Advances in the identification of deciduous molar tooth germs.

In forensic anthropology, correct identification of human deciduous teeth is of paramount importance for age-at-death estimation and relies on detailed anatomical descriptions. Yet literature is scarce on indications: details on the morphology of molar tooth germs of fetuses and newborns, developing from multiple mineralized centers that will eventually coalesce, are scant. This paper presents new anatomical elements for practitioners to identify human molar tooth germs at early developmental stages. 126 deciduous molars from 22 modern skeletons of fetuses and newborns (with a known age-at-death ranging between 0 days and 2 months and 21 days postnatal), without reported or observed dental pathological signs, were selected from the Collezione Antropologica LABANOF (CAL) documented skeletal collection. Gross anatomical descriptions of the morphology and configuration of the centers were provided, considering the number of mineralized centers, the shape and the outline of the occlusal plane at different stages. Three different developmental stages were observed in the maxillary first and second molar and the mandibular first molar, whereas in the mandibular second molar four stages were observed. For each stage, we provide additional detailed morphological descriptions, sketches outlining the shape of the tooth germ, and a picture of the tooth; also, indications for siding the teeth are presented. This information can be used by forensic anthropologists and odontologists for a proper identification when tooth germs are not found in anatomical connection within the dental sockets. Further analyses that encompass more age groups on a larger sample would allow to map the entire crown development of deciduous molars.

Regulation of root patterns in mammalian teeth.

Mammalian teeth have diverse pattern of the crown and root. The patterning mechanism of the root position and number is relatively unknown compared to that of the crown. The root number does not always match to the cusp number, which has prevented the complete understanding of root patterning. In the present study, to elucidate the mechanism of root pattern formation, we examined (1) the pattern of cervical tongues, which are tongue-like epithelial processes extending from cervical loops, (2) factors influencing the cervical tongue pattern and (3) the relationship among patterns of cusp, cervical tongue and root in multi-rooted teeth. We found a simple mechanism of cervical tongue formation in which the lateral growth of dental mesenchyme in the cuspal region pushes the cervical loop outward, and the cervical tongue appears in the intercuspal region subsequently. In contrast, when lateral growth was physically inhibited, cervical tongue formation was suppressed. Furthermore, by building simple formulas to predict the maximum number of cervical tongues and roots based on the cusp pattern, we demonstrated a positive relationship among cusp, cervical tongue and root numbers. These results suggest that the cusp pattern and the lateral growth of cusps are important in the regulation of the root pattern.

New regression formula to estimate the prenatal crown formation time of human deciduous central incisors derived from a Roman Imperial sample (Velia, Salerno, Italy, I-II cent. CE).

The characterization and quantification of human dental enamel microstructure, in both permanent and deciduous teeth, allows us to document crucial growth parameters and to identify stressful events, thus contributing to the reconstruction of the past life history of an individual. Most studies to date have focused on the more accessible post-natal portion of the deciduous dental enamel, even though the analysis of prenatal enamel is pivotal in understanding fetal growth, and reveals information about the mother's health status during pregnancy. This contribution reports new data describing the prenatal enamel development of 18 central deciduous incisors from the Imperial Roman necropolis of Velia (I-II century CE, Salerno, Italy). Histomorphometrical analysis was performed to collect data on prenatal crown formation times, daily secretion rates and enamel extension rates. Results for the Velia sample allowed us to derive a new regression formula, using a robust statistical approach, that describes the average rates of deciduous enamel formation. This can now be used as a reference for pre-industrial populations. The same regression formula, even when daily incremental markings are difficult to visualize, may provide a clue to predicting the proportion of infants born full term and pre-term in an archaeological series.

Primary tooth size asymmetry in twins and singletons.

