet101 for age estimation. An age threshold was also employed to evaluate the two CNN models. Results: The VGG16 network outperformed the ResNet101 network in terms of prediction performance. However, the model effect of VGG16 was less favorable than that in other age ranges in the 15–17 age group. The VGG16 network model prediction results for the younger age groups were acceptable. In the 6-to 8-year-old group, the accuracy of the VGG16 model can reach up to 93.63%, which was higher than the 88.73% accuracy of the ResNet101 network. The age threshold also implies that VGG16 has a smaller age-difference error. Conclusions: This study demonstrated that VGG16 performed better when dealing with DA estimation via OPGs than the ResNet101 network on a wholescale. CNNs such as VGG16 hold great promise for future use in clinical practice and forensic sciences. [ABSTRACT FROM AUTHOR]

The Keratinocyte differentiation factor 1 (KDF1) is reported to take part in tooth formation in humans, but the dental phenotype of Kdf1 mutant mice has not been understood. Additionally, the role of the KDF1 gene in dental hard tissue development is rarely known. In this study, we constructed a Kdf1 missense mutation knock-in mouse model through CRISPR/Cas9 gene-editing technology. Enamel samples from wildtypes (WT) and Kdf1 homozygous mutants (HO) were examined using micro-computed tomography (micro-CT), scanning electron microscopy (SEM), an atomic force microscope (AFM) and Raman microspectroscopy. The results showed that a novel Kdf1 missense mutation (c. 908G>C, p.R303P) knock-in mice model was constructed successfully. The enamel of HO mice incisors appeared chalky and defective, exposing the rough interior of the inner enamel and dentin. Micro-CT showed that HO mice had lower volume and mineral density in their tooth enamel. In addition, declined thickness was found in the unerupted enamel layer of incisors in the HO mice. Using SEM and AFM, it was found that enamel prisms in HO mice enamel were abnormally and variously shaped with loose decussating crystal arrangement, meanwhile the enamel rods were partially fused and collapsed, accompanied by large gaps. Furthermore, misshapen nanofibrous apatites were disorderly combined with each other. Raman microspectroscopy revealed a compromised degree of order within the crystals in the enamel after the Kdf1 mutation. To conclude, we identified enamel structure defects in the Kdf1 missense mutation knock-in mice, which displayed fragmentary appearance, abnormally shaped prism structure, decreased mineral density, altered crystal ordering degree and chemical composition of the enamel layer. This may support the potential role of the KDF1 gene in the natural development of enamel. [ABSTRACT FROM AUTHOR]

Tooth germs undergo a series of dynamic morphologic changes through bud, cap, and bell stages, in which odontogenic epithelium continuously extends into the underlying mesenchyme. During the transition from the bud stage to the cap stage, the base of the bud flattens and then bends into a cap shape whose edges are referred to as "cervical loops." Although genetic mechanisms for cap formation have been well described, little is understood about the morphogenetic mechanisms. Computer modeling and cell trajectory tracking have suggested that the epithelial bending is driven purely by differential cell proliferation and adhesion in different parts of the tooth germ. Here, we show that, unexpectedly, inhibition of cell proliferation did not prevent bud-to-cap morphogenesis. We quantified cell shapes and actin and myosin distributions in different parts of the tooth epithelium at the critical stages and found that these are consistent with basal relaxation in the forming cervical loops and basal constriction around enamel knot at the center of the cap. Inhibition of focal adhesion kinase, which is required for basal constriction in other systems, arrested the molar explant morphogenesis at the bud stage. Together, these results show that the bud-to-cap transition is largely proliferation independent, and we propose that it is driven by classic actomyosin-driven cell shape-dependent mechanisms. We discuss how these results can be reconciled with the previous models and data.

FGF signaling plays a critical role in tooth development, and mutations in modulators of this pathway produce a number of striking phenotypes. However, many aspects of the role of the FGF pathway in regulating the morphological features and the mineral quality of the dentition remain unknown. Here, we used transgenic mice overexpressing the FGF negative feedback regulator Sprouty4 under the epithelial keratin 14 promoter (K14-Spry4) to achieve downregulation of signaling in the epithelium. This led to highly penetrant defects affecting both cusp morphology and the enamel layer. We characterized the phenotype of erupted molars, identified a developmental delay in K14-Spry4 transgenic embryos, and linked this with changes in the tooth developmental sequence. These data further delineate the role of FGF signaling in the development of the dentition and implicate the pathway in the regulation of tooth mineralization. © 2019 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Objective To investigate the dynamic expression profile of mdanoma associated antigen D1 (Mage-D1) at different tooth development stages in mice after birth, and explore the effect of Mage-D1 knockout on tooth development in vivo. Methods C57-BL/6 wild-type suckling mice were grouped according to their age (1, 4, 7, 11 and 15 d after birth, n=3 in each group). Tissue samples were prepared and the developmental dynamics of the first mandibular molars in mice were observed by HE staining. Immunohistochemical staining was used to observe the expression of Mage-D1 as well as the homologous box genes Dlx1 and Msx1 at different stages of postnatal tooth development in mice. The Mage-D1 homozygous knockout mice (KO) and wild-type mice (WT) aged 1 and 3 months (n=6 in each group) were selected for phenotypic observation, and micro-CT scanning for mandible was performed to analyze the data of teeth and mandible. Results Morphological observation showed that on postnatal day 1, the first mandibular molar was in the late bell-shaped stage without obvious hard tissue formation; on day 4, dentin and enamel alternatively formed and crown began to develop; on day 7, epithelial root sheaths shaped and root development initiated; on day 11, the enamel and crown dentin were almost completed, with the root about half developed; on day 15, the root of the tooth further developed, reaching more than 2/3, and the cementum was formed. Mage-D1 and the homeobox genes Dlx1 and Msx1 were positively expressed in both ameloblasts and odontoblasts of mice throughout the period from 1 d to 15 d after birth. As compared with WT mice, the body weight of KO mice was increased (P < 0.05); though the number of teeth was the same in the 2 groups, the teeth appearance in KO group was slightly different, with incisors less stained and molars more easily worn. Scanning results indicated no significant difference in the density of teeth and alveolar bone. Conclusion Mage-D1, Dlx1 and Msx1 may contribute together to the formation of tooth enamel and dentin in mice, and the mice with Mage-D1 gene knockout present obesity signs and altered tooth phenotype.

The development of tooth is a highly complex procedure and mastered by specific genetic programs. Genetic alterations, environmental factors, and developmental timing can disturb the execution of these programs, and result in various dental anomalies like hypodontia/oligodontia, and supernumerary teeth, which are commonly seen in our clinical practice. Advances in molecular research enabled the identification of various genes involved in the pathogenesis of dental anomalies. In the near future, it will help provide a more accurate diagnosis and biological-based treatment for these anomalies. In this article, we present the molecular phenomenon of tooth development and the genetics of various dental anomalies.

