The Role of GH/IGF Axis in Dento-Alveolar Complex from Development to Aging and Therapeutics: A Narrative Review.

The GH/IGF axis is a major regulator of bone formation and resorption and is essential to the achievement of normal skeleton growth and homeostasis. Beyond its key role in bone physiology, the GH/IGF axis has also major pleiotropic endocrine and autocrine/paracrine effects on mineralized tissues throughout life. This article aims to review the literature on GH, IGFs, IGF binding proteins, and their respective receptors in dental tissues, both epithelium (enamel) and mesenchyme (dentin, pulp, and tooth-supporting periodontium). The present review re-examines and refines the expression of the elements of the GH/IGF axis in oral tissues and their in vivo and in vitro mechanisms of action in different mineralizing cell types of the dento-alveolar complex including ameloblasts, odontoblasts, pulp cells, cementoblasts, periodontal ligament cells, and jaw osteoblasts focusing on cell-specific activities. Together, these data emphasize the determinant role of the GH/IGF axis in physiological and pathological development, morphometry, and aging of the teeth, the periodontium, and oral bones in humans, rodents, and other vertebrates. These advancements in oral biology have elicited an enormous interest among investigators to translate the fundamental discoveries on the GH/IGF axis into innovative strategies for targeted oral tissue therapies with local treatments, associated or not with materials, for orthodontics and the repair and regeneration of the dento-alveolar complex and oral bones.

Variation in mineral, organic, and water volumes at the neonatal line and in pre- and postnatal enamel.

OBJETIVES: The neonatal line (NNL) in enamel is hypomineralized, but quantitative data on the enamel component volumes of the NNL are lacking. This study aimed at quantifying the variation in the mineral, organic, and water volumes at the NNL and in pre- and postnatal enamel. MATERIALS AND METHODS: In buccal enamel longitudinal ground sections of exfoliated primary incisors (upper and lower; n = 17), the enamel component volumes were quantified at five histological sites (located at 40 µm intervals along a transversal line): the NNL, two sites in prenatal enamel, and two sites in postnatal enamel. Mineral volume was quantified using microradiography, and non-mineral volumes were quantified using polarizing microscopy. RESULTS: Differences in component volumes between the NNL and pre- and postnatal enamel had high effect sizes (Hedge's G ranging from 0.89, for the water volume, to 1.88, for the mineral volume; power > 90 %). The distance from the NNL correlated with the normalized component volume: r = 0.459, 95 % CI = 0.274/0.612 (mineral); r = -0.504; 95 % CI= -0.328/-0.647 (organic), and r = -0.294; 95 % CI= -0.087/-0.476 (water). Approaching the NNL from postnatal enamel, the percentage differences in component volumes were: -1.93 to -3.22 % for the mineral volume, +21.26 to +35.42 % for the organic volume, and +3.86 to +6.03 % for the water volume. Towards postnatal enamel, the percentage differences had the opposite trend. CONCLUSIONS: The enamel NNL is slightly hypomineralized with an increased organic volume one order of magnitude higher than the percentage differences in both mineral and water volumes.

Esmalte en terminología: concordancia y propuesta para terminologia anatomica, histologica y embryologica / Enamel in terminology: concordance and proposal for anatomical, histological and embryological terminology

El esmalte dental es una estructura considerada en Terminologia Anatomica, Histologica y Embryologica. Los términos incluidos relacionados a él presentan tres raíces asociadas: enamel, amelo y adamantino. El objetivo de este artículo es analizar la etimología de estas raíces y presentar propuestas en latín y español que unifiquen las denominaciones relacionadas al esmalte dentario. En la búsqueda fueron utilizaron diccionarios médicos, terminológicos y etimológicos. Los resultados obtenidos señalan tres orígenes diferentes para cada raíz: adamantino es un término grecolatino relacionado a la extrema dureza de una estructura; enamel tiene un origen anglo-normando y antiguo alto germánico relacionado a la acción de fundir, derretir, y el aspecto estético que esto produce en un material; y amelo, el cual en sí mismo está relacionado a la botánica mientras que el término completo ameloblasto, fue acuñado por un académico estadounidense. Si bien la etimología de adamantino y enamel señalan características de firmeza y estética, respectivamente, en el desarrollo de la propuesta se decidió mantener el término latinizado enamelum y su adjetivo enameleus, eliminando las otras dos raíces relacionadas al esmalte dentario. Se realizó la propuesta para los cambios en los términos del idioma latín y su traducción al español, acorde a los requerimientos de la FIPAT para Terminologia Anatomica, Histologica y Embryologica, con el fin de unificar la denominación de los términos asociados y derivados del esmalte dental, para fines académicos y de investigación.

