RESUMEN
The morphological and possible functional interactions between the connective tissue and enamel organ cells were examined during the maturation phase of enamel formation, using immunohistochemical techniques. Decalcified mandibular sections (10 µm) including incisors were used from Wistar rats ages 10-12 weeks. Sections were incubated with one or two primary antibodies targeting cell cytoskeleton (vimentin, α-actin, α-tubulin), dendritic marker (OX6), gap junctions (cx-43), enzymes (nitric-oxide synthase (nos1) and cyclooxygenase (cox1)), and the ion transporters (Na+/H+ exchanger (NHE1) and Na+/Ca2+ exchanger (NCX)) for 24 h, before incubation with the appropriate conjugated fluorescent secondary antibodies. Sections were examined by fluorescence microscopy. Haematoxylin-eosin slides were also employed. Cellular heterogeneity and morphological modulations were identified within enamel organ cells and connective tissue covering suggesting complex cellular interactions and indicating a new functional concept and possible complementary role during enamel maturation. Also, some ion transportation activity, and nos1 and cox1 signalling pathways have been identified, indicating intercellular communication between these regions. A hypothesis is suggested, to explain the morphological modulation of ameloblasts and papillary cells during enamel maturation which functions to increase the transporting membrane surface area to accomplish faster and bulker ion transportation to achieve controlled pH and to direct Ca2+ towards enamel.
Asunto(s)
Tejido Conectivo/anatomía & histología , Tejido Conectivo/fisiología , Órgano del Esmalte/anatomía & histología , Órgano del Esmalte/crecimiento & desarrollo , Epitelio/anatomía & histología , Epitelio/fisiología , Animales , Ciclooxigenasa 1/metabolismo , Incisivo/citología , Masculino , Mandíbula/citología , Modelos Biológicos , Óxido Nítrico Sintasa/metabolismo , Ratas WistarRESUMEN
We cryopreserved mouse tooth germs with widely open cervical margins of the enamel organ to overcome difficulties in cryoprotectant permeation and tested their efficacy by transplanting them into recipient mice. The upper right first molar germs of 8-day-old donor mice were extracted and categorized into the following four groups according to cryopreservation time: no cryopreservation, 1 week, 1 month, and 3 months. The donor tooth germs were transplanted into the upper right first molar germ sockets of the 8-day-old recipient mice. The upper left first molars of the recipient mice were used as controls. The outcome of the transplantation was assessed at 1, 2, and 3 weeks after transplantation. Stereomicroscopic evaluation revealed that most of the transplanted teeth erupted by 3 weeks after transplantation. Micro-computed tomography analysis revealed root elongation in the transplanted groups as well as in the controls. There was no significant difference between the cryopreserved and non-cryopreserved transplanted teeth, but the roots of the cryopreserved teeth were significantly shorter than those of the control teeth. Histological examination revealed root and periodontal ligament formations in all the transplanted groups. These results suggest that the transplantation of cryopreserved tooth germs facilitates subsequent root elongation and tooth eruption.