OBJECTIVES: To explore asymmetry values of antimeric deciduous tooth crown dimensions in three types of twins: monozygotic (MZ), dizygotic same-sex (DZ) and opposite-sex (OS) vs. single-born controls. SETTING AND SAMPLE POPULATION: Mesiodistal and labio-lingual crown dimensions of second deciduous molars and mesiodistal canine and first molar crown dimensions of 2159 children at 6-12 years of age were evaluated, originating from the US cross-sectional Collaborative Perinatal Study from the 1970s, including altogether MZ (n = 28), DZ same-sex (n = 33) and OS (n = 39) pairs. Single born (n = 1959) were used as controls. MATERIAL AND METHODS: Dental casts were measured for comparison of variance relationships calculated from antimeric teeth, exhibiting fluctuating (FA), and directional (DA) asymmetry using anova. RESULTS: Significant differences appeared in MZ and OS girls in DA of deciduous canines, which gain size in the first and second trimester, and deciduous second molars, which finally stop crown growth during the early post-natal period. Significantly, increased FA values appeared for lower deciduous canines and second molars, indicating greatest environmental stress in OS girls, MZ girls and DZ boys. Twin girls had more fluctuating and directional crown asymmetry than twin boys, but in some dimensions, the twins were more symmetric than controls. CONCLUSIONS: Transmembrane hormonal influence between opposite-sex twins, and late gestational stress factors, caused by placental malfunction and/or monochorionicity, may be involved in asymmetric growth of antimers, during critical periods of crown size gain.

Suppression of epithelial differentiation by Foxi3 is essential for molar crown patterning.

Epithelial morphogenesis generates the shape of the tooth crown. This is driven by patterned differentiation of cells into enamel knots, root-forming cervical loops and enamel-forming ameloblasts. Enamel knots are signaling centers that define the positions of cusp tips in a tooth by instructing the adjacent epithelium to fold and proliferate. Here, we show that the forkhead-box transcription factor Foxi3 inhibits formation of enamel knots and cervical loops and thus the differentiation of dental epithelium in mice. Conditional deletion of Foxi3 (Foxi3 cKO) led to fusion of molars with abnormally patterned shallow cusps. Foxi3 was expressed in the epithelium, and its expression was reduced in the enamel knots and cervical loops and in ameloblasts. Bmp4, a known inducer of enamel knots and dental epithelial differentiation, downregulated Foxi3 in wild-type teeth. Using genome-wide gene expression profiling, we showed that in Foxi3 cKO there was an early upregulation of differentiation markers, such as p21, Fgf15 and Sfrp5. Different signaling pathway components that are normally restricted to the enamel knots were expanded in the epithelium, and Sostdc1, a marker of the intercuspal epithelium, was missing. These findings indicated that the activator-inhibitor balance regulating cusp patterning was disrupted in Foxi3 cKO. In addition, early molar bud morphogenesis and, in particular, formation of the suprabasal epithelial cell layer were impaired. We identified keratin 10 as a marker of suprabasal epithelial cells in teeth. Our results suggest that Foxi3 maintains dental epithelial cells in an undifferentiated state and thereby regulates multiple stages of tooth morphogenesis.

Development and evolution of dentition pattern and tooth order in the skates and rays (batoidea; chondrichthyes).

Shark and ray (elasmobranch) dentitions are well known for their multiple generations of teeth, with isolated teeth being common in the fossil record. However, how the diverse dentitions characteristic of elasmobranchs form is still poorly understood. Data on the development and maintenance of the dental patterning in this major vertebrate group will allow comparisons to other morphologically diverse taxa, including the bony fishes, in order to identify shared pattern characters for the vertebrate dentition as a whole. Data is especially lacking from the Batoidea (skates and rays), hence our objective is to compile data on embryonic and adult batoid tooth development contributing to ordering of the dentition, from cleared and stained specimens and micro-CT scans, with 3D rendered models. We selected species (adult and embryonic) spanning phylogenetically significant batoid clades, such that our observations may raise questions about relationships within the batoids, particularly with respect to current molecular-based analyses. We include developmental data from embryos of recent model organisms Leucoraja erinacea and Raja clavata to evaluate the earliest establishment of the dentition. Characters of the batoid dentition investigated include alternate addition of teeth as offset successional tooth rows (versus single separate files), presence of a symphyseal initiator region (symphyseal tooth present, or absent, but with two parasymphyseal teeth) and a restriction to tooth addition along each jaw reducing the number of tooth families, relative to addition of successor teeth within each family. Our ultimate aim is to understand the shared characters of the batoids, and whether or not these dental characters are shared more broadly within elasmobranchs, by comparing these to dentitions in shark outgroups. These developmental morphological analyses will provide a solid basis to better understand dental evolution in these important vertebrate groups as well as the general plesiomorphic vertebrate dental condition.

YAP overexpression affects tooth morphogenesis and enamel knot patterning.

