Notum regulates the cusp and root patterns in mouse molar.

Notum is a direct target of Wnt/ß-catenin signaling and plays a crucial role as a Wnt inhibitor within a negative feedback loop. In the tooth, Notum is known to be expressed in odontoblasts, and severe dentin defects and irregular tooth roots have been reported in Notum-deficient mice. However, the precise expression pattern of Notum in early tooth development, and the role of Notum in crown and root patterns remain elusive. In the present study, we identified a novel Notum expression in primary enamel knot (EK), secondary EKs, and dental papilla during tooth development. Notum-deficient mice exhibited enlarged secondary EKs, resulting in broader cusp tips, altered cusp patterns, and reduced concavity in crown outline. These alterations in crown outline led to a reduction in cervical tongue length, thereby inducing root fusion in Notum-deficient mice. Overall, these results suggest that the secondary EK size, regulated by the Wnt/Notum negative feedback loop, has a significant impact on the patterns of crown and root during tooth morphogenesis.

The human EDAR 370V/A polymorphism affects tooth root morphology potentially through the modification of a reaction-diffusion system.

Morphological variations in human teeth have long been recognized and, in particular, the spatial and temporal distribution of two patterns of dental features in Asia, i.e., Sinodonty and Sundadonty, have contributed to our understanding of the human migration history. However, the molecular mechanisms underlying such dental variations have not yet been completely elucidated. Recent studies have clarified that a nonsynonymous variant in the ectodysplasin A receptor gene (EDAR 370V/A; rs3827760) contributes to crown traits related to Sinodonty. In this study, we examined the association between the EDAR polymorphism and tooth root traits by using computed tomography images and identified that the effects of the EDAR variant on the number and shape of roots differed depending on the tooth type. In addition, to better understand tooth root morphogenesis, a computational analysis for patterns of tooth roots was performed, assuming a reaction-diffusion system. The computational study suggested that the complicated effects of the EDAR polymorphism could be explained when it is considered that EDAR modifies the syntheses of multiple related molecules working in the reaction-diffusion dynamics. In this study, we shed light on the molecular mechanisms of tooth root morphogenesis, which are less understood in comparison to those of tooth crown morphogenesis.

Crown tissue proportions and enamel thickness distribution in the Middle Pleistocene hominin molars from Sima de los Huesos (SH) population (Atapuerca, Spain).

Dental enamel thickness, topography, growth and development vary among hominins. In Homo, the thickness of dental enamel in most Pleistocene hominins display variations from thick to hyper-thick, while Neanderthals exhibit proportionally thinner enamel. The origin of the thin trait remains unclear. In this context, the Middle Pleistocene human dental assemblage from Atapuerca-Sima de los Huesos (SH) provides a unique opportunity to trace the evolution of enamel thickness in European hominins. In this study, we aim to test the hypothesis if the SH molar sample approximates the Neanderthal condition for enamel thickness and/or distribution. This study includes 626 molars, both original and comparative data. We analysed the molar inner structural organization of the original collections (n = 124), belonging to SH(n = 72) and modern humans from Spanish origin (n = 52). We compared the SH estimates to those of extinct and extant populations of the genus Homo from African, Asian and European origin (estimates extracted from literature n = 502). The comparative sample included maxillary and mandibular molars belonging to H. erectus, East and North African Homo, European Middle Pleistocene Homo, Neanderthals, and fossil and extant H. sapiens. We used high-resolution images to investigate the endostructural configuration of SH molars (tissue proportions, enamel thickness and distribution). The SH molars exhibit on average thick absolute and relative enamel in 2D and 3D estimates, both in the complete crown and the lateral enamel. This primitive condition is shared with the majority of extinct and extant hominin sample, except for Neanderthals and some isolated specimens. On the contrary, the SH molar enamel distribution maps reveal a distribution pattern similar to the Neanderthal signal (with thicker enamel on the lingual cusps and more peripherally distributed), compared to H. antecessor and modern humans. Due to the phylogenetic position of the SH population, the thick condition in molars could represent the persistence of the plesiomorphic condition in this group. Still, more data is needed on other Early and Middle Pleistocene populations to fully understand the evolutionary meaning of this trait.