Vertebrate skin appendages are incredibly diverse. This diversity, which includes structures such as scales, feathers, and hair, likely evolved from a shared anatomical placode, suggesting broad conservation of the early development of these organs. Some of the earliest known skin appendages are dentine and enamel‐rich tooth‐like structures, collectively known as odontodes. These appendages evolved over 450 million years ago. Elasmobranchs (sharks, skates, and rays) have retained these ancient skin appendages in the form of both dermal denticles (scales) and oral teeth. Despite our knowledge of denticle function in adult sharks, our understanding of their development and morphogenesis is less advanced. Even though denticles in sharks appear structurally similar to oral teeth, there has been limited data directly comparing the molecular development of these distinct elements. Here, we chart the development of denticles in the embryonic small‐spotted catshark (Scyliorhinus canicula) and characterize the expression of conserved genes known to mediate dental development. We find that shark denticle development shares a vast gene expression signature with developing teeth. However, denticles have restricted regenerative potential, as they lack a sox2+ stem cell niche associated with the maintenance of a dental lamina, an essential requirement for continuous tooth replacement. We compare developing denticles to other skin appendages, including both sensory skin appendages and avian feathers. This reveals that denticles are not only tooth‐like in structure, but that they also share an ancient developmental gene set that is likely common to all epidermal appendages. Highlights: Despite the structural similarity between shark denticles and oral teeth, their early development has not been comprehensively compared. We demonstrate that developmental gene expression patterns are broadly conserved between these units. We also describe a key distinction in the expression of sox2, observed during the development of shark teeth, but absent from developing denticles. This is indicative of the role of sox2 in maintaining the continuous replacement of shark teeth—a characteristic that is not exhibited by denticles. [ABSTRACT FROM AUTHOR]

The dental anomaly fused teeth is defined as the union of two or more teeth. Its aetiology is unclear; to date no studies have investigated its genetic background. Therefore, this study, on the basis of a hypothesised genetic component, investigated the genetic background of patients with fused teeth using whole exome sequencing. Fifteen individuals from six families, including members with and without fused teeth, provided saliva samples that were analysed using whole exome sequencing. Patients with other congenital diseases were excluded from this study. Rare variants were extracted from the sequencing data and filtered by family grouping to identify candidate variants. As a result, ERCC6, OBSCN, SLC27A3, and KIF25 were identified as candidate variants. Our sequencing analysis identified four candidate gene variants associated with fused teeth, which now require further investigation. A genetic basis for the anomaly appears likely. This may assist in understanding the aetiology of fused teeth, which in turn supports better oral care and treatment, as well as future regenerative medicine and gene therapy. [ABSTRACT FROM AUTHOR]

Purpose of Review: Genetic studies in humans and animal models have improved our understanding of the role of numerous genes in the etiology of nonsyndromic tooth agenesis (TA). The purpose of this review is to discuss recently identified genes potentially contributing to TA. Recent Findings: Despite research progress, understanding the genetic factors underlying nonsyndromic TA has been challenging given the genetic heterogeneity, variable expressivity, and incomplete penetrance of putatively pathogenic variants often observed associated with the condition. Next-generation sequencing technologies have provided a platform for novel gene and variant discoveries and informed paradigm-shifting concepts in the etiology of TA. Summary: This review summarizes the current knowledge on genes and pathways related to nonsyndromic TA with a focus on recently identified genes/variants. Evidence suggesting possible multi-locus variation in TA is also presented. [ABSTRACT FROM AUTHOR]

This study aimed to reveal the effects of SET domain bifurcated 1 (SETDB1) on epithelial cells during tooth development. We generated conditional knockout mice (Setdb1 fl/fl,Keratin14 -Cre+ mice), in which Setdb1 was deleted only in epithelial cells. At embryonic day 14.5 (E14.5), immunofluorescence staining was performed to confirm the absence of SETDB1 within the epithelium of tooth embryos from Setdb1 fl/fl,Keratin14 -Cre+ mice. Mouse embryos were harvested after reaching embryonic day 13.5 (E13.5), and sections were prepared for histological analysis. To observe tooth morphology in detail, electron microscopy and micro-CT analysis were performed at postnatal months 1 (P1M) and 6 (P6M). Tooth embryos were harvested from postnatal day 7 (P7) mice, and the epithelial components of the tooth embryos were isolated and examined using quantitative RT-PCR for the expression of genes involved in tooth development. Setdb1 fl/fl,Keratin14 -Cre+ mice exhibited enamel hypoplasia, brittle and fragile dentition, and significant abrasion. Coronal sections displayed abnormal ameloblast development, including immature polarization, and a thin enamel layer that detached from the dentinoenamel junction at P7. Electron microscopic analysis revealed characteristic findings such as an uneven surface and the absence of an enamel prism. The expression of Msx2 , Amelogenin (Amelx), Ameloblastin (Ambn), and Enamelin (Enam) was significantly downregulated in the epithelial components of tooth germs in Setdb1 fl/fl,Keratin14 -Cre+ mice. These results indicate that SETDB1 in epithelial cells is important for tooth development and clarify the relationship between the epigenetic regulation of SETDB1 and amelogenesis imperfecta for the first time. • Loss of Setdb1 in epithelial-derived cells induce impaired tooth development. • Loss of Setdb1 in mice exhibited enamel hypoplasia and hypomineralization. • Loss of Setdb1 downregulates the expression of genes important for tooth development. • Setdb1 may be the gene responsible for amelogenesis imperfecta. [ABSTRACT FROM AUTHOR]

Context: Radiographs have an essential role in Chronological Age (CA) estimation and are being used for dental age (DA) determination. Aims: Detecting the validity of Nolla's method (NM) for the age assessment of Kurdish Iraqi children (KIC). Methods and Material: A retrospective study was performed using orthopantomographs (OPGs) of 354 subjects aged from 4 to 13 years (178 boys and 176 girls) and their recording files. Subjects were divided into nine study groups: 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 years old. The chronological age (CA) was subtracted from the DA to find the validity of NM; the positive results indicated the overestimation of age, whereas the negative results indicated for underestimation. The data were recorded through a digitalised system using Microsoft Excel worksheet and analysed by Statistical Package for the Social Sciences (SPSS, version 25) programme using the dependent T-test and graphical analysis. The level of P value used in this study was set at < 0.05. Results: The DA is underestimated in ages 9 to 13 in boys and girls. The highest difference in DA–CA was at the age of 9 years (−0.146 ± 0.162). Conclusions: NM for age estimation was slightly overestimated in age groups of 4, 5, 6, 7, and 8 years in boys and girls without statistically significant differences. However, this method underestimated the ages of KIC ranging from 9 to 13 years significantly.