Teeth develop through distinct morphological stages. At the cap stage, a compactly clustered and concentrically arranged cell mass, the enamel knot, appears at the tip of the enamel organ. Cells in this knot express sets of key molecules, and as such have been proposed to act as a signaling center directing tooth morphogenesis and tooth cusp formation. YAP is a transcriptional co-activator of the Hippo signaling pathway that is essential for the proper regulation of organ growth. In this study, we analyzed the tooth phenotype in transgenic mice that overexpressed a constitutively active form of YAP in the dental epithelium. We found that overexpression of YAP resulted in deformed tooth morphogenesis with widened dental lamina. In addition, the enamel knot was mislocated to the upper portion of the enamel organ, where it remained devoid of proliferating cells and contained apoptotic cells with intense Edar transcripts and reduced E-cadherin expression. Interestingly, some signaling molecules, such as Shh, Fgf4, and Wnt10a, were not expressed in this mislocated enamel knot, but remained at the tip of the enamel organ. Analysis of these data suggests that the signaling center is induced by reciprocal epithelial-mesenchymal interactions, and its induction may be independent of the enamel knot.

Three-dimensional analysis of molar development in the mouse from the cap to bell stage.

During four days of prenatal development in the mouse, the morphology of the first lower molar moves from the early cap to the bell stage. Five phenomena characterize this period: growth of the tooth germ; development of the cervical loop; histogenesis of the enamel organ; folding of the epithelial-mesenchymal junction associated with cusp formation; and change in cellular heterogeneity in the mesenchyme. All these processes are controlled by epithelial-mesenchymal interactions. These complex histo-morphogenetic events have been documented using histological sections and 3D reconstructions. When combined with functional tests in vitro, this approach allowed searching for possible relationships between simultaneous changes occurring in both the epithelial and ecto-mesenchymal compartments. Parallel changes that occur in the two tissues could result from different mechanisms, as illustrated by the increasing number of pre-odontoblasts and pre-ameloblasts during crown growth. Cell division was involved mainly in the ecto-mesenchyme, while proliferation and cell re-organization occurred in the inner dental epithelium. 3D reconstructions also raised still unsolved questions, such as the possible relationship between cusp size and spatial specification of cell kinetic parameters, changes in cell position within the inner dental epithelium, and tracing cell migration in the mesenchyme during development.

Size and shape variability in human molars during odontogenesis.

Under the patterning cascade model (PCM) of cusp development inspired by developmental genetic studies, it is predicted that the location and the size of later-forming cusps are more variable than those of earlier-forming ones. Here we assessed whether differences in the variability among cusps in total and each particular crown component (enamel-dentin junction [EDJ], outer enamel surface [OES], and cement-enamel junction [CEJ]) could be explained by the PCM, using human maxillary permanent first molars (UM1) and second deciduous molars (um2). Specimens were µCT-scanned, and 3D models of EDJ and OES were reconstructed. Based on these models, landmark-based 3D geometric morphometric analyses were conducted. Size variability in both tooth types was generally consistent with the above prediction, and the differences in size variation among cusps were smaller for the crown components completed in later stages of odontogenesis. With a few exceptions, however, the prediction was unsupported regarding shape variability, and UM1 and um2 showed different patterns. Our findings suggested that the pattern of size variability would be caused by temporal factors such as the order of cusp initiation and the duration from the beginning of mineralization to the completion of crown formation, whereas shape variability may be affected by both topographic and temporal factors.

Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.

BACKGROUND AND OBJECTIVE: Some clinical cases of hypoplastic tooth root are congenital. Because the formation of Hertwig's epithelial root sheath (HERS) is an important event for root development and growth, we have considered that understanding the HERS developmental mechanism contributes to elucidate the causal factors of the disease. To find integrant factors and phenomenon for HERS development and growth, we studied the proliferation and mobility of the cervical loop (CL). MATERIAL AND METHODS: We observed the cell movement of CL by the DiI labeling and organ culture system. To examine cell proliferation, we carried out immunostaining of CL and HERS using anti-Ki67 antibody. Cell motility in CL was observed by tooth germ slice organ culture using green fluorescent protein mouse. We also examined the expression of paxillin associated with cell movement. RESULTS: Imaging using DiI labeling showed that, at the apex of CL, the epithelium elongated in tandem with the growth of outer enamel epithelium (OEE). Cell proliferation assay using Ki67 immunostaining showed that OEE divided more actively than inner enamel epithelium (IEE) at the onset of HERS formation. Live imaging suggested that mobility of the OEE and cells in the apex of CL were more active than in IEE. The expression of paxillin was observed strongly in OEE and the apex of CL. CONCLUSION: The more active growth and movement of OEE cells contributed to HERS formation after reduction of the growth of IEE. The expression pattern of paxillin was involved in the active movement of OEE and HERS. The results will contribute to understand the HERS formation mechanism and elucidate the cause of anomaly root.