Asunto(s)
Diente Molar/crecimiento & desarrollo , Erupción Dental/fisiología , Germen Dentario/crecimiento & desarrollo , Germen Dentario/cirugía , Raíz del Diente/crecimiento & desarrollo , Proceso Alveolar/crecimiento & desarrollo , Animales , Criopreservación/métodos , Órgano del Esmalte/crecimiento & desarrollo , Maxilar/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ligamento Periodontal/crecimiento & desarrollo , Periodoncio/crecimiento & desarrollo , Regeneración/fisiología , Anomalías Dentarias/cirugía , Alveolo Dental/crecimiento & desarrollo , Microtomografía por Rayos X/métodosRESUMEN
Hyaluronan (HA) is a major constituent molecule in most extracellular matrices and is synthesized by Hyaluronan synthase (Has). In the present study, we examined expression patterns of Has1, -2, -3 mRNA in developing mouse molar and incisor tooth germs from embryonic day (E) 11.5 to postnatal day (P) 7, focusing on Hertwig's epithelial root sheath (HERS) and the apical bud in particular. Has1 mRNA expression was not detected in all tooth germs examined. Has2 mRNA was expressed in the surrounding mesenchyme from E12.0 to 18.0 in both molar and incisor tooth germs, but disappeared after birth. Meanwhile, Has3 mRNA was exclusively expressed within the enamel organ, especially in the inner enamel epithelium (IEE), stellate reticulum (SR), and stratum intermedium (SI) until the early bell stage at E16.0. Has3 mRNA disappeared as IEE differentiated into differentiating ameloblasts (dABs), but remained in SI until the root developmental stage of the molar tooth germ at P7. Has3 mRNA was also expressed in HERS until P7. In incisors, Has3 mRNA was expressed in the apical bud, especially in the transit-amplifying (TA) cell region from E16.0 to P7, and in the papillary layer (PL) adjacent to the mature enamel. These gene expression patterns suggested that Has3 is the main control factor for prenatal and postnatal HA synthesis of the tooth germ, and may in part regulate crown and root formation of the tooth germ, maintenance of stem cell niches in the apical bud as well as mineral transport in PL.
Asunto(s)
Desarrollo Embrionario/genética , Glucuronosiltransferasa/genética , Germen Dentario/crecimiento & desarrollo , Animales , Órgano del Esmalte/crecimiento & desarrollo , Órgano del Esmalte/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glucuronosiltransferasa/biosíntesis , Hialuronano Sintasas , Ácido Hialurónico/metabolismo , Hibridación in Situ , Incisivo/crecimiento & desarrollo , Incisivo/metabolismo , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Ratones , Diente Molar/crecimiento & desarrollo , Diente Molar/metabolismo , Odontogénesis/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Germen Dentario/metabolismoRESUMEN
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.
Asunto(s)
Ghrelina/biosíntesis , Ghrelina/metabolismo , Diente Molar/metabolismo , Ameloblastos/citología , Ameloblastos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Esmalte Dental/citología , Esmalte Dental/embriología , Esmalte Dental/metabolismo , Órgano del Esmalte/embriología , Órgano del Esmalte/crecimiento & desarrollo , Órgano del Esmalte/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos ICR , Diente Molar/citología , Diente Molar/efectos de los fármacos , Diente Molar/crecimiento & desarrollo , Odontoblastos/citología , Odontoblastos/metabolismo , Odontogénesis/efectos de los fármacos , Odontogénesis/fisiología , Embarazo , Germen Dentario/embriología , Germen Dentario/crecimiento & desarrollo , Germen Dentario/metabolismo , Raíz del Diente/embriología , Raíz del Diente/crecimiento & desarrollo , Raíz del Diente/metabolismoRESUMEN
The purpose of this study was to identify the major molecular components in the secretory and maturation stages of amelogenesis through transcriptome analyses. Ameloblasts (40 sections per age group) were laser micro-dissected from Day 5 (secretory stage) and Days 11-12 (maturation stage) first molars. PolyA+ RNA was isolated from the lysed cells, converted to cDNA, and amplified to generate a cDNA library. DNA sequences were obtained using next generation sequencing and analyzed to identify genes whose expression had increased or decreased at least 1.5-fold in maturation stage relative to secretory stage ameloblasts. Among the 9198 genes that surpassed the quality threshold, 373 showed higher expression in secretory stage, while 614 genes increased in maturation stage ameloblasts. The results were cross-checked against a previously published transcriptome generated from tissues overlying secretory and maturation stage mouse incisor enamel and 34 increasing and 26 decreasing expressers common to the two studies were identified. Expression of F2r, which encodes protease activated receptor 1 (PAR1) that showed 10-fold higher expression during the secretory stage in our transcriptome analysis, was characterized in mouse incisors by immunohistochemistry. PAR1 was detected in secretory, but not maturation stage ameloblasts. We conclude that transcriptome analyses are a good starting point for identifying genes/proteins that are critical for proper dental enamel formation and that PAR1 is specifically expressed by secretory stage ameloblasts.