Crystallographic texture and mineral concentration quantification of developing and mature human incisal enamel.

For human dental enamel, what is the precise mineralization progression spatially and the precise timing of mineralization? This is an important question in the fundamental understanding of matrix-mediated biomineralization events, but in particular because we can use our understanding of this natural tissue growth in humans to develop biomimetic approaches to repair and replace lost enamel tissue. It is important to understand human tissues in particular since different species have quite distinct spatial and temporal progression of mineralization. In this study, five human central incisors at different stages of enamel maturation/mineralization were spatially mapped using synchrotron X-ray diffraction and X-ray microtomography techniques. From the earliest developmental stage, two crystallite-orientation populations coexist with angular separations between the crystallite populations of approximately 40° varying as a function of position within the tooth crown. In general, one population had significantly lower texture magnitude and contributed a higher percentage to the overall crystalline structure, compared to the other population which contributed only 20-30% but had significantly higher texture magnitude. This quantitative analysis allows us to understand the complex and co-operative structure-function relationship between two populations of crystallites within human enamel. There was an increase in the mineral concentration from the enamel-dentin junction peripherally and from the incisal tip cervically as a function of maturation time. Quantitative backscattered-electron analyses showed that mineralization of prism cores precedes that of prism boundaries. These results provide new insights into the precise understanding of the natural growth of human enamel.

Transmission electron microscopical study of teenage crown dentin on the nanometer scale.

This is the first transmission electron microscopic study of teenage crown dentin on the nanometer scale. Samples for TEM were prepared by mechanical thinning and chemical polishing that allowed obtaining the electron transparent foils. It was firstly shown that human dentin possesses the layered morphology: the layers are oriented normally to the main axis of a tooth and have the thickness of ~50nm. HA inorganic phase of teenage crown dentin is in the amorphous state. The cellular structure, which was formed from collagen fibers (diameter is ~5nm), are observed near DEJ region in teenage dentin, whereas bioorganic phase of teenage crown dentin near the pulp camera does not contain the collagen fibers. Cracks in dentin thin foils have sharp tips, but big angles of opening (~30°) with plastic zone ahead crack tip. It means that young crown human dentin exhibits ductile or viscous-elastic fracture behavior on the nanometer scale.

Epiprofin Regulates Enamel Formation and Tooth Morphogenesis by Controlling Epithelial-Mesenchymal Interactions During Tooth Development.

The synchronization of cell proliferation and cytodifferentiation between dental epithelial and mesenchymal cells is required for the morphogenesis of teeth with the correct functional shapes and optimum sizes. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and is essential for ameloblast and odontoblast differentiation. Epfn deficiency results in the lack of enamel and ironically the formation of extra teeth. We investigated the mechanism underlying the functions of Epfn in tooth development through the creation of transgenic mice expressing Epfn under the control of an epithelial cell-specific K5 promoter (K5-Epfn). We found that these K5-Epfn mice developed abnormally shaped incisors and molars and formed fewer molars in the mandible. Remarkably, ameloblasts differentiated ectopically and enamel was formed on the lingual side of the K5-Epfn incisors. By contrast, ameloblasts and enamel were found only on the labial side in wild-type mice, as Follistatin (Fst) expressed in the lingual side inhibits BMP4 signaling necessary for ameloblast differentiation. We showed that Epfn transfection into the dental epithelial cell line SF2 abrogated the inhibitory activity of Fst and promoted ameloblast differentiation of SF2 cells. We found that Epfn induced FGF9 in dental epithelial cells and this dental epithelial cell-derived FGF9 promoted dental mesenchymal cell proliferation via the FGF receptor 1c (FGFR1c). Taken together, these results suggest that Epfn preserves the balance between cell proliferation and cytodifferentiation in dental epithelial and mesenchymal cells during normal tooth development and morphogenesis. © 2016 American Society for Bone and Mineral Research.

In Vitro Assessment of Enamel Permeability in Primary Teeth with and without Early Childhood Caries Using Laser Scanning Confocal Microscope.