Ectodysplasin (Eda) plays important roles in both shaping the developing tooth and establishing the number of teeth within the tooth row. Sonic hedgehog (Shh) has been shown to act downstream of Eda and is involved in the initiation of tooth development. Eda−/− mice possess hypoplastic and hypomineralized incisors and show changes in tooth number in the molar region. In the present study we used 3D reconstruction combined with expression analysis, cell lineage tracing experiments, and western blot analysis in order to investigate the formation of the incisor germs in Eda−/− mice. We show that a lack of functional Eda protein during early stages of incisor tooth germ development had minimal impact on development of the early expression of Shh in the incisor, a region proposed to mark formation of a rudimental incisor placode and act as an initiating signalling centre. In contrast, deficiency of Eda protein had a later impact on expression of Shh in the primary enamel knot of the functional tooth. Eda−/− mice had a smaller region where Shh was expressed, and a reduced contribution from Shh descendant cells. The reduction in the enamel knot led to the formation of an abnormal enamel organ creating a hypoplastic functional incisor. Eda therefore appears to influence the spatial formation of the successional signalling centres during odontogenesis.

Abstract Background Recently, the dental age estimation method developed by Cameriere has been widely recognized and accepted. Although machine learning (ML) methods can improve the accuracy of dental age estimation, no machine learning research exists on the use of the Cameriere dental age estimation method, making this research innovative and meaningful. Aim The purpose of this research is to use 7 lower left permanent teeth and three models [random forest (RF), support vector machine (SVM), and linear regression (LR)] based on the Cameriere method to predict children's dental age, and compare with the Cameriere age estimation. Subjects and methods This was a retrospective study that collected and analyzed orthopantomograms of 748 children (356 females and 392 males) aged 5–13 years. Data were randomly divided into training and test datasets in an 80–20% proportion for the ML algorithms. The procedure, starting with randomly creating new training and test datasets, was repeated 20 times. 7 permanent developing teeth on the left mandible (except wisdom teeth) were recorded using the Cameriere method. Then, the traditional Cameriere formula and three models (RF, SVM, and LR) were used to estimate the dental age. The age prediction accuracy was measured by five indicators: the coefficient of determination (R2), mean error (ME), root mean square error (RMSE), mean square error (MSE), and mean absolute error (MAE). Results The research showed that the ML models have better accuracy than the traditional Cameriere formula. The ME, MAE, MSE, and RMSE values of the SVM model (0.004, 0.489, 0.392, and 0.625, respectively) and the RF model (− 0.004, 0.495, 0.389, and 0.623, respectively) were lower with the highest accuracy. In contrast, the ME, MAE, MSE and RMSE of the European Cameriere formula were 0.592, 0.846, 0.755, and 0.869, respectively, and those of the Chinese Cameriere formula were 0.748, 0.812, 0.890 and 0.943, respectively. Conclusions Compared to the Cameriere formula, ML methods based on the Cameriere’s maturation stages were more accurate in estimating dental age. These results support the use of ML algorithms instead of the traditional Cameriere formula.

The dental pulp plays a crucial role in the long-termmaintenance of tooth function. The progress of endodontic treatment and pulp tissue regeneration engineering has made pulp-regeneration therapy promising in clinical practice. However, the mechanisms of pulp regeneration and the role of dental stem cells in development and regeneration have not been fully elucidated. Bridging the gaps between clinical operation and basic research is urgently needed. With the application of single-cell sequencing technology in dental research, the landscapes ofhuman dental pulp cellshave begunbeingoutlined. However, the specific cellular heterogeneity of dental pulp cells, especially that of dental stem cells, at different spatial and temporal levels, is still unclear. In this study, we used single-cell RNA sequencing analysis of pulp samples at four different developmental stages and combined the findings with immunohistochemical staining to explore the developmentof dental pulp andstemcells. The results revealed temporal changes in the proportion of pulp cells during development. For example, mononuclear phagocytes accounted for a higher proportion in early samples. Odontoblasts identified by DMP1 had a higher expression of ion channel-related and neurodevelopment-related genes. Subpopulations were identified in fibroblasts, odontoblasts, and mesenchymal stem cells.We identified a subclass of odontoblasts that expresses DGKI and RRBP1 present in early developmental samples.Apopulation of earliermesenchymal stemcells expressed the SEPTIN gene, which may have greater proliferative and differentiation potential. Furthermore, dental pulp stemcells can differentiate into two directions:mineralization andmyogenesis. In summary, the specific cellular heterogeneity of dental pulp cells was revealed at different spatial and temporal levels. These findings may shed light on the mechanismof tooth development. The gene expression profile of developing pulp cells may help to select cells for regenerative engineering and improve the success of dental pulp regeneration. [ABSTRACT FROM AUTHOR]

Context: Radiographs have an essential role in Chronological Age (CA) estimation and are being used for dental age (DA) determination. Aims: Detecting the validity of Nolla's method (NM) for the age assessment of Kurdish Iraqi children (KIC). Methods and Material: A retrospective study was performed using orthopantomographs (OPGs) of 354 subjects aged from 4 to 13 years (178 boys and 176 girls) and their recording files. Subjects were divided into nine study groups: 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 years old. The chronological age (CA) was subtracted from the DA to find the validity of NM; the positive results indicated the overestimation of age, whereas the negative results indicated for underestimation. The data were recorded through a digitalised system using Microsoft Excel worksheet and analysed by Statistical Package for the Social Sciences (SPSS, version 25) programme using the dependent T-test and graphical analysis. The level of P value used in this study was set at < 0.05. Results: The DA is underestimated in ages 9 to 13 in boys and girls. The highest difference in DA--CA was at the age of 9 years (-0.146 ± 0.162). Conclusions: NM for age estimation was slightly overestimated in age groups of 4, 5, 6, 7, and 8 years in boys and girls without statistically significant differences. However, this method underestimated the ages of KIC ranging from 9 to 13 years significantly. [ABSTRACT FROM AUTHOR]