Asunto(s)
Ameloblastos/metabolismo , Amelogénesis/genética , Proteínas del Esmalte Dental/genética , Perfilación de la Expresión Génica , Transcriptoma/genética , Animales , Órgano del Esmalte/crecimiento & desarrollo , Secuenciación de Nucleótidos de Alto Rendimiento , RatonesRESUMEN
BACKGROUND: N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development. METHODOLOGY/PRINCIPAL FINDINGS: The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. µCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ. CONCLUSIONS/SIGNIFICANCE: The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.
Asunto(s)
Ameloblastos/metabolismo , Amelogénesis/genética , Cadherinas/genética , Esmalte Dental/metabolismo , Órgano del Esmalte/metabolismo , Ameloblastos/citología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Cadherinas/deficiencia , Cadherinas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Movimiento Celular , Esmalte Dental/citología , Esmalte Dental/crecimiento & desarrollo , Órgano del Esmalte/citología , Órgano del Esmalte/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Dureza , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Cultivo Primario de Células , Transducción de Señal , Propiedades de SuperficieRESUMEN
In mouse tooth development, the roots of the first lower molar develop after crown formation to form 2 cylindrical roots by post-natal day 5. This study compared the morphogenesis and cellular events of the mesial-root-forming (MRF) and bifurcation-forming (BF) regions, located in the mesial and center of the first lower molar, to better define the developmental mechanisms involved in multi-rooted tooth formation. We found that the mesenchyme in the MRF showed relatively higher proliferation than the bifurcation region. This suggested that spatially regulated mesenchymal proliferation is required for creating cylindrical root structure. The mechanism may involve the mesenchyme forming a physical barrier to epithelial invagination of Hertwig's epithelial root sheath. To test these ideas, we cultured roots in the presence of pharmacological inhibitors of microtubule and actin polymerization, nocodazole and cytochalasin-D. Cytochalasin D also inhibits proliferation in epithelium and mesenchyme. Both drugs resulted in altered morphological changes in the tooth root structures. In particular, the nocodazole- and cytochalasin-D-treated specimens showed a loss of root diameter and formation of a single-root, respectively. Immunolocalization and three-dimensional reconstruction results confirmed these mesenchymal cellular events, with higher proliferation in MRF in multi-rooted tooth formation.
Asunto(s)
Mesodermo/citología , Diente Molar/crecimiento & desarrollo , Morfogénesis/fisiología , Raíz del Diente/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Recuento de Células , Proliferación Celular/efectos de los fármacos , Citocalasina D/farmacología , Saco Dental/citología , Saco Dental/crecimiento & desarrollo , Órgano del Esmalte/citología , Órgano del Esmalte/crecimiento & desarrollo , Epitelio/efectos de los fármacos , Epitelio/crecimiento & desarrollo , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Antígeno Ki-67/análisis , Mesodermo/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Diente Molar/citología , Diente Molar/efectos de los fármacos , Morfogénesis/efectos de los fármacos , Nocodazol/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Odontogénesis/efectos de los fármacos , Odontogénesis/fisiología , Técnicas de Cultivo de Órganos , Raíz del Diente/citología , Raíz del Diente/efectos de los fármacos , Moduladores de Tubulina/farmacologíaRESUMEN
Signals of perlecan, an extracellular matrix molecule, which accumulates within the intercellular spaces of the stellate reticulum of the enamel organ, are mediated by at least two receptors, dystroglycan (DG) and integrin ß1, in a case-dependent manner in various events in embryogenesis and pathogenesis. This study aims to understand the expression profiles of these two perlecan receptors at both protein and gene levels in murine enamel organ development. Before birth, α-DG was immunolocalized in stellate reticulum cells, in which perlecan was colocalized, while integrin ß1 was mainly distributed in the peripheral enamel organ cells as well as the dental mesenchymal cells. On and after postnatal Day 1, the expression of α-DG was dramatically decreased in the stellate reticulum, while integrin ß1 was enhanced around blood vessels within the enamel organ. Furthermore, biosyntheses of α-DG and integrin ß1 by dental epithelial and pulp mesenchymal cells were confirmed in vitro by using immunofluorescence and reverse-transcriptase polymerase chain reaction. The results suggest that DG is a perlecan receptor that specifically functions in the stellate reticulum of the embryonic stage, but that dental epithelial and mesenchymal cells are maturated by capturing perlecan signals differentially through integrin ß1.