OBJECTIVE: To compare the permeability of the enamel of primary teeth from individuals free of Early Childhood Caries (ECC) with that from individuals affected with ECC by assessment of dye penetration using Laser Scanning Confocal Microscope (LSCM). STUDY DESIGN: Experimental in vitro study. Exfoliated primary maxillary anterior teeth (n = 44) were collected and divided into two groups (n=22 per group): samples with ECC (Group 1) and without ECC (Group 2). The samples were immersed in Rhodamine B dye solution for 1 day, cut longitudinally into 3 sections, observed using LSCM. Dye penetration depths in the incisal, middle, cervical thirds and on labial, lingual surfaces were recorded. Data were analyzed by the Mann-Whitney test (α = 5%, p < .005). RESULTS: The overall mean penetration depth for group 1 (100.6 µm ± 58.48 µm) was significantly higher than that of group 2 (31.55 µm ± 23.40 µm, p < .000). Mean penetration depth in the incisal, middle, and cervical thirds and on the labial and lingual surfaces of group 1 also presented significantly higher scores than in group 2 (p < .005). CONCLUSION: There was significantly more dye penetration in the ECC group than in the non-ECC group. This could be related to a higher level of enamel permeability in teeth affected with ECC.

Characterization of mesenchymal progenitor cells in the crown and root pulp of primary teeth.

The existence of progenitor/mesenchymal stem cells (MSCs) was demonstrated previously in human primary/deciduous teeth. In this study, we examined dental pulp cells from root portion (root cells) of primary teeth without discernible root resorption and compared them with pulp cells from the crown portion (crown cells). Root cells and crown cells were characterized and compared to each other based on progenitor/MSC characteristics and on their generation efficiency of induced pluripotent stem (iPS) cells. Root cells and crown cells included cells manifesting typical progenitor/MSC properties such as osteogenic and adipogenic differentiation potential and clonogenicity. Interestingly, root cells showed a higher expression level of embryonic stem cell marker, KLF4, than crown cells. Moreover, the number of colony-forming unit-fibroblast and cell proliferation rate were higher for root cells than crown cells, and the efficiency of generating iPS cells from root cells was approximately four times higher than that from crown cells. Taken together, these results suggest that root cells from primary teeth show the MSC-like properties and thus could be a potent alternative source for iPS cell generation and the subsequent transplantation therapy.

In vivo scanning electron microscope assessment of enamel permeability in primary teeth with and without early childhood caries.

BACKGROUND: Over the years, certain primary teeth have been shown to be highly sensitive to dental caries, while others have remained caries-free. It has been hypothesized that this may be attributed to differences in the permeability of the enamel surface. AIM: The aim of the study was to evaluate the hypothesized differences in the permeability of primary tooth enamel in children with and those without Severe Early Childhood Caries (S-ECC) using scanning electron microscopy. MATERIALS AND METHODS: Sixteen children between 3 and 6 years of age were randomly selected and divided into two groups: Group 1, children without S-ECC (n = 8), and Group 2, children with S-ECC (n = 8). In each child, 4 teeth (the maxillary right and left central and lateral incisors) were subjected to evaluation. An impression was made with polyvinylsiloxane impression material, and scanning electron microscopy was used to inspect the negative replicas for droplets. RESULTS: The results indicated higher significance when individual regions (cervical, middle, and incisal thirds) in the two groups were evaluated and compared. Similarly, the overall results showed high statistical significance between S-ECC and non-S-ECC teeth. CONCLUSION: There could be a positive relationship between the permeability of tooth enamel and the development of caries, which needs further research.

Hydrogen peroxide penetration into the pulp chamber and dental permeability after bleaching.