Dental enamel is a structure considered in Anatomical, Histological and Embryological Terminology. The included terms related to it have three associated roots: enamel, amelo and adamantino. The objective of this article is to analyze the etymology of these roots and present proposals in Latin and Spanish that unify the denominations related to tooth enamel. In the search, medical, terminological and etymological dictionaries were used. The results obtained indicate three different origins for each root: adamantine is a Greco-Roman term related to the extreme hardness of a structure; enamel has an Anglo-Norman and ancient high Germanic origin related to the action of melting, melting, and the aesthetic appearance that this produces in a material; and amelo, which in itself is related to botany while the full term ameloblast, was coined by an American academic. Although the etymology of adamantine and enamel indicate characteristics of firmness and aesthetics, respectively, in the development of the proposal it was decided to maintain the Latinized term enamelum and its adjective enameleus, eliminating the other two roots related to dental enamel. The proposal was made for changes in the terms of the Latin language and its translation into Spanish, according to the requirements of the FIPAT for Anatomical, Histological and Embryological Terminology, in order to unify the denomination of the associated terms and derivatives of dental enamel, for academic and research purposes.

Surface analysis of etched enamel modified during the prenatal period.

Structural changes in the enamel surface subjected to induced demineralization and assessment of the influence of prenatal administration of ß-hydroxy ß-methylbutyrate (HMB) on enamel resistance were investigated. The examination was conducted on five sets of teeth from one-day-old spiny mice (Acomys cahirinus), one from the control and four from the experimental groups. Surface structure, molecular arrangement and crystalline organization of offspring's enamel both before and after etching were studied. Obtained results revealed that the physical and molecular arrangements of enamel were altered after the prenatal supplementation, and significantly affected its final structure and resistance against acid action. The enamel of incisors from the offspring which mothers were supplemented with HMB in a high dose (0.2 g/kgbw) and in the late period of gestation (26th-39th day) showed the highest endurance against acid treatment demonstrating only vestigial changes in their surface structure after acid action. Comparing to the remaining experimental groups, it was characterized by a reduced roughness and fractal dimension, significantly lower degree of demineralization and simultaneous lack of notable differences in the Raman spectra before and after acid etching. The results suggest that an increased enamel resiliency was the effect of a relatively high degree of mineralization and higher organization of the surface.

[Knockdown of MSX2 gene inhibits the expression of enamel matrix proteins and the enamel mineralization in mouse ameloblasts].

Objective To study the effect of muscle segment homeodomain homeobox 2 (MSX2) on the expression of enamel matrix protein and the formation of enamel. Methods Immunohistochemical staining was used to detect the expression of MSX2 in mouse tooth embryos and its localization in ameloblasts. The short hairpin RNA (shRNA) of the MSX2 gene was designed and synthesized, and then the annealed double stranded DNA was constructed into the pGMLV-SC5 RNAi lentivirus vector, and finally it was packaged with lentivirus. The lentivirus was used to infect ameloblasts. Real-time fluorescent quantitative PCR was performed to screen the best interference fragment, and detect the mRNAs of amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam), amelotin (Amtn) and kallikrein 4 (Klk4). The embryos were isolated for 18.5 days and then infected with RNAi recombinant lentivirus targeting MSX2. The tooth germ was implanted under the renal capsule of the mouse. Ten weeks later, the tissue was harvested to separate and observe the tooth form and contour. Results MSX2 was expressed in the secretory phase and maturation phase of mouse ameloblasts, but the expression signal was weaker in the secretory phase and was stronger in the mature stage. The lentivirus of MSX2-shRNA targeting MSX2 gene we constructed inhibited the expression of Amelx and Klk4 mRNAs. The RNAi lentivirus targeting MSX2 gene infected the tooth enamel and led to a decrease in the degree of enamel mineralization. Conclusion The MSX2 gene is expressed in ameloblasts. The knockdown of MSX2 can inhibit the expression of enamel matrix proteins and the enamel mineralization.

Cell cycle of the enamel knot during tooth morphogenesis.