Objective: Tooth morphogenesis and the formation of hard tissues have been reported to be closely related to circadian rhythms. This study investigates the spatiotemporal expression and relationship of p75NTR with core clock genes, mineralization-related or odontogenesis-related genes, and aims to derive the potential role of p75NTR in regulating circadian rhythm and incrementality growth line formation during tooth development. Materials and methods: The dynamic morphology of the rat dental germ was observed at seven stages (E14.5 d, E16.5 d, E18.5 d, P.N. 4 d, P.N. 7 d, P.N. 10 d, and P.N. 15 d). Next, the expressions of p75NTR and other target factors were traced. The ectomesenchymal stem cells (EMSCs) were isolated from the E18.5d rat dental germs and synchronized using 50% of fetal bovine serum. Then, they were cultured in light/light (L.L.), dark/dark (D.D.), and light/dark (L.D.) conditions for 48 h. The total RNA was collected every 4 h, and the circadian rhythm dynamics of target factors were observed. To reveal the mechanism further, p75NTR was down-regulated in p75NTRExIII−/− mice and up-regulated in immortalized mouse dental apical papilla progenitor cells. The change tendencies of other target factors were also detected. Results: The clock genes Bmal1, Clock, Per1, and Per2 were all expressed in tooth germs before the formation of dental hard tissues and demonstrated a regular oscillating expression pattern in EMSCs from dental germs. Their expression was affected by the L.D. stimulus, and most of them were promoted by D.D. conditions. p75NTR presented a similar expression pattern and a positive or negative relationship with most clock genes, mineralization-related and odontogenesis-related factors, such as brain and muscle ARNT-like protein-1 (Bmal1), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), MSH-like 1 (MSX1), dentin matrix acidic phosphoprotein 1 (Dmp1), and dentin sialophosphoprotein (Dspp). Moreover, the arrangement, morphology, and even boundary in pre-odontoblast/pre-ameloblast layers were disordered in the p75NTRExIII−/− mice. Conclusion: Circadian rhythm was found to affect tooth development. p75NTR might play a crucial role in regulating clock genes in the mineralization and formation of the dental hard tissues. p75NTR is actively involved in the odontoblast-ameloblast junction and cell polarity establishment during tooth morphogenesis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Tooth regeneration is one of the hot issues in dentistry. The development of tooth regeneration is based on the in-depth understanding of the mechanism of tooth germ development. Previous studies have focused on the exploration of intracellular signaling regulatory networks related to tooth development, but studies on extracellular regulatory molecules have been scarce. As an important component of extracellular matrix and stem cell microenvironment, proteoglycans are involved in signal transduction and mediate the balance between of self-renewal and differentiation of stem cells. OBJECTIVE: To review the research progress on proteoglycans in tooth development and its roles in mediating stem cell homeostasis and the mechanisms involved, and to prospect its future research focus. METHODS: PubMed, Web of Science and CNKI databases were searched for the articles concerning proteoglycans in regulating stem cell self-renewal homeostasis and tooth development with the search terms of “proteoglycans, glycosaminoglycans, stem cell homeostasis, tooth development, tooth renewal, cell signaling, heparan sulfate, chondroitin sulfate” in English and Chinese. After primarily screening based on the inclusion and exclusion criteria, the articles with high relevance were included for review. RESULTS AND CONCLUSION: As an important component of extracellular matrix and stem cell microenvironment, proteoglycans are extensively involved in the development of tooth germ and affect the destination of dental stem cells and mesenchymal stem cells. Clarifying the mechanism of proteoglycan in tooth germ development will be beneficial to establishing and perfecting the intracellular and extracellular regulatory signal network related to tooth development and provide a new idea for the study of biological tooth regeneration. [ABSTRACT FROM AUTHOR]

Background: The comparison of the two Willems dental age estimation methods (gender-specific (Willems I) and non-gender-specific (Willems II)) has not been fully investigated. Here we aimed to explore the applicability of the Willems dental age estimation in an Eastern Chinese population, which may cast light on the field of dental age estimation. Methods: A total of 1211 oral panoramic radiographs (582 boys and 629 girls) of the Chinese Han population aged 11–16 years old were collected. Dental ages (DAs) were calculated using the Willems method. Statistical significance was set at a p-value < 0.05. Age differences between chronological age (CA) and dental age were analyzed by paired t-tests and mean absolute error (MAE). Results: The differences between CA and DA determined by the Willems I method were + 0.44 and + 0.09 years for boys and girls, respectively. When using the Willems II method, these differences were + 0.57 and − 0.09. The MAEs of the Willems I method between DA and CA were 0.95 and 1.00 years in boys and girls, respectively. For Willems II, MAEs were 1.02 and 1.00 years in boys and girls. Conclusions: This study showed that the Willems I method was more accurate than the Willems II method in the boys' group for predicting age from a whole scale. In comparison, Willems II is more competitive in the girls' group. Neither method may be satisfactory for 11-to-16-year-old teenagers in Eastern China. [ABSTRACT FROM AUTHOR]

Rodents are characterized by continuously renewing incisors whose growth is fueled by epithelial and mesenchymal stem cells housed in the proximal compartments of the tooth. The epithelial stem cells reside in structures known as the labial (toward the lip) and lingual (toward the tongue) cervical loops (laCL and liCL, respectively). An important feature of the rodent incisor is that enamel, the outer, highly mineralized layer, is asymmetrically distributed, because it is normally generated by the laCL but not the liCL. Here, we show that epithelial-specific deletion of the transcription factor Islet1 (Isl1) is sufficient to drive formation of ectopic enamel by the liCL stem cells, and also that it leads to production of altered enamel on the labial surface. Molecular analyses of developing and adult incisors revealed that epithelial deletion of Isl1 affected multiple, major pathways: Bmp (bone morphogenetic protein), Hh (hedgehog), Fgf (fibroblast growth factor), and Notch signaling were upregulated and associated with liCL-generated ectopic enamel; on the labial side, upregulation of Bmp and Fgf signaling, and downregulation of Shh were associated with premature enamel formation. Transcriptome profiling studies identified a suite of differentially regulated genes in developing Isl1 mutant incisors. Our studies demonstrate that ISL1 plays a central role in proper patterning of stem cell-derived enamel in the incisor and indicate that this factor is an important upstream regulator of signaling pathways during tooth development and renewal. © 2017 American Society for Bone and Mineral Research.

Aim: This study investigates the accuracy of an apex locator in mature and immature teeth with divergent root canals ex vivo., Materials and Methods: Fifty extracted premolar teeth were utilized for the study, with 25 mature teeth (Nolla stage 9 or 10) and 25 immature teeth (Nolla stages ≤ 8). The discrepancies between the actual length (AL) and the electronic length (EL), measured using a Bingo 1020 apex locator, were compared and analyzed. Statistical analysis included Pearson's correlation analysis, a T-test for independent samples, and multiple linear regression. Statistical significance was set at p < 0.05., Results: The difference between AL and EL in immature teeth was significantly higher than in mature premolar teeth. Nonetheless, patient gender or jaw type (upper/lower) did not affect the accuracy of electronic apex locator (EAL) measurements., Conclusion: EAL readings are less accurate in immature teeth than in mature teeth. Supplementary measures, such as radiographic length determination and wet-dry paper point tests, are required to confirm the working length for improved treatment outcomes and patient compliance., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Hadassah Medical Center Ethics Committee issued approval HMO-21-0583. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Kaufman et al.)

Background: Mineral metabolism is critical for proper development of hard tissues of the skeleton and dentition. The dentoalveolar complex includes the following 4 mineralized tissues: enamel, dentin, cementum, and alveolar bone. Developmental processes of these tissues are affected by inherited disorders that disrupt phosphate and pyrophosphate homeostasis, although manifestations are distinct from those in the skeleton., Types of Studies Reviewed: The authors discuss original data from experiments and comparative analyses and review articles describing effects of inherited phosphate and pyrophosphate disorders on dental tissues. A particular emphasis is placed on how new therapeutic approaches for these conditions may affect oral health and dental treatments of affected patients., Results: Disorders of phosphate and pyrophosphate metabolism can lead to reduced mineralization (hypomineralization) or inappropriate (ectopic) calcification of soft tissues. Disruptions in phosphate levels in X-linked hypophosphatemia and hyperphosphatemic familial tumoral calcinosis and disruptions in pyrophosphate levels in hypophosphatasia and generalized arterial calcification of infancy contribute to dental mineralization defects. Traditionally, there have been few options to ameliorate dental health problems arising from these conditions. New antibody and enzyme replacement therapies bring possibilities to improve oral health in affected patients., Practical Implications: Research over the past 2 decades has exponentially expanded the understanding of mineral metabolism, and has led to novel treatments for mineralization disorders. Newly implemented and emerging therapeutic strategies affect the dentoalveolar complex and interact with aspects of oral health care that must be considered for dental treatment, clinical trial design, and coordination of multidisciplinary care teams., Competing Interests: Disclosures None of the authors reported any disclosures., (Copyright © 2024 American Dental Association. Published by Elsevier Inc. All rights reserved.)