This study sought to quantify the concentration of hydrogen peroxide (HP) in the pulp chamber and evaluate changes on dental permeability after bleaching with 3 HP concentrations (10%, 35%, and 50%). This study was divided into 2 experiments and the bleaching treatments consisted of 3 applications of HP for 30 minutes during a single session. The first experiment tested HP penetration into the pulp chamber of 4 experimental groups (n = 10) of bovine crowns, which were divided by HP concentration: an unbleached control group (0% HP), 10% HP, 35% HP, and 50% HP. Acetate buffer solution was placed into the pulp chamber and after each application of HP. This solution was collected to determine spectrophotometrically the concentration of HP that reached the pulp chamber. The second experiment evaluated dental permeability. Bovine crowns were divided into 3 groups (n = 10). The crowns were connected to a permeability device and the initial permeability was measured at 10 psi. Three different concentrations of HP gels (10%, 35% and 50%) were applied to the buccal enamel surfaces and the dental permeability was measured after the first, second, and third applications of HP. The data were analyzed by 2-way ANOVA and Tukey test (P ≤ 0.05). All concentrations of HP reached the pulp chamber, although no significant differences were noted between the 3 concentrations tested (P > 0.05). However, the increase of dental permeability in the group that received 50% HP was significantly higher than the 10% HP group (P < 0.05). The results indicate that the HP bleaching treatments increased dental permeability.

CryoTEM study of effects of phosphorylation on the hierarchical assembly of porcine amelogenin and its regulation of mineralization in vitro.

Amelogenin, the major extracellular enamel matrix protein, plays a critical role in regulating the growth and organization of enamel. Assembly and mineralization of full-length native (P173) and recombinant (rP172) porcine amelogenins were studied by cryogenic Transmission Electron Microscopy (cryoTEM). The cryoTEM revealed that both native and recombinant porcine amelogenins undergo step-wise self-assembly. Although the overall structural organization of P173 and rP172 oligomers was similar and resembled oligomers of murine recombinant amelogenin rM179, there were subtle differences suggesting that a single phosphorylated serine present in P173 might affect amelogenin self-assembly. Our mineralization studies demonstrated that both P173 and rP172 oligomers stabilize initial mineral clusters. Importantly, however, rP172 regulated the organization of initial mineral clusters into linear chains and guided the formation of parallel arrays of elongated mineral particles, which are the hallmark of enamel structural organization. These results are similar to those obtained previously using full-length recombinant murine amelogenin (Fang et al., 2011a). In contrast to that seen with rP172, phosphorylated P173 strongly inhibits mineralization for extended periods of time. We propose that these differences might be due to the differences in the structural organization and charge distribution between P173 and rP172. Overall our studies indicate that self-assembly of amelogenin and the mechanisms of its control over mineralization might be universal across different mammalian species. Our data also provide new insight into the effect of phosphorylation on amelogenin self-assembly and its regulation of mineralization.

Establishment of crown-root domain borders in mouse incisor.

Teeth are composed of two domains, the enamel-covered crown and the enamel-free root. The understanding of the initiation and regulation of crown and root domain formation is important for the development of bioengineered teeth. In most teeth the crown develops before the root, and erupts to the oral cavity whereas the root anchors the tooth to the jawbone. However, in the continuously growing mouse incisor the crown and root domains form simultaneously, the crown domain forming the labial and the root domain the lingual part of the tooth. While the crown-root border on the incisor distal side supports the distal enamel extent, reflecting an evolutionary diet adaptation, on the incisor mesial side the root-like surface is necessary for the attachment of the interdental ligament between the two incisors. Therefore, the mouse incisor exhibits a functional distal-mesial asymmetry. Here, we used the mouse incisor as a model to understand the mechanisms involved in the crown-root border formation. We analyzed the cellular origins and gene expression patterns leading to the development of the mesial and distal crown-root borders. We discovered that Barx2, En1, Wnt11, and Runx3 were exclusively expressed on the mesial crown-root border. In addition, the distal border of the crown-root domain might be established by cells from a different origin and by an early Follistatin expression, factor known to be involved in the root domain formation. The use of different mechanisms to establish domain borders gives indications of the incisor functional asymmetry.

Ameloblastin in Hertwig's epithelial root sheath regulates tooth root formation and development.

Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig's epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-2'-deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21(Cip1) and p27(Kip1) was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.

[Expression of transient receptor potential vanilloid 3 ion channel protein in human odontoblasts].