Asunto(s)
Distroglicanos/metabolismo , Órgano del Esmalte/metabolismo , Expresión Génica , Integrina beta1/metabolismo , Animales , Línea Celular , Distroglicanos/genética , Órgano del Esmalte/citología , Órgano del Esmalte/embriología , Órgano del Esmalte/crecimiento & desarrollo , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Incisivo/citología , Incisivo/embriología , Incisivo/crecimiento & desarrollo , Incisivo/metabolismo , Integrina beta1/genética , Mesodermo/citología , Ratones Endogámicos ICR , Diente Molar/citología , Diente Molar/embriología , Diente Molar/crecimiento & desarrollo , Diente Molar/metabolismo , Especificidad de ÓrganosRESUMEN
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.
Asunto(s)
Ameloblastos/citología , Proteínas del Esmalte Dental/genética , Regulación del Desarrollo de la Expresión Génica , Odontogénesis/genética , Raíz del Diente/crecimiento & desarrollo , Ameloblastos/metabolismo , Animales , Animales Recién Nacidos , Bromodesoxiuridina , Diferenciación Celular , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas del Esmalte Dental/antagonistas & inhibidores , Proteínas del Esmalte Dental/metabolismo , Órgano del Esmalte/anatomía & histología , Órgano del Esmalte/crecimiento & desarrollo , Órgano del Esmalte/metabolismo , Silenciador del Gen , Ratones , Ratones Endogámicos C57BL , Diente Molar/anatomía & histología , Diente Molar/crecimiento & desarrollo , Diente Molar/metabolismo , Técnicas de Cultivo de Órganos , ARN Interferente Pequeño/genética , Corona del Diente/anatomía & histología , Corona del Diente/crecimiento & desarrollo , Corona del Diente/metabolismo , Raíz del Diente/anatomía & histología , Raíz del Diente/metabolismoRESUMEN
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.
Asunto(s)
Órgano del Esmalte/embriología , Odontogénesis/fisiología , Corona del Diente/embriología , Germen Dentario/embriología , Raíz del Diente/embriología , Animales , Movimiento Celular/fisiología , Proliferación Celular , Esmalte Dental/citología , Esmalte Dental/embriología , Esmalte Dental/crecimiento & desarrollo , Órgano del Esmalte/citología , Órgano del Esmalte/crecimiento & desarrollo , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Proteínas Fluorescentes Verdes , Antígeno Ki-67/análisis , Sustancias Luminiscentes , Ratones , Diente Molar/embriología , Diente Molar/crecimiento & desarrollo , Técnicas de Cultivo de Órganos , Paxillin/análisis , Corona del Diente/citología , Corona del Diente/crecimiento & desarrollo , Germen Dentario/citología , Germen Dentario/crecimiento & desarrollo , Raíz del Diente/citología , Raíz del Diente/crecimiento & desarrolloRESUMEN
Hertwig's epithelial root sheath (HERS), a bilayered epithelial cell sheath located at the cervical loop of the enamel organ in a developing tooth, is at the forefront of root formation. However, little is known about the exact mechanisms that regulate the development of HERS. The neuropeptide vasoactive intestinal peptide (VIP) is involved in the development of various tissues and cells. In this study, we investigated the roles of VIP in HERS development. VIP-immunoreactive nerve fibers were found in the dental pulp and around the root apex of the tooth, while the expression of VIP receptor 1 (VPAC1) was observed in HERS. The expression level of VPAC1 correlated with the development of HERS and was elevated at postnatal days 14 and 21. Using ex vivo cultures of neonatal tooth germs, VIP enhanced the elongation and proliferation of HERS. In vitro, VIP also promoted the proliferation of cells from the HERS-derived cell line, HERS01a cells, and upregulated the mRNA expression of cytokeratin 14 and vimentin (typical molecular markers of HERS) in these cells. These results suggest that VIP may be an essential factor for HERS development.