Mouse genetic background influences the dental phenotype.

Dental enamel covers the crown of the vertebrate tooth and is considered to be the hardest tissue in the body. Enamel develops during secretion of an extracellular matrix by ameloblast cells in the tooth germ, prior to eruption of the tooth into the oral cavity. Secreted enamel proteins direct mineralization patterns during the maturation stage of amelogenesis as the tooth prepares to erupt. The amelogenins are the most abundant enamel proteins and are required for normal enamel development. Phenotypic differences were observed between incisors from individual Amelx (amelogenin) null mice that had a mixed 129xC57BL/6J genetic background and between inbred wild-type (WT) mice with different genetic backgrounds (C57BL/6J, C3H/HeJ, FVB/NJ). We hypothesized that this could be due to modifier genes, as human patients with a mutation in an enamel protein gene causing the enamel defect amelogenesis imperfecta (AI) can also have varied appearance of dentitions within a kindred. Enamel density measurements varied for all WT inbred strains midway during incisor development. Enamel thickness varied between some WT strains, and, unexpectedly, dentin density varied extensively between incisors and molars of all WT and Amelx null strains studied. WTFVB/NJ incisors were more similar to those of Amelx null mice than to those of the other WT strains in terms of incisor height/width ratio and pattern of enamel mineralization. Strain-specific differences led to the conclusion that modifier genes may be implicated in determining both normal development and severity of enamel appearance in AI mouse models and may in future studies be related to phenotypic heterogeneity within human AI kindreds reported in the literature.

The expression pattern of FHL2 during mouse molar development.

Four and a half LIM domains 2 (FHL2) functions as a transcriptional co-activator or co-repressor in a cell-type-specific manner. As a positive regulator, FHL2 plays an important role in osteoblast differentiation and bone formation. Our previous study showed that FHL2 was expressed in odontoblasts in mature human teeth under normal and pathological conditions. The purpose of this study was to investigate the spatial-temporal expression patterns of FHL2 at different stages of mouse molar development by immunohistochemistry. Our results showed that at the bud and cap stage, FHL2 was expressed both in enamel organ and the underlying mesenchyme. At the early bell stage, FHL2 appeared in the inner and outer enamel epithelium, stratum intermedium and the secondary enamel knot. Positive staining gradually converged at the cusps of dental papilla. At the late bell stage, FHL2 was expressed in the terminal differentiated ameloblasts and odontoblasts and stratum intermedium. At the postnatal day, FHL2 was detected in the secretory and mature ameloblasts and odontoblasts and mature enamel, and gradually appeared at Hertwig's epithelial root sheath and periodontal tissues. The spatial-temporal expression patterns of FHL2 from the bud stage to the postnatal day (13.5) suggested that during tooth development, FHL2 might play an important role in ameloblast and odontoblast differentiation, secretion of enamel and dentin matrix, mineralization of enamel, molar crown morphogenesis, as well as root formation.

Unicuspid and bicuspid tooth crown formation in squamates.

The molecular and developmental factors that regulate tooth morphogenesis in nonmammalian species, such as snakes and lizards, have received relatively little attention compared to mammals. Here we describe the development of unicuspid and bicuspid teeth in squamate species. The simple, cone-shaped tooth crown of the bearded dragon and ball python is established at cap stage and fixed in shape by the differentiation of cells and the secretion of dental matrices. Enamel production, as demonstrated by amelogenin expression, occurs relatively earlier in squamate teeth than in mouse molars. We suggest that the early differentiation in squamate unicuspid teeth at cap stage correlates with a more rudimentary tooth crown shape. The leopard gecko can form a bicuspid tooth crown despite the early onset of differentiation. Cusp formation in the gecko does not occur by the folding of the inner enamel epithelium, as in the mouse molar, but by the differential secretion of enamel. Ameloblasts forming the enamel epithelial bulge, a central swelling of cells in the inner enamel epithelium, secrete amelogenin at cap stage, but cease to do so by bell stage. Meanwhile, other ameloblasts in the inner enamel epithelium continue to secrete enamel, forming cusp tips on either side of the bulge. Bulge cells specifically express the gene Bmp2, which we suggest serves as a pro-differentiation signal for cells of the gecko enamel organ. In this regard, the enamel epithelial bulge of the gecko may be more functionally analogous to the secondary enamel knot of mammals than the primary enamel knot.