OBJECTIVE: To investigate the expression of transient receptor potential vanilloid 3 (TRPV3) ion channel protein in human odontoblasts (OD). METHODS: Twenty intact and healthy third molars extracted for orthodontic purpose were included. The quality of dental tissue sections was determined through HE staining, and the OD layer was further determined by dentin sialophosphoproteins (DSPP) antibody staining, and finally the expression of TRPV3 ion channel protein in human dental pulp tissue was examined by TRPV3 ion channel protein-specific antibody. The expression of TRPV3 channel proteins in human OD at different part of dental pulp was compared using Image Pro Plus (IPP) and SPSS software. RESULTS: TRPV3 channel protein expressed on the cell body of OD in the coronal and root pulp, and the expression in the coronal pulp was significantly higher than that in the root pulp. The TRPV3 protein also expressed at the odontoblastic process, with the higher expression in the crown (IA = 2516 ± 162) than in the root (IA = 2224 ± 150) and external root (IA = 2121 ± 92) (P < 0.05), but the expression between the lateral root area and external root area was not significantly different (P > 0.05). CONCLUSIONS: Human odonoblasts expressed TRPV3 ion channel protein and the expression level was different at different part of dental pulp OD.

Direct measurement of stain retention in third molars.

AIM: To directly determine the mass of dye retained in teeth following exposure to aqueous solutions of Rhodamine B and to correlate tooth color modifications. MATERIALS AND METHODS: Extracted third molars (25) were selected and sectioned at the cementoenamel junction for coronal staining. Pulp tissue was removed and teeth sonicated to remove debris. Teeth were kept in deionized water for 12 hours and subsequently weighed. They were then stained for 4 hours in 5 ml of Rhodamine B dye at two different concentrations. The samples were then subjected to two 8 hours rinses in deionized water. The tooth shade was recorded with a commercially available intraoral spectrophotometer (Vita Easyshade Compact, Vita Zahnfabrik, Bad Säckingen, Germany) at baseline (T1), after dye immersion (T2), and after water rinsing (T3). A standard absorption curve was then used to calculate the dye mass in the rinse solutions as well as the post- treatment stain solutions. All solution optical absorption curves were recorded using a laboratory research spectrophotometer (Cary 300, Agilent, USA). The mass of dye in each solution was then calculated from the standard curve relating optical absorption to aqueous dye concentration. RESULTS: An average change in the CIE (a) values of 8.0 ± 0.3 were observed for concentrations of Rhodamine B similar to the optical appearance of wine or other darkly colored juices while an increase of 10× in concentration gave values too high to measure using a standard intraoral spectrophotometer. By measuring the optical absorbance of the staining solutions before and after the staining process, we were able to measure dye retention of 54 ± 26 micrograms per gram of tooth. CONCLUSION: While no significant correlation could be found between the amount of stain retention in the dentition and the tooth shade due to the high uncertainties in the spectroscopic measurements, we were able to show that this method should admit such comparisons for future research. CLINICAL SIGNIFICANCE: The development of a reliable chromophore infiltration model may provide standardized and reproducible results in evaluating tooth whitening efficacy.

Permanent tooth mineralization in bonobos (Pan paniscus) and chimpanzees (P. troglodytes).

The timing of tooth mineralization in bonobos (Pan paniscus) is virtually uncharacterized. Analysis of these developmental features in bonobos and the possible differences with its sister species, the chimpanzee (P. troglodytes), is important to properly quantify the normal ranges of dental growth variation in closely related primate species. Understanding this variation among bonobo, chimpanzee and modern human dental development is necessary to better contextualize the life histories of extinct hominins. This study tests whether bonobos and chimpanzees are distinguished from each other by covariance among the relative timing and sequences of tooth crown initiation, mineralization, root extension, and completion. Using multivariate statistical analyses, we compared the relative timing of permanent tooth crypt formation, crown mineralization, and root extension between 34 P. paniscus and 80 P. troglodytes mandibles radiographed in lateral and occlusal views. Covariance among our 12 assigned dental scores failed to statistically distinguish between bonobos and chimpanzees. Rather than clustering by species, individuals clustered by age group (infant, younger or older juvenile, and adult). Dental scores covaried similarly between the incisors, as well as between both premolars. Conversely, covariance among dental scores distinguished the canine and each of the three molars not only from each other, but also from the rest of the anterior teeth. Our study showed no significant differences in the relative timing of permanent tooth crown and root formation between bonobos and chimpanzees.