Asunto(s)
Órgano del Esmalte/citología , Células Epiteliales/metabolismo , Péptido Intestinal Vasoactivo/fisiología , Animales , Línea Celular , Proliferación Celular , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/crecimiento & desarrollo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Queratina-14/genética , Queratina-14/metabolismo , Masculino , Ratones , Ratones SCID , ARN Mensajero/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Raíz del Diente/citología , Raíz del Diente/efectos de los fármacos , Raíz del Diente/crecimiento & desarrollo , Péptido Intestinal Vasoactivo/farmacología , Vimentina/genética , Vimentina/metabolismoRESUMEN
Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1-4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination.
Asunto(s)
Transportador de Glucosa de Tipo 1/metabolismo , Glucosa/farmacocinética , Diente Molar/metabolismo , Odontogénesis , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Órgano del Esmalte/embriología , Órgano del Esmalte/crecimiento & desarrollo , Órgano del Esmalte/metabolismo , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos ICR , Diente Molar/embriología , Diente Molar/crecimiento & desarrollo , Floretina/farmacología , Embarazo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Germen Dentario/embriología , Germen Dentario/crecimiento & desarrollo , Germen Dentario/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: It is well known that tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial-mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. MATERIAL AND METHODS: The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF-soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5'-bromo-2'-deoxyuridine (BrdU) assay in an organ-culture system. RESULTS: HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ-culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down-regulated when antibody against HGF receptor was present in the culture medium. CONCLUSION: Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development.
Asunto(s)
Factor de Crecimiento de Hepatocito/fisiología , Diente Molar/crecimiento & desarrollo , Odontogénesis/efectos de los fármacos , Raíz del Diente/crecimiento & desarrollo , Animales , Antimetabolitos , Bromodesoxiuridina , Recuento de Células , Técnicas de Cultivo de Célula , Línea Celular , Proliferación Celular/efectos de los fármacos , Cemento Dental/citología , Cemento Dental/efectos de los fármacos , Dentina/citología , Dentina/efectos de los fármacos , Órgano del Esmalte/citología , Órgano del Esmalte/crecimiento & desarrollo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Factor de Crecimiento de Hepatocito/farmacología , Inmunohistoquímica , Ratones , Diente Molar/citología , Diente Molar/efectos de los fármacos , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas c-met/análisis , Ápice del Diente/citología , Ápice del Diente/efectos de los fármacos , Ápice del Diente/crecimiento & desarrollo , Germen Dentario/citología , Germen Dentario/crecimiento & desarrollo , Raíz del Diente/citología , Raíz del Diente/efectos de los fármacosRESUMEN
BACKGROUND AND OBJECTIVE: The tooth root is one of the critical parts to maintain tooth function; however, the molecular mechanisms of root development remain unknown. We aimed to identify specific factors for root morphogenesis using a newly developed experimental system. MATERIAL AND METHODS: Tentative cementoblasts and periodontal ligament cells from mouse mandibular molars were isolated using laser capture microdissection. More than 500 cementoblasts and periodontal ligament cells were separately captured. After RNA extraction and amplification, mRNA expression in isolated cementoblasts was compared with that of periodontal ligament cells by cDNA microarray analysis. Then, putative cementoblast-specific genes were subjected to in situ hybridization analysis to confirm the results in mouse mandible. RESULTS: Approximately 2000 genes were differentially expressed between these tissues. Among those genes, zinc finger helicase (ZFH), also termed chromodomain-helicase-DNA-binding protein 3 (Chd3), was one of the highly expressed transcripts in tentative cementoblasts. In situ hybridization revealed that ZFH/Chd3 was strongly expressed in Hertwig's epithelial root sheath rather than in cementum. Moreover, its expression disappeared when root formation was advanced in the first molar. In contrast, Chd3 was continuously expressed in dental epithelial cells of the cervical loop, in which root extension is never terminated. CONCLUSION: These results suggest that ZFH/Chd3 might play an important role in tooth root development and subsequent cementogenesis.