Enamel knot (EK) is known to be a central organ in tooth development, especially for cusp patterning. To trace the exact position and movement among the inner dental epithelium (IDE) and EK cells, and to monitor the relationship between the EK and cusp patterning, it is essential that we understand the cell cycle status of the EK in early stages of tooth development. In this study, thymidine analogous (IdU, BrdU) staining was used to evaluate the cell cycle phase of the primary EK at the early casp stage (E13.0) and the gerbil embryo (E19) in a developing mouse embryo. The centerpiece of this study was to describe the cell cycle phasing and sequencing during proliferation in the IDE according to the expression of IdU and BrdU following their injection at calculated time points. The interval time between IdU injection and BrdU injection was set at 4 h. As a result, the cell cycle in the IDE of the mouse and gerbil was found to be synchronous. Conversely, the cell cycle in primary EKs of mice was much longer than that of the IDE. Therefore, the difference of cell cycle of the IDE and the EK is related to the diversity of cusp patterning and would provide a new insight into tooth morphogenesis.

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.

Isl1 Controls Patterning and Mineralization of Enamel in the Continuously Renewing Mouse Incisor.

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.

A cytoplasmic role of Wnt/ß-catenin transcriptional cofactors Bcl9, Bcl9l, and Pygopus in tooth enamel formation.

Wnt-stimulated ß-catenin transcriptional regulation is necessary for the development of most organs, including teeth. Bcl9 and Bcl9l are tissue-specific transcriptional cofactors that cooperate with ß-catenin. In the nucleus, Bcl9 and Bcl9l simultaneously bind ß-catenin and the transcriptional activator Pygo2 to promote the transcription of a subset of Wnt target genes. We showed that Bcl9 and Bcl9l function in the cytoplasm during tooth enamel formation in a manner that is independent of Wnt-stimulated ß-catenin-dependent transcription. Bcl9, Bcl9l, and Pygo2 localized mainly to the cytoplasm of the epithelial-derived ameloblasts, the cells responsible for enamel production. In ameloblasts, Bcl9 interacted with proteins involved in enamel formation and proteins involved in exocytosis and vesicular trafficking. Conditional deletion of both Bcl9 and Bcl9l or both Pygo1 and Pygo2 in mice produced teeth with defective enamel that was bright white and deficient in iron, which is reminiscent of human tooth enamel pathologies. Overall, our data revealed that these proteins, originally defined through their function as ß-catenin transcriptional cofactors, function in odontogenesis through a previously uncharacterized cytoplasmic mechanism, revealing that they have roles beyond that of transcriptional cofactors.

Murine matrix metalloproteinase-20 overexpression stimulates cell invasion into the enamel layer via enhanced Wnt signaling.

Matrix metalloproteinase-20 (MMP20) is expressed by ameloblasts in developing teeth and MMP20 mutations cause enamel malformation. We established a stably transfected Tet-Off Mmp20-inducible ameloblast-lineage cell line and found that MMP20 expression promoted cell invasion. Previously, we engineered transgenic mice (Tg) that drive Mmp20 expression and showed that Mmp20(+/+)Tg mice had soft enamel. Here we asked if Mmp20 overexpression disrupts ameloblast function. Incisors from Mmp20(+/+) mice expressing the Mmp20 Tg had a striking cell infiltrate which nearly replaced the entire enamel layer. A thin layer of enamel-like material remained over the dentin and at the outer tooth surface, but between these regions were invading fibroblasts and epithelial cells that surrounded ectopic bone-like calcifications. Mmp20(+/+)Tg mice had decreased enamel organ cadherin levels compared to the Mmp20 ablated and WT mice and, instead of predominantly locating adjacent to the ameloblast cell membrane, ß-catenin was predominantly present within the nuclei of invading cells. Our data suggest that increased cadherin cleavage by transgenic MMP20 in the WT background releases excess ß-catenin, which translocates to ameloblast nuclei to promote cell migration/invasion. Therefore, we conclude that MMP20 plays a role in normal ameloblast migration through tightly controlled Wnt signaling and that MMP20 overexpression disrupts this process.

Altered distribution of Ghrelin protein in mice molar development.

OBJECTIVE: Ghrelin, an appetite-stimulating hormone, plays diverse regulatory functions in cell growth, proliferation, differentiation and apoptosis during mammalian development. There is limited information currently available regarding Ghrelin expression during mammalian tooth development, thus we aimed to establish the spatiotemporal expression of Ghrelin during murine molar odontogenesis. DESIGN: Immunohistochemistry was performed to detect the expression pattern of Ghrelin in mandible molar from E15.5 to PN7 during murine tooth development. RESULTS: The results showed that Ghrelin initially expressed in the inner enamel epithelium and the adjacent mesenchymal cells below, further with persistent expression in the ameloblasts and odontoblasts throughout the following developmental stages. In addition, Ghrelin was also present in Hertwig's epithelial root sheath at the beginning of tooth root formation. CONCLUSIONS: These results suggest that Ghrelin was present in tooth organs throughout the stages of tooth development, especially in ameloblasts and odontoblasts with little spatiotemporal expression differences. However, the potential regulatory roles of this hormone in tooth development still need to be validated by functional studies.