Single-cell (sc) omics has become a powerful tool to unravel a tissue’s cell landscape across health and disease. In recent years, sc transcriptomic interrogation has been applied to a variety of tooth tissues of both human and mouse, which has considerably advanced our fundamental understanding of tooth biology. Now, an overarching and integrated bird’s-view of the human and mouse tooth sc transcriptomic landscape would be a powerful multi-faceted tool for dental research, enabling further decipherment of tooth biology and development through constantly progressing state-of-the-art bioinformatic methods as well as the exploration of novel hypothesis-driven research. To this aim, we re-assessed and integrated recently published scRNA-sequencing datasets of different dental tissue types (healthy and diseased) from human and mouse to establish inclusive tooth sc atlases, and applied the consolidated data map to explore its power. For mouse tooth, we identified novel candidate transcriptional regulators of the ameloblast lineage. Regarding human tooth, we provide support for a developmental connection, not advanced before, between specific epithelial compartments. Taken together, we established inclusive mouse and human tooth sc atlases as powerful tools to potentiate innovative research into tooth biology, development and disease. The maps are provided online in an accessible format for interactive exploration.

Background: Dental age estimation is important in clinical practice for orthodontic treatment, forensic sciences and radiology. The Nolla (1960) classification scheme is one of the most frequently utilized estimation tools in teaching and clinical practice. However, the applicability of the Nolla (1960) classification scheme in determining age in a select KwaZulu-Natal population of South Africa is unknown. Therefore, this study aimed to determine the applicability of the Nolla Classification Scheme on a select South African sample of KwaZulu-Natal and develop a regression model to extend the Nolla (1960) maturation and age norm tables beyond 17 years. Materials and methods: 840 digital panoramic radiographs of individuals aged between 5 and 25 years met the inclusion criteria. The 8 maxillary and 8 mandibular teeth in the left quadrant was analysed using the Nolla (1960) Classification Scheme. The paired T-test, Wilcoxon and Polynomial Regression Analysis were performed. Results: The original Nolla (1960) method over-estimated age for the entire sample in this study by −0.39 and −0.57 years in females and males, respectively. While, the modified Nolla (1960), highlighted an overall under-estimation in females, but over-estimation in males aged between 18.00 and 18.99 years. Conclusion: The Nolla Classification scheme over- or under-estimated dental age with the select population group, therefore population-specific tables were developed for the South African Black and Indian population groups of KwaZulu-Natal. This may increase the accuracy of age estimation of the selected South African sample within the KwaZulu-Natal region.

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta (TGF-β) family, are critical for the control of developmental processes such as dorsal-ventral axis formation, somite and tooth formation, skeletal development, and limb formation. Despite Oplegnathus having typical healing beak-like teeth and tooth development showing a trend from discrete to healing, the potential role of BMPs in the development of the beak-like teeth is incompletely understood. In the present study, 19 and 16 BMP genes were found in O. fasciatus and O. punctatus, respectively, and divided into the BMP2/4/16, BMP5/6/7/8, BMP9/10, BMP12/ 13/14, BMP3/15 and BMP11 subfamilies. Similar TGFb and TGF_β gene domains and conserved protein motifs were found in the same subfamily; furthermore, two common tandem repeat genes (BMP9 and BMP3a-1) were identified in both Oplegnathus fasciatus and Oplegnathus punctatus. Selection pressure analysis revealed 13 amino acid sites in the transmembrane region of BMP3, BMP7, and BMP9 proteins of O. fasciatus and O. punctatus, which may be related to the diversity and functional differentiation of genes within the BMP family. The qPCR-based developmental/temporal expression patterns of BMPs showed a trend of high expression at 30 days past hatching (dph), which exactly corresponds to the ossification period of the bones and beak-like teeth in Oplegnathus. Tissue-specific expression was found for the BMP4 gene, which was upregulated in the epithelial and mesenchymal tissues of the beak-like teeth, suggesting that it also plays a regulatory role in the development of the beak-like teeth in O. punctatus. Our investigation not only provides a scientific basis for comprehensively understanding the BMP gene family but also helps screen the key genes responsible for beak-like tooth healing in O. punctatus and sheds light on the developmental regulatory mechanism. [ABSTRACT FROM AUTHOR]

We hypothesized that odontoblasts release exosomes as well as dental pulp cells and focused on the exosome membrane marker CD63. Odontoblasts are well-differentiated mesenchymal cells that produce dentin. Dental pulp, a tissue complex formed with odontoblasts, releases exosomes to epithelial cells and stimulates their differentiation to ameloblasts. However, the localization of CD63 in differentiated odontoblasts is poorly understood. Therefore, herein, we aimed to reveal the expression of CD63 in odontoblasts during tooth development. We first investigated the localization of CD63 in mouse incisors and molars using immunofluorescence. In adult mouse incisors, the anti-CD63 antibody was positive in mature odontoblasts and dental pulp cells but not in pre-odontoblasts along the ameloblasts in the apical bud. Additionally, the anti-CD63 antibody was observed as a vesicular shape in the apical area of odontoblast cytosol and inside Tomes' fibers. The anti-CD63 antibody-positive vesicles were also observed using immunoelectron microscopy. Moreover, during mouse mandibular molar tooth morphogenesis (E16 to postnatal 6 weeks), labeling of anti-CD63 antibody was positive in the odontoblasts at E18. In contrast, the anti-CD63 antibody was positive in the dental pulp after postnatal day 10. Furthermore, anti-CD63 antibody was merged with the multivesicular body marker Rab7 in dental pulp tissues but not with the lysosome marker Lamp1. Finally, we determined the effect of a ceramide-generation inhibitor GW4869 on the mouse organ culture of tooth germ in vitro. After 28 days of GW4869 treatment, both CD63 and Rab7 were negative in Tomes' fibers, but were positive in control odontoblasts. These results suggest that CD63-positive vesicular organelles are important for mouse tooth morphogenesis. [ABSTRACT FROM AUTHOR]