Developmentally regulated expression of intracellular Fgf11-13, hormone-like Fgf15 and canonical Fgf16, -17 and -20 mRNAs in the developing mouse molar tooth.

OBJECTIVE: To investigate and compare the cellular expression of non-secreted Fgf11-14 and secreted Fgf15-18 and -20 mRNAs during tooth formation. MATERIALS AND METHODS: mRNA expression was analyzed from the morphological initiation of the mouse mandibular first molar development to the onset of crown calcification using sectional in situ hybridization. RESULTS: This study found distinct, differentially regulated expression patterns for the Fgf11-13, -15-17 and -20, in particular in the epithelial-mesenchymal interface, whereas Fgf14 and 18 mRNAs were not detected. Fgf11, -15, -16, -17 and -20 were seen in the epithelium, whereas Fgf12 and -13 signals were restricted to the mesenchymal tissue component of the tooth. Fgf11 was observed in the putative epithelial signaling areas, the tertiary enamel knots and enamel free areas of the calcifying crown. Fgf15, Fgf17 and -20 were transiently colocalized in the thickened dental epithelium at E11.5. Later Fgf15 and -20 were exclusively expressed in the epithelial enamel knot signaling centers. In contrast, Fgf13 was present in the dental mesenchyme including odontoblasts cell lineage, whereas Fgf12 appeared transiently in the preodontoblasts. CONCLUSIONS: The expression of the Fgf11-13, -15, -17 and -20 in the epithelial signaling centers and/or epithelial-mesenchymal interfaces at key stages of the tooth formation suggest important functions in odontogenesis. Future analyses of the transgenic mice will help elucidate in vivo functions of the studied Fgfs during odontogenesis and whether any of the functions of the tooth expressed epithelial and mesenchymal Fgfs of different sub-families are redundant.

The morphology and mineralization of dental hard tissue in the offspring of passive smoking rats.

OBJECTIVE: To study the effects of maternal passive smoking on the morphology and mineralization of dental hard tissue in offspring rats. DESIGN: We have established a maternal passive smoking model. Offspring rats were sacrificed on the 20th day of gestation (E20) or the 3rd (D3) or 10th day (D10) after birth. We observed hard tissue morphology using Haematoxylin-Eosin (H&E) staining sections, used micro computer tomography (Micro-CT) to measure hard tissue thickness and volume on the mandibular first molars of the offspring rats, and used Micro-CT and energy dispersive X-ray spectroscopy with scanning electron microscopy (SEM/EDS) to determine the hard tissue mineral density and the ratio of calcium atom number/calcium atom+phosphorus atom number (Ca(2+)/P(3-)+Ca(2+)). RESULTS: Overall, the development of dental hard tissue was delayed in the offspring of passive smoking rats. The thickness and volume of hard tissue were lower in the offspring of the maternal passive smoking group than in the offspring of the control group. Mineral density of the hard tissue and the ratio of (Ca(2+)/P(3-)+Ca(2+)) were also reduced in the offspring of the maternal passive smoking group. CONCLUSION: Maternal passive smoking inhibits the morphological development and mineralization level of hard tissue on the mandibular first molars of offspring rats.

Regulation of dental enamel shape and hardness.