Tooth and scale morphogenesis in shark: an alternative process to the mammalian enamel knot system.

BACKGROUND: The gene regulatory network involved in tooth morphogenesis has been extremely well described in mammals and its modeling has allowed predictions of variations in regulatory pathway that may have led to evolution of tooth shapes. However, very little is known outside of mammals to understand how this regulatory framework may also account for tooth shape evolution at the level of gnathostomes. In this work, we describe expression patterns and proliferation/apoptosis assays to uncover homologous regulatory pathways in the catshark Scyliorhinus canicula. RESULTS: Because of their similar structural and developmental features, gene expression patterns were described over the four developmental stages of both tooth and scale buds in the catshark. These gene expression patterns differ from mouse tooth development, and discrepancies are also observed between tooth and scale development within the catshark. However, a similar nested expression of Shh and Fgf suggests similar signaling involved in morphogenesis of all structures, although apoptosis assays do not support a strictly equivalent enamel knot system in sharks. Similarities in the topology of gene expression pattern, including Bmp signaling pathway, suggest that mouse molar development is more similar to scale bud development in the catshark. CONCLUSIONS: These results support the fact that no enamel knot, as described in mammalian teeth, can be described in the morphogenesis of shark teeth or scales. However, homologous signaling pathways are involved in growth and morphogenesis with variations in their respective expression patterns. We speculate that variations in this topology of expression are also a substrate for tooth shape evolution, notably in regulating the growth axis and symmetry of the developing structure.

The enamel knot-like structure is eternally maintained in the apical bud of postnatal mouse incisors.

OBJECTIVES: The boundary where inner and outer enamel epithelia meet is referred to as the cervical loop (CL) in molar tooth germs. In contrast, rodent incisors are continuously growing: the labial side of the teeth is covered with enamel (crown-analog), and the lingual side is covered with cementum (root-analog). These results in the appearance of CL in the frontal sections apart from the apical end. However, many researchers have used the term "labial CL" to indicate the apical end of incisors. DESIGN: This study investigated the gene expression patterns for the enamel knot signaling center in tooth morphogenesis to clarify the difference between "labial CL" and molar CL. We examined the three-dimensional expression patterns of markers for the enamel knot including fibroblast growth factor 4 (Fgf4), sonic hedgehog (Shh), Msx2, and P21 in frontal sections of murine mandibular incisors. RESULTS: Serial frontal sections from the apical end of the postnatal incisor clearly demonstrated the existence of enamel knot-like structures composed of supra-inner enamel epithelium and stellate reticulum in the "labial CL". This structure includes the expression of all examined markers for enamel knots such as Fgf4, Shh, Msx2, and P21. CONCLUSIONS: The molar tooth germ-like structure is maintained indefinitely in the "labial CL". Therefore, the "labial CL" is not equivalent to the molar CL. The existence of an EK-like structure in the apical end of incisors implies that the usage of "labial CL" may be inappropriate for indicating this region. The "apical bud" could be used as an alternative term.

Prenatal factors associated with the neonatal line thickness in human deciduous incisors.

The neonatal line (NNL) is used to distinguish developmental events observed in enamel which occurred before and after birth. However, there are few studies reporting relationship between the characteristics of the NNL and factors affecting prenatal conditions. The aim of the study was to determine prenatal factors that may influence the NNL thickness in human deciduous teeth. The material consisted of longitudinal ground sections of 60 modern human deciduous incisors obtained from full-term healthy children with reported birth histories and prenatal factors. All teeth were sectioned in the labio-lingual plane using diamond blade (Buechler IsoMet 1000). Final specimens were observed using scanning electron microscopy at magnifications 320×. For each tooth, linear measurements of the NNL thickness were taken on its labial surface at the three levels from the cemento-enamel junction. The difference in the neonatal line thickness between tooth types and between males and females was statistically significant. A multiple regression analyses confirmed influence of two variables on the NNL thickness standardised on tooth type and the children's sex (z-score values). These variables are the taking of an antispasmodic medicine by the mother during pregnancy and the season of the child's birth. These two variables together explain nearly 17% of the variability of the NNL. Children of mothers taking a spasmolytic medicine during pregnancy were characterised by a thinner NNL compared with children whose mothers did not take such medication. Children born in summer and spring had a thinner NNL than children born in winter. These results indicate that the prenatal environment significantly contributes to the thickness of the NNL influencing the pace of reaching the post-delivery homeostasis by the newborn's organism.