The development of tooth is a highly complex procedure and mastered by specific genetic programs. Genetic alterations, environmental factors, and developmental timing can disturb the execution of these programs, and result in various dental anomalies like hypodontia/oligodontia, and supernumerary teeth, which are commonly seen in our clinical practice. Advances in molecular research enabled the identification of various genes involved in the pathogenesis of dental anomalies. In the near future, it will help provide amore accurate diagnosis and biological-based treatment for these anomalies. In this article, we present the molecular phenomenon of tooth development and the genetics of various dental anomalies. [ABSTRACT FROM AUTHOR]

Human dental follicle cells (DFCs) as periodontal progenitor cells are used for studies and research in regenerative medicine and not only in dentistry. Even if innovative regenerative therapies in medicine are often considered the main research area for dental stem cells, these cells are also very useful in basic research and here, for example, for the elucidation of molecular processes in the differentiation into mineralizing cells. This article summarizes the molecular mechanisms driving osteogenic differentiation of DFCs. The positive feedback loop of bone morphogenetic protein (BMP) 2 and homeobox protein DLX3 and a signaling pathway associated with protein kinase B (AKT) and protein kinase C (PKC) are presented and further insights related to other signaling pathways such as the WNT signaling pathway are explained. Subsequently, some works are presented that have investigated epigenetic modifications and non-coding ncRNAs and their connection with the osteogenic differentiation of DFCs. In addition, studies are presented that have shown the influence of extracellular matrix molecules or fundamental biological processes such as cellular senescence on osteogenic differentiation. The putative role of factors associated with inflammatory processes, such as interleukin 8, in osteogenic differentiation is also briefly discussed. This article summarizes the most important insights into the mechanisms of osteogenic differentiation in DFCs and is intended to be a small help in the direction of new research projects in this area. [ABSTRACT FROM AUTHOR]

The development of dentition begins in the embryonic oral cavity and progresses in the branchial arches and alveolar bone. Continuous cellular and molecular crosstalk occurs during crown formation, after which the tooth germ begins to migrate apically through the alveolar process into the oral cavity. It eventually comes in contact with its antagonist in the contralateral jaw to establish functional occlusion. Any defect in either step can result in delayed tooth development, the spectrum of which varies from a congenitally missing tooth to an impacted tooth (infraocclusion) with an eruption problem, both of which can impair oral function. Congenitally missing teeth or eruption problems may result from genetic mutations. Several different mutations have been identified, each causing a distinct phenotype. Thus, it is imperative that medical providers understand the fundamentals of these genetic principles that govern such dental diseases. In this review, we focus on several diseases, including congenitally missing teeth and tooth eruption problems. We review these diseases with aspect to their association with a particular syndrome, as well as independently in a non-syndromic capacity. We also review previously identified genetic mutations and discuss the possible mechanisms that cause individual phenotypes by analyzing previous investigations. We also discuss future prospects of how genetic diagnosis and precision medicine could impact the clinical environment in the field of dentistry. Present study has been carried out in accordance with The Code of Ethics of the World Medical Association and approved by Institutional Review Board of Osaka University Graduate School of Dentistry. [ABSTRACT FROM AUTHOR]

Background and Objectives: The objective of this study was to evaluate the effects of bisphosphonate (BP) administration on tooth growth, using CT-data of a minipig animal model investigation. Materials and Methods: Tooth growth was evaluated in minipigs, with eight animals receiving weekly zoledronate (ZOL) and three animals serving as the control group. Tooth growth was evaluated at the right 2nd molar (M2) in the maxilla. A computed tomography-based measuring method was applied to evaluate tooth growth in the coronal-apical, buccal-oral and mesial-distal axis. Results: ZOL-administration was found to impact tooth growth in all evaluated measuring axes, with the highest effect observed in the coronal-apical axis. Conclusions: Detrimental effects of BP administration on growing teeth have been reported by a number of investigators. The results of this investigation demonstrate that intravenous ZOL affects the growth of the whole tooth within a short period of administration. With BPs being administered to a growing number of pediatric patients, further studies should be conducted to qualify and quantify the effects of BPs on developing teeth. [ABSTRACT FROM AUTHOR]

Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5′-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type–specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type–specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future. [ABSTRACT FROM AUTHOR]

Background: Sturgeons belong to an early‐branching lineage often used as a proxy of ancestor‐like traits of ray‐finned fishes. However, many features of this lineage, such as the transitory presence and the eventual loss of dentition, exemplify specializations that, in fact, provide important information on lineage‐specific evolutionary dynamics. Results: Here, we introduce a detailed overview of the dentition during the development of the sterlet sturgeon. The dentition is composed of tooth fields at oral, palatal, and anterior pharyngeal regions. Oral fields are single‐rowed, non‐renewed and are shed early. Palatal and pharyngeal fields are multi‐rowed and renewed from the adjacent superficial epithelium without the presence of the successional dental lamina. The early loss of oral fields and subsequent establishment of palatal and pharyngeal fields leads to a translocation of the functional dentition from the front to the rear of the oropharyngeal cavity until the eventual loss of all teeth. Conclusions: Our survey shows the sterlet dentition as a dynamic organ system displaying differential composition at different time points in the lifetime of this fish. These dynamics represent a conspicuous feature of sturgeons, unparalleled among extant vertebrates, and appropriate to scrutinize developmental and evolutionary underpinnings of vertebrate odontogenesis. Key Findings: The dentition is a highly dynamic organ system during the sterlet sturgeon ontogeny.Oral teeth are single‐rowed, non‐renewed and shed early, while palatal and pharyngeal teeth are multi‐rowed, renewed and shed much later.Early loss of oral teeth and persistence of palatal and pharyngeal teeth leads to the anterior‐to‐posterior translocation of the sterlet functional dentition.Developmental dynamics of the dentition reflect the dietary demands of the sterlet ontogenetic stages. [ABSTRACT FROM AUTHOR]

Abstract Background The aims of this study were to create a method for estimation of dental age in Saudi children and adolescents based on the Willems model developed using the Belgian Caucasian (BC) reference data and to compare the ability of the two models to predict age in Saudi children. Methods Development of the seven lower left permanent mandibular teeth was staged in 1146 panoramic radiographs from healthy Saudi children (605 male, 541 female) without missing permanent teeth and without all permanent teeth fully developed (except third molars). The data were used to validate the Willems BC model and to construct a Saudi Arabian-specific (Willems SA) model. The mean error, mean absolute error, and root mean square error obtained from both validations were compared to quantify the variance in errors in the sample. Results The overall mean error for the Willems SA method was 0.023 years (standard deviation, ± 0.55), indicating no systematic underestimation or overestimation of age. For girls, the error using the Willems SA method was significantly lower but still negligible at 0.06 years. A small but statistically significant difference in total mean absolute error (11 days) was found between the Willems BC and Willems SA models when the data were compared independent of sex. The overall mean absolute error for girls was slightly lower for the Willems BC method than for the Willems SA method (1.33 years vs. 1.37 years). Conclusions The difference in ability to predict dental age between the Willems BC and Willems SA methods is very small, indicating that the data from the BC population can be used as a reference in the Saudi population.