Asunto(s)
ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Odontogénesis/genética , Raíz del Diente/crecimiento & desarrollo , Ameloblastos/fisiología , Animales , Técnicas de Cultivo de Célula , Ensamble y Desensamble de Cromatina/genética , Cemento Dental/fisiología , Órgano del Esmalte/crecimiento & desarrollo , Células Epiteliales/fisiología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Incisivo/crecimiento & desarrollo , Captura por Microdisección con Láser , Masculino , Mandíbula/citología , Ratones , Ratones Endogámicos ICR , Diente Molar/crecimiento & desarrollo , Morfogénesis/genética , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Ligamento Periodontal/citología , Germen Dentario/crecimiento & desarrolloRESUMEN
The mouse third molar (M3) develops postnatally and is thus a unique model for studying the integration of a non-mineralized tooth with mineralized bone. This study assessed the morphogenesis of the mouse M3, related to the alveolar bone, comparing M3 development with that of the first molar (M1), the most common model in odontogenesis. The mandibular M3 was evaluated from initiation to eruption by morphology and by assessing patterns of proliferation, apoptosis, osteoclast distribution, and gene expression. Three-dimensional reconstruction and explant cultures were also used. Initiation of M3 occurred perinatally, as an extension of the second molar (M2) which grew into a region of soft mesenchymal tissue above the M2, still far away from the alveolar bone. The bone-free M3 bud gradually became encapsulated by bone at the cap stage at postnatal day 3. Osteoclasts were first visible at postnatal day 4 when the M3 came into close contact with the bone. The number of osteoclasts increased from postnatal day 8 to postnatal day 12 to form a space for the growing tooth. The M3 had erupted by postnatal day 26. The M3, although smaller than the M1, passed through the same developmental stages over a similar time span but showed differences in initiation and in the timing of bone encapsulation.
Asunto(s)
Mandíbula/crecimiento & desarrollo , Tercer Molar/crecimiento & desarrollo , Morfogénesis/fisiología , Odontogénesis/fisiología , Fosfatasa Ácida/análisis , Proceso Alveolar/anatomía & histología , Proceso Alveolar/crecimiento & desarrollo , Animales , Apoptosis/fisiología , Biomarcadores/análisis , Resorción Ósea/patología , Resorción Ósea/fisiopatología , Proliferación Celular , Órgano del Esmalte/anatomía & histología , Órgano del Esmalte/crecimiento & desarrollo , Factor 4 de Crecimiento de Fibroblastos/análisis , Proteínas Hedgehog/análisis , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Hibridación in Situ , Isoenzimas/análisis , Mandíbula/anatomía & histología , Ratones , Diente Molar/anatomía & histología , Diente Molar/crecimiento & desarrollo , Tercer Molar/anatomía & histología , Osteoblastos/fisiología , Osteoclastos/fisiología , Osteogénesis/fisiología , Antígeno Nuclear de Célula en Proliferación/análisis , Fosfatasa Ácida Tartratorresistente , Técnicas de Cultivo de Tejidos , Calcificación de Dientes/fisiología , Erupción Dental/fisiología , Germen Dentario/anatomía & histología , Germen Dentario/crecimiento & desarrollo , Raíz del Diente/anatomía & histología , Raíz del Diente/crecimiento & desarrolloRESUMEN
PURPOSE: Previous studies have evaluated the presence of serotonin in the dental epithelia and mesenchyme during odontogenesis, suggesting its participation in tooth development. MATERIALS AND METHODS: Here, we used fluoxetine, a selective serotonin re-uptake inhibitor, at a dose of 10 mg/kg, administered for 20 days during pregnancy in 12 Wistar rats to examine the influence of this drug on the development of the enamel organ of the upper first molars of rat fetuses at 17 days of intra-uterine life (i.u.l.), and at one, five and ten days postpartum. The pregnant rats were anesthetized with xylazine at 10 mg/kg and ketamine at 25 mg/kg. The fetuses were removed and beheaded; their jaws were removed, and the upper jaws were exposed. The tissues were fixed in Bouin's fixative, decalcified in 5% nitric acid for 4 - 12 h, conventionally processed for microscopy, and embedded in paraffin. Serial sections of approximately 5 mum were obtained and stained with hematoxylin and eosin, as well as periodic acid-Schiff. RESULTS AND CONCLUSION: Morphological analysis showed no structural changes in the experimental group compared to the controls, suggesting that, at the dose used, fluoxetine does not interfere with serotonin-mediated development of the enamel organ or the process of amelogenesis.