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

Neonatal line on fetus and infant teeth: An indicator of live birth and mode of delivery.

BACKGROUND: The neonatal line (NL) is an important issue in forensic odontology. It is the sign of a developmental birth defect, which is caused by the effect of metabolic stress on tooth structures when the fetus passes to extrauterine life. AIMS: The aim of this research is to determine the existence and thickness of NL in teeth, as it is a legal necessity to indicate the signs of viability at birth in a forensic examination of a fetus or infant case. STUDY DESIGN AND SUBJECTS: This research was conducted on 48 lower left and right lateral teeth, which were taken from 24 autopsy cases (46% female and 54% male). Left lateral teeth were sectioned in a vertical plane and right lateral teeth were sectioned in a horizontal plane. The NL thickness was measured with a scanning electron microscope (SEM). These cases comprised three conditions as: 70.3% normal birth, 16.7% caesarean sections, and 12.5% still birth cases under the legal and ethical permission. OUTCOME MEASURES: The mean NL thickness of normal birth cases was higher than caesarean cases as 7.7µm and 2.5µm, respectively. RESULTS AND CONCLUSIONS: The results showed a statistical significance between all birth conditions. NL does not exist in still birth cases (p<0.001). Not only is the presence of NL a sign of live birth, but also its thickness is an indicator of the delivery mode where NL thickness of normal birth was found thicker than the caesarean cases.

Developmental regulations of Perp in mice molar morphogenesis.

Teraspanin transmembrane protein, Perp (P53 apoptosis effector related to PMP22), which is found in the plasma membrane as a component of the desmosome, is reported to be involved in the morphogenesis of the epithelium and the enamel formation of the incisor. However, its expression pattern and signaling regulation during molar development have not been elucidated in detail. We have examined the precise expression patterns of Perp in developing lower molars and employed the knock-down of Perp by antisense oligodeoxynucleotide treatment during in vitro organ cultivation at embryonic day 13 to define the precise developmental function of Perp. Perp was expressed mainly in the dental lamina and stellate reticulum regions at the bud and cap stages. After Perp knock-down, the tooth germ showed disruption of the dental lamina and stellate reticulum with altered apoptosis and proliferation. The changed expression levels of related signaling molecules from the enamel knot and desmosome were evaluated by real-time quantitative polymerase chain reaction. A renal capsule transplantation method was employed to examine the effects of Perp knock-down on molar crown development. Ultrastructural observations revealed that enamel was deposited more densely in an irregular pattern in the cusp region, and that dentin was hypo-mineralized after Perp knock-down at the cap stage. Thus, Perp might play important roles in the formation and integration of stellate reticulum, dental lamina structure and enamel formation through signaling interactions with the enamel knot and desmosome-related signaling molecules at the cap stage of lower molar development.

Enamelin is critical for ameloblast integrity and enamel ultrastructure formation.

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam-/- mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam-/- mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using ß-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam-/- background did not fully recover enamel formation while a medium expresser in the Enam+/- background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation.

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.

c-kit positive cells and networks in tooth germs of human midterm fetuses.

Numerous studies have attempted to characterize the dental pulp stem cells. However, studies performed on prenatal human tissues have not been performed to evaluate the in situ characterization and topography of progenitor cells. We aimed to perform such a study using of antibodies for CD117/c-kit and multiplex antibody for Ki67+ caspase 3. Antibodies were applied on samples dissected from five human midterm fetuses. Positive CD117/c-kit labeling was found in mesenchymal derived tissues, such as the dental follicle and the dental papilla. The epithelial tissues, that is, dental lamina, enamel organ and oral epithelia, also displayed isolated progenitor cells which were CD117/c-kit positive. Interestingly, CD117/c-kit positive cells of mesenchymal derived tissues extended multiple prolongations building networks; the most consistent of such networks were those of the dental follicle and the perivascular networks of the dental papilla. However, the mantle of the dental papilla was also positive for CD117/c-kit positive stromal networks. The CD117/c-kit cell populations building networks appeared mostly with a Ki67 negative phenotype. The results suggest that CD117/c-kit progenitor cells of the prenatal tooth germ tissues might be involved in intercellular signaling.