An important event in organogenesis is the formation of signaling centers, which are clusters of growth factor-secreting cells. In the case of tooth development, sequentially formed signaling centers known as the initiation knot (IK) and the enamel knot (EK) regulate morphogenesis. However, despite the importance of signaling centers, their origin, as well as the fate of the cells composing them, remain open questions. Here, using lineage tracing of distinct epithelial populations, we found that the EK of the mouse incisor is derived de novo from a group of SRY-box 2 (Sox2)-expressing cells in the posterior half of the tooth germ. Specifically, EK progenitors are located in the posterior ventral basal layer, as demonstrated by DiI labeling of cells. Lineage tracing the formed EK with ShhCreER , which encodes an inducible Cre recombinase under the control of the Sonic hedgehog promoter, at subsequent developmental stages showed that, once formed, some EK cells in the incisor give rise to differentiated cells, whereas in the molar, EK cells give rise to the buccal secondary EK. This work thus establishes the developmental origin as well as the fate of the EK and reveals two strategies for the emergence of serially formed signaling centers: one through de novo establishment and the other by incorporation of progeny from previously formed signaling centers.

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

The developing tooth offers a model for the study of ectodermal appendage organogenesis. The signaling networks that regulate tooth development have been intensively investigated, but how cell biological responses to signaling pathways regulate dental morphogenesis remains an open question. The increasing use of ex vivo imaging techniques has enabled live tracking of cell behaviors over time in high resolution. While recent studies using these techniques have improved our understanding of tooth morphogenesis, important gaps remain that require additional investigation. In addition, some discrepancies have arisen between recent studies, and resolving these will advance our knowledge of tooth development. Developmental Dynamics 246:442-450, 2016. © 2017 Wiley Periodicals, Inc.

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) - dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 10(6)/cm(2)) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation.

The tooth shape of sharks and mice are regulated by a similar signaling center despite their teeth having very different geometries.

Development of tooth shape is regulated by the enamel knot signalling centre, at least in mammals. Fgf signalling regulates differential proliferation between the enamel knot and adjacent dental epithelia during tooth development, leading to formation of the dental cusp. The presence of an enamel knot in non-mammalian vertebrates is debated given differences in signalling. Here, we show the conservation and restriction of fgf3, fgf10, and shh to the sites of future dental cusps in the shark (Scyliorhinus canicula), whilst also highlighting striking differences between the shark and mouse. We reveal shifts in tooth size, shape, and cusp number following small molecule perturbations of canonical Wnt signalling. Resulting tooth phenotypes mirror observed effects in mammals, where canonical Wnt has been implicated as an upstream regulator of enamel knot signalling. In silico modelling of shark dental morphogenesis demonstrates how subtle changes in activatory and inhibitory signals can alter tooth shape, resembling developmental phenotypes and cusp shapes observed following experimental Wnt perturbation. Our results support the functional conservation of an enamel knot-like signalling centre throughout vertebrates and suggest that varied tooth types from sharks to mammals follow a similar developmental bauplan. Lineage-specific differences in signalling are not sufficient in refuting homology of this signalling centre, which is likely older than teeth themselves.

X-linked hypohidrotic ectodermal dysplasia (XLHED), caused by a genetic deficiency of ectodysplasin A1 (EDA1), is a rare developmental disorder of ectodermal derivatives such as hair, sweat glands, and teeth. The absence of sweat glands and perspiration can evoke life-threatening hyperthermia. As molecular genetic findings are not always conclusive, the concentrations of circulating EDA1 may help to distinguish between total and partial EDA1 deficiencies. We previously treated nine male patients with obvious signs of XLHED with a recombinant EDA1 replacement protein, Fc-EDA, either shortly after birth (n = 3) or by prenatal administration in gestational week 26 and beyond (n = 6). Here, we present the long-term follow-up for up to six years. In patients who had received Fc-EDA after birth, neither sweat glands nor sweating ability were detected at the age of 12–60 months. In contrast, prenatal EDA1 replacement resulted in ample sweat gland development and pilocarpine-inducible sweating in all treated subjects, who also attained more permanent teeth than their untreated affected relatives. Normal perspiration has persisted for six years in the two oldest boys treated repeatedly with Fc-EDA in utero. When they had a sauna, adequate thermoregulation was evidenced. Lower sweat production after single prenatal dosing may indicate a dose–response relationship. The absence of circulating EDA1 in five prenatally treated subjects proved that these children would have been unable to perspire if they had been left untreated. The sixth infant was shown to produce an EDA1 molecule that, albeit interacting with its cognate receptor, cannot activate EDA1 signaling. In conclusion, a causal treatment of XLHED before birth is feasible.

Heat shock proteins (HSPs) are a class of molecular chaperones with expression increased in response to heat or other stresses. HSPs regulate cell homeostasis by modulating the folding and maturation of intracellular proteins. Tooth development is a complex process that involves many cell activities. During tooth preparation or trauma, teeth can be damaged. The damaged teeth start their repair process by remineralizing and regenerating tissue. During tooth development and injury repair, different HSPs have different expression patterns and play a special role in odontoblast differentiation and ameloblast secretion by mediating signaling pathways or participating in protein transport. This review explores the expression patterns and potential mechanisms of HSPs, particularly HSP25, HSP60 and HSP70, in tooth development and injury repair.

The Keratinocyte differentiation factor 1 (KDF1) is reported to take part in tooth formation in humans, but the dental phenotype of Kdf1 mutant mice has not been understood. Additionally, the role of the KDF1 gene in dental hard tissue development is rarely known. In this study, we constructed a Kdf1 missense mutation knock-in mouse model through CRISPR/Cas9 gene-editing technology. Enamel samples from wildtypes (WT) and Kdf1 homozygous mutants (HO) were examined using micro-computed tomography (micro-CT), scanning electron microscopy (SEM), an atomic force microscope (AFM) and Raman microspectroscopy. The results showed that a novel Kdf1 missense mutation (c. 908G>C, p.R303P) knock-in mice model was constructed successfully. The enamel of HO mice incisors appeared chalky and defective, exposing the rough interior of the inner enamel and dentin. Micro-CT showed that HO mice had lower volume and mineral density in their tooth enamel. In addition, declined thickness was found in the unerupted enamel layer of incisors in the HO mice. Using SEM and AFM, it was found that enamel prisms in HO mice enamel were abnormally and variously shaped with loose decussating crystal arrangement, meanwhile the enamel rods were partially fused and collapsed, accompanied by large gaps. Furthermore, misshapen nanofibrous apatites were disorderly combined with each other. Raman microspectroscopy revealed a compromised degree of order within the crystals in the enamel after the Kdf1 mutation. To conclude, we identified enamel structure defects in the Kdf1 missense mutation knock-in mice, which displayed fragmentary appearance, abnormally shaped prism structure, decreased mineral density, altered crystal ordering degree and chemical composition of the enamel layer. This may support the potential role of the KDF1 gene in the natural development of enamel.