Asunto(s)
Órgano del Esmalte/anatomía & histología , Órgano del Esmalte/efectos de los fármacos , Fluoxetina/administración & dosificación , Inhibidores Selectivos de la Recaptación de Serotonina/administración & dosificación , Amelogénesis/efectos de los fármacos , Amelogénesis/fisiología , Animales , Órgano del Esmalte/crecimiento & desarrollo , Femenino , Modelos Animales , Embarazo , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
Purpose: Previous studies have evaluated the presence of serotonin in the dental epithelia and mesenchyme during odontogenesis, suggesting its participation in tooth development. Materials and methods: Here, we used fluoxetine, a selective serotonin re-uptake inhibitor, at a dose of 10 mg/kg, administered for 20 days during pregnancy in 12 Wistar rats to examine the influence of this drug on the development of the enamel organ of the upper first molars of rat fetuses at 17 days of intra-uterine life (i.u.l.), and at one, five and ten days postpartum. The pregnant rats were anesthetized with xylazine at 10 mg/kg and ketamine at 25 mg/kg. The fetuses were removed and beheaded; their jaws were removed, and the upper jaws were exposed. The tissues were fixed in Bouin's fixative, decalcified in 5 percent nitric acid for 4 - 12 h, conventionally processed for microscopy, and embedded in paraffin. Serial sections of approximately 5 mm were obtained and stained with hematoxylin and eosin, as well as periodic acid-Schiff. Results and conclusion: Morphological analysis showed no structural changes in the experimental group compared to the controls, suggesting that, at the dose used, fluoxetine does not interfere with serotonin-mediated development of the enamel organ or the process of amelogenesis.
Asunto(s)
Animales , Femenino , Embarazo , Ratas , Órgano del Esmalte/anatomía & histología , Órgano del Esmalte/efectos de los fármacos , Fluoxetina/administración & dosificación , Inhibidores Selectivos de la Recaptación de Serotonina/administración & dosificación , Amelogénesis/efectos de los fármacos , Amelogénesis/fisiología , Órgano del Esmalte/crecimiento & desarrollo , Modelos Animales , Distribución Aleatoria , Ratas WistarRESUMEN
The implantation of cultured dental cell-cell re-associations allows for the reproduction of fully formed teeth, crown morphogenesis, epithelial histogenesis, mineralized dentin and enamel deposition, and root-periodontium development. Since vascularization is critical for organogenesis and tissue engineering, this work aimed to study: (a) blood vessel formation during tooth development, (b) the fate of blood vessels in cultured teeth and re-associations, and (c) vascularization after in vivo implantation. Ex vivo, blood vessels developed in the dental mesenchyme from the cap to bell stages and in the enamel organ, shortly before ameloblast differentiation. In cultured teeth and re-associations, blood-vessel-like structures remained in the peridental mesenchyme, but never developed into dental tissues. After implantation, both teeth and re-associations became revascularized, although later in the case of the re-associations. In implanted re-associations, newly formed blood vessels originated from the host, allowing for their survival, and affording conditions organ growth, mineralization, and enamel secretion.