Influence of in-office whitening gel pH on hydrogen peroxide diffusion through enamel and color changes in bovine teeth.

PURPOSE: To assess the influence of in-office whitening gel pH on whitening efficiency. METHODS: Hydrogen peroxide diffusion and color changes on bovine teeth were assessed. Three gels with close hydrogen peroxide concentrations but with various pH levels were tested: Zoom 2 (Discus Dental), Opalescence Endo and Opalescence Boost (Ultradent). The pH levels were respectively: 3.0, 5.0 and 7.0. Thirty enamel slices and tooth crowns were used for both studies (n = 10 per group per study). Hydrogen peroxide diffusion through the enamel slices and the tooth crowns was spectrophotometrically recorded every 10 minutes for 1 hour to calculate the diffusion coefficients. Color changes were spectrophotometrically recorded every 10 minutes for 1 hour and quantified in term of CIE-Lab. RESULTS: The hydrogen peroxide diffusion coefficient through enamel ranged from 5.12 +/- 0.82 x 10(-9) cm2 s(-1) for pH 3 to 5.19 +/- 0.92 x 10(-9) cm2 S(-1) for pH 7. Through tooth crowns it ranged from 4.80 +/- 1.75 x 10(-10) cm2 s(-1) for pH 5 to 4.85 +/- 1.82 x 10(-10) cm2 s(-1) for pH 3. After 1 hour, the deltaE varied from 5.6 +/- 4.0 for pH 7 to 7.0 +/- 5.0 for pH 3 on enamel slices and from 3.9 +/- 2.5 for pH 5 to 4.9 +/- 3.5 for pH 7 on tooth crowns. There was no statistically significant difference between groups for both parameters.

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.

Expression patterns of Sema3F, PlexinA4, -A3, Neuropilin1 and -2 in the postnatal mouse molar suggest roles in tooth innervation and organogenesis.

OBJECTIVE: Semaphorins form a family of axon wiring molecules but still little is known about their role in tooth formation. A class 3 semaphorin, Semaphorin3F (Sema3F), besides acting as a chemorepellant for different types of axons, controls a variety of non-neuronal developmental processes. MATERIALS AND METHODS: Cellular mRNA expression patterns of Sema3F as well as neuropilin 1 (Npn1), neuropilin 2 (Npn2), plexinA3 and plexinA4 receptors were analyzed by sectional in situ hybridization in the mouse molar tooth during postnatal days 0-7. The expression of the receptors was studied in PN5 trigeminal ganglia. RESULTS: Sema3F, Npn1, -2 and plexinA4 exhibited distinct, spatiotemporally changing expression patterns, whereas plexinA3 was not observed in the tooth germs. Besides being expressed in the base of the dental mesenchyme Sema3F, like plexinA4, Npn1 and -2, was present in the ameloblast cell lineage. Npn1 and Npn2 were additionally seen in the pulp horns and endothelial cells and like PlexinA4 in the developing alveolar bone. Npn1, plexinA3 and -A4 were observed in trigeminal ganglion neurons. CONCLUSIONS: Sema3F may act as a tooth target-derived axonal chemorepellant controlling establishment of the tooth nerve supply. Furthermore, Sema3F, like Npn1, -2 and plexinA4 may serve non-neuronal functions by controlling the development of the ameloblast cell lineage. Moreover, Npn1 and Npn2 may regulate dental vasculogenesis and, together with PlexinA4, alveolar bone formation. Further analyses such as investigation of transgenic mouse models will be required to elucidate in vivo signaling functions of Sema3F and the receptors in odontogenesis.

Morphogenetic roles of perlecan in the tooth enamel organ: an analysis of overexpression using transgenic mice.

Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin ß1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-ß1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.