Epithelial-mesenchymal interactions guide tooth development through its early stages and establish the morphology of the dentin surface upon which enamel will be deposited. Starting with the onset of amelogenesis beneath the future cusp tips, the shape of the enamel layer covering the crown is determined by five growth parameters: the (1) appositional growth rate, (2) duration of appositional growth (at the cusp tip), (3) ameloblast extension rate, (4) duration of ameloblast extension, and (5) spreading rate of appositional termination. Appositional growth occurs at a mineralization front along the ameloblast distal membrane in which amorphous calcium phosphate (ACP) ribbons form and lengthen. The ACP ribbons convert into hydroxyapatite crystallites as the ribbons elongate. Appositional growth involves a secretory cycle that is reflected in a series of incremental lines. A potentially important function of enamel proteins is to ensure alignment of successive mineral increments on the tips of enamel ribbons deposited in the previous cycle, causing the crystallites to lengthen with each cycle. Enamel hardens in a maturation process that involves mineral deposition onto the sides of existing crystallites until they interlock with adjacent crystallites. Neutralization of acidity generated by hydroxyapatite formation is a key part of the mechanism. Here we review the growth parameters that determine the shape of the enamel crown as well as the mechanisms of enamel appositional growth and maturation.

Formation of the tooth-bone interface.

Not only are teeth essential for mastication, but also missing teeth are considered a social handicap due to speech and aesthetic problems, with a resulting high impact on emotional well-being. Several treatment procedures are currently available for tooth replacement with mostly inert prosthetic materials and implants. Natural tooth substitution based on copying the developmental process of tooth formation is particularly challenging and creates a rapidly developing area of molecular dentistry. In any approach, functional interactions among the tooth, the surrounding bone, and the periodontium must be established. Therefore, recent research in craniofacial genetics searches for mechanisms responsible for correct cell and tissue interactions, not only within a specific structure, but also in the context of supporting structures. A tooth crown that is not functionally anchored to roots and bone is useless. This review aims to summarize the developmental and tissue homeostatic aspects of the tooth-bone interface, from the initial patterning toward tooth eruption and lifelong interactions between the tooth and its surrounding alveolar bone.

Temporal and spatial expression of TGF-beta2 in tooth crown development in mouse first lower molar.

Transforming Growth Factor beta2 (TGF-beta2) is involved in the regulation of many important cellular processes during tooth development. In this study we systematically characterized the expression pattern of TGF-beta2 in vivo and further analyzed its possible roles during different developmental stages of mouse first lower molar using immunofluorescence histochemical method with confocal microscopy. TGF-beta2 signaling was detected in different developing stages in both dental epithelium and surrounding dental mesenchyme. For the first time, we found that the basement membrane and epithelial cells in the basal layer showed no immunostaining from embryonic day 11 to 13; the primary enamel knot and secondary enamel knot exhibited pronounced immunostaining with different expression patterns at embryonic day 14 and 16. In addition, the mature ameloblast lost immunoreactivity, but the secretory ameloblast still exhibited positive immunoreaction at day 2 of postnatal development. Collectively, the temporospatial distribution patterns of TGF- beta2, especially in the basement membrane, epithelial cells in the basal layer, enamel knot, mature odontoblast and ameloblast, suggested a close association between TGF-beta2 signaling and tooth crown development, and indicated that TGF-beta2 might participate in tooth initiation, epithelial morphogenesis, formation of dentine matrix, and ameloblast differentiation.

Epithelial-mesenchymal cell ratios can determine the crown morphogenesis of dental pulp stem cells.

Although dental pulp stem cells (DPSC) have been isolated from adult dental pulp tissues, knowledge on how to use them to make teeth lags behind. To date, little is known about the effects of epithelial-mesenchymal cell ratios on the bioengineered odontogenesis mediated by DPSCs. In this study, we investigated the effects of apical bud cells (ABC) from dental epithelial stem cell niche of rat incisors on the differentiation and morphogenesis of molar DPSCs at different proportions (DPSC/ABC cell ratios=1:10, 1:3, 1:1, 3:1, 10:1, respectively). In vitro mixed DPSCs/ABCs at 1:1, 1:3, and 3:1 ratios displayed several crucial characteristics of odontoblast/ameloblast lineages, as indicated by accelerated mineralization, upregulated alkaline phosphatase activity, protein/gene expression for dentin sialophosphoprotein and ameloblastin. In vivo transplantation of reassociated DPSC and ABC pellets at different ratios was also carried out. Histological analyses demonstrated that only DPSC/ABC recombinants at 1:1 ratio generated typical molar crown-shaped structures, whereas recombinations at other ratios presented an atypical crown morphogenesis with unbalanced distribution of amelogenesis and dentinogenesis. Together, these findings revealed that the proportions of dental epithelial and mesenchymal cell populations can determine the odontogenic differentiation of DPSCs/ABCs in vitro as well as the bioengineered tooth morphogenesis in vivo.