Asunto(s)
Neovascularización Fisiológica/fisiología , Odontogénesis/fisiología , Ingeniería de Tejidos , Diente/irrigación sanguínea , Ameloblastos/fisiología , Amelogénesis/fisiología , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Diferenciación Celular/fisiología , Colágeno Tipo IV/análisis , Dentinogénesis/fisiología , Órgano del Esmalte/crecimiento & desarrollo , Epitelio/crecimiento & desarrollo , Mesodermo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Desnudos , Morfogénesis/fisiología , Técnicas de Cultivo de Órganos , Periodoncio/crecimiento & desarrollo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Diente/trasplante , Calcificación de Dientes/fisiología , Corona del Diente/crecimiento & desarrollo , Germen Dentario/crecimiento & desarrollo , Raíz del Diente/crecimiento & desarrollo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/análisisRESUMEN
Tissue engineering can now reproduce tooth from postnatal tooth cells. However, crown formation is not accurately reconstituted, even when the complex structure of the enamel dentin is reproduced. Here, we showed that a tissue-engineered (TE) tooth, exhibiting morphogenesis according to regular crown-cusp pattern formation, was produced by embryonic tooth germ cells in a three-dimensional scaffold. Heterogeneous cells dissociated from embryonic day 14 (E14) mice tooth germs were seeded on a scaffold and implanted under a kidney capsule in adult mice. The developmental process of the implants was examined for up to 14 d. At 5 d, the cells had formed initial tooth germ, followed by enamel-covered dentin tissue formed symmetrically. To study the developmental process, we examined the growth pattern using 5-bromo-2'-deoxyuridine (BrdU)-labeling analysis. The initial cell-proliferation patterns of the TE teeth were similar to that at the cap and early bell stages in natural teeth. This was particularly true in the cervical loop, which showed a similar distribution pattern of BrdU-positive cells in TE- and natural teeth. These results suggested that even when embryonic tooth germs are dissociated, the single cells can reconstitute tooth, and that enamel organ morphogenesis proceeds as in natural teeth.
Asunto(s)
Técnicas de Cultivo de Célula , Odontogénesis/fisiología , Ácido Poliglicólico/química , Ingeniería de Tejidos/métodos , Germen Dentario/citología , Animales , Antimetabolitos , Bromodesoxiuridina , Proliferación Celular , Dentina/crecimiento & desarrollo , Órgano del Esmalte/crecimiento & desarrollo , Epitelio/crecimiento & desarrollo , Masculino , Mesodermo/fisiología , Ratones , Trasplante de Tejidos , Corona del Diente/crecimiento & desarrollo , Germen Dentario/crecimiento & desarrolloRESUMEN
OBJECTIVES: The aim of this study is to clarify the functional significance of heat-shock protein (HSP)-25 during tooth development. DESIGN: We compared the expression of HSP-25 in the dental epithelial and mesenchymal cells with their proliferative activity during odontogenesis in rat molars on postnatal days 1-100 by immunohistochemistry using anti-HSP-25 and anti-5-bromo-2'-deoxyuridine (BrdU) for cell proliferation assay. RESULTS: On day 1, BrdU-immunoreactive cells were densely located in the inner enamel epithelium in the cervical loop and intercusped areas and the dental pulp adjacent to them, whereas HSP-25-immunoractivity (IR) was restricted to the cusped area where odontoblasts and ameloblasts had already differentiated. Subsequently, BrdU-IR shifted in the apical direction to be localized around Hertwig's epithelial root sheath during days 5-30, never overlapping with concomitantly apically-shifted HSP-25-IR. On days 60-100, BrdU-immunoreactive cells were hardly recognizable in the dental pulp, where HSP-25-IR was exclusively localized in the odontoblast layer. Furthermore, the odontoblast- and ameloblast-lineage cells exhibited two steps in the expression of HSP-25 throughout the postnatal stages: first, dental epithelial and pulpal mesenchymal cells showed a weak IR for HSP-25 after the cessation of their proliferative activity, and subsequently odontoblasts and ameloblasts consistently expressed an intense HSP-25-IR. CONCLUSION: Odontoblast- and ameloblast-lineage cells acquire HSP-25-IR after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. The consistent expression of HSP-25-IR in the formative cells may be involved in the maintenance of their functional integrity.