Simple Summary: In modern humans, congenital absence of one or more permanent teeth has a prevalence of 22.6% when considering the third molars and of 6.4% when not. Its high prevalence, in conjunction with evolutionary findings pinpointing to a steady reduction in teeth number, raises the question whether the congenital absence of teeth in modern humans is an evolutionary trend rather than an anomaly. Previous studies have shown that modern humans with less teeth also have smaller faces; however, the association between teeth number and craniofacial morphology remains unclear. Here, we show that less teeth are associated with a flatter profile and a decreased facial height. These findings support the claim of a broader relationship between number of teeth and overall craniofacial development and have evolutionary implications, since face reduction comprises also an evolutionary trend in humans. One of the most common dental anomalies in humans is the congenital absence of teeth, referred to as tooth agenesis. The association of tooth agenesis to craniofacial morphology has been previously investigated but remains unclear. We investigated this association by applying geometric morphometric methods in a large sample of modern humans. In line with previous studies, we report here that a reduced teeth number is linked to a less convex profile, as well as to a shorter face. The effects were similar for males and females; they increased as the severity of the tooth agenesis increased and remained unaltered by the inclusion of third molars and of allometry in the analysis. Furthermore, in cases with tooth agenesis only in the maxilla, there was no detectable effect in mandibular shape, whereas maxillary shape was affected independently of the location of missing teeth. The robustness of the present sample along with the shape analysis and the statistical approach applied, allowed for thorough testing of various contributing factors regarding the presence but also the magnitude of effects. The present findings suggest a relationship between number of teeth and overall craniofacial development and have evolutionary implications. [ABSTRACT FROM AUTHOR]

Objectives: We set out to investigate whether a hybrid stem‐like p‐EMT phenotype develops during murine molar enamel development in vivo. Setting and Sample Population: Histology specimens incorporating molar tooth buds harvested from mice at post‐natal day 4 (P4) were included in our studies. Materials and Methods: We employed double immunofluorescence staining to analyze the simultaneous expression of the epithelial marker E‐cadherin and the mesenchymal marker N‐cadherin in histology specimens with tooth buds harvested from P4 mice. Moreover, we evaluated the expression of the core master stem cell markers Oct4 and Sox2, as well as the mature ameloblast marker amelogenin. Results: Here we document the co‐expression of E‐cadherin and N‐cadherin in a sub‐population of pre‐ameloblasts in the inner enamel epithelium suggestive of the presence of a hybrid epithelial/mesenchymal phenotype resulting from p‐EMT. Moreover, the core stem cell factors Oct4 and Sox2 colocalized with E‐cadherin expressing pre‐ameloblasts, whereas the mesenchymal marker N‐cadherin was expressed specifically by amelogenin–positive mature secretory ameloblasts. Conclusions: The differentiation of E‐cadherin–positive pre‐ameloblasts towards N‐cadherin–positive mature secretory ameloblasts transition through a previously unidentified epithelial/mesenchymal stage derived through p‐EMT, co‐expressing the master transcription factors Oct4 and Sox2. [ABSTRACT FROM AUTHOR]

FGF8, which is specifically expressed in the dental epithelium prior to the E12.5 bud stage, is a key player during odontogenesis, being responsible for the initiation of tooth development. Here, to investigate the impact of persistent FGF8 signaling on tooth development, we forcibly activated FGF8 signaling in the dental epithelium after the bud stage by generating K14-Cre;R26R-Fg8 mice. We found that a unique type of fused supernumerary incisors is formed, although morphologically resembling the features of type II dens invaginatus in humans. Further analysis revealed that ectopically activated epithelial FGF8 alters the cell fate of the incisor lingual outer enamel epithelium, endowing it with odontogenic potential by the activation of several key tooth genes, including Pitx2, Sox2, Lef-1, p38, and Erk1/2, and induces de novo formation of an extra incisor crown lingually in parallel to the original one, leading to the formation of an extra incisor crown and fused with the original incisor eventually. Meanwhile, the overdosed epithelial FGF8 signaling dramatically downregulates the expression of mesenchymal Bmp4, leading to severely impaired enamel mineralization. Based on the location of the extra incisors, we propose that they are likely to be rescued replacement teeth. Our results further demonstrate the essential role of FGF8 signaling for tooth initiation and the establishment of progenitor cells of dental epithelial stem cells during development. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 8 (FGF8) is known to be a potent stimulator of canonical Wnt/β-catenin activity, an essential factor for tooth development. In this study, we analyzed the effects of co-administration of FGF8 and a CHIR99021 (GSK3β inhibitor) on differentiation of dental mesenchymal cells into odontoblasts. Utilizing Cre-mediated EGFP reporter mice, dentin matrix protein 1 (Dmp1) expression was examined in mouse neonatal molar tooth germs. At birth, expression of Dmp1-EGFP was not found in mesenchymal cells but rather epithelial cells, after which Dmp1-positive cells gradually emerged in the mesenchymal area along with disappearance in the epithelial area. Primary cultured mesenchymal cells from neonatal tooth germ specimens showed loss of Dmp1-EGFP positive signals, whereas addition of Wnt3a or the CHIR99021 significantly regained Dmp1 positivity within approximately 2 weeks. Other odontoblast markers such as dentin sialophosphoprotein (Dspp) could not be clearly detected. Concurrent stimulation of primary cultured mesenchymal cells with the CHIR99021 and FGF8 resulted in significant upregulation of odonto/osteoblast proteins. Furthermore, increased expression levels of runt-related transcription factor 2 (Runx2), osterix, and osteocalcin were also observed. The present findings indicate that coordinated action of canonical Wnt/β-catenin and FGF8 signals is essential for odontoblast differentiation of tooth germs in mice. [ABSTRACT FROM AUTHOR]

The primary cilium is a nonmotile microtubule-based organelle in most vertebrate cell types. The primary cilium plays a critical role in tissue development and homeostasis by sensing and transducing various signaling pathways. Ciliary proteins such as intraflagellar transport (IFT) proteins as well as ciliary motor proteins, kinesin and dynein, comprise a bidirectional intraflagellar transport system needed for cilia formation and function. Mutations in ciliary proteins that lead to loss or dysfunction of primary cilia cause ciliopathies such as Jeune syndrome and Ellis–van Creveld syndrome and cause abnormalities in tooth development. These diseases exhibit severe skeletal and craniofacial dysplasia, highlighting the significance of primary cilia in skeletal development. Cilia are necessary for the propagation of hedgehog, transforming growth factor β, platelet-derived growth factor, and fibroblast growth factor signaling during osteogenesis and chondrogenesis. Ablation of ciliary proteins such as IFT80 or IFT20 blocks cilia formation, which inhibits osteoblast differentiation, osteoblast polarity, and alignment and reduces bone formation. Similarly, cilia facilitate chondrocyte differentiation and production of a cartilage matrix. Cilia also play a key role in mechanosensing and are needed for increased bone formation in response to mechanical forces. [ABSTRACT FROM AUTHOR]