RESUMEN
The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-ß1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-ß1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-ß1 stimulation. These results suggested that TGF-ß1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.
Asunto(s)
Ameloblastos/metabolismo , Germen Dentario/metabolismo , Factores Generales de Transcripción/metabolismo , Transcripción Genética , Ameloblastos/efectos de los fármacos , Animales , Diferenciación Celular , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones Noqueados , Morfogénesis , Fosforilación , Ratas , Transducción de Señal , Proteínas Smad Reguladas por Receptores/metabolismo , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Factores Generales de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
OBJECTIVE: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). METHODOLOGY: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic ï¬broblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. RESULTS: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). CONCLUSION: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Compuestos de Calcio/farmacología , Cerámica/farmacología , Materiales de Obturación del Conducto Radicular/farmacología , Silicatos/farmacología , Células Madre/efectos de los fármacos , Materiales Biocompatibles/farmacología , Supervivencia Celular/efectos de los fármacos , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Factor 2 de Crecimiento de Fibroblastos/análisis , Factor 2 de Crecimiento de Fibroblastos/efectos de los fármacos , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Ensayo de Materiales , Neovascularización Fisiológica/efectos de los fármacos , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/análisis , Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Reproducibilidad de los Resultados , Estadísticas no Paramétricas , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacosRESUMEN
Abstract Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.
Asunto(s)
Humanos , Materiales de Obturación del Conducto Radicular/farmacología , Células Madre/efectos de los fármacos , Cerámica/farmacología , Silicatos/farmacología , Compuestos de Calcio/farmacología , Inductores de la Angiogénesis/farmacología , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Materiales Biocompatibles/farmacología , Ensayo de Materiales , Factor de Crecimiento Derivado de Plaquetas/análisis , Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Supervivencia Celular/efectos de los fármacos , Reproducibilidad de los Resultados , Factor 2 de Crecimiento de Fibroblastos/análisis , Factor 2 de Crecimiento de Fibroblastos/efectos de los fármacos , Estadísticas no Paramétricas , Neovascularización Fisiológica/efectos de los fármacos , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Citometría de FlujoRESUMEN
Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without ß-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by ß-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that ß-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.
Asunto(s)
Diferenciación Celular , Sulfatos de Condroitina/metabolismo , Embrión de Mamíferos/citología , Diente Molar/embriología , Odontoblastos/citología , Odontoblastos/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Himecromona/análogos & derivados , Himecromona/farmacología , Inmunohistoquímica , Hibridación in Situ , Ratones Endogámicos ICR , Odontoblastos/efectos de los fármacos , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Germen Dentario/ultraestructuraRESUMEN
OBJECTIVE: F-spondin is an extracellular matrix (ECM) protein that belongs to the thrombospondin type I repeat superfamily and is a negative regulator of bone mass. We have previously shown that f-spondin is specifically expressed in the dental follicle (DF), which gives rise to the periodontal ligament (PDL) during the tooth root formation stage. To investigate the molecular mechanism of PDL formation, we investigated the function of f-spondin in DF differentiation. DESIGN: The expression patterning of f-spondin in the developing tooth germ was compared with that of periodontal ligament-related genes, including runx2, type I collagen and periostin, by in situ hybridization analysis. To investigate the function of f-spondin during periodontal ligament formation, an f-spondin adenovirus was infected into the bell stage of the developing tooth germ, and the effect on dental differentiation was analyzed. RESULTS: F-spondin was specifically expressed in the DF of the developing tooth germ; by contrast, type I collagen, runx2 and periostin were expressed in the DF and in the alveolar bone. F-spondin-overexpresssing tooth germ exhibited a reduction in gene expression of periostin and type I collagen in the DF. By contrast, the knockdown of f-spondin in primary DF cells increased the expression of these genes. Treatment with recombinant f-spondin protein functionally inhibited periostin expression induced by transforming growth factor-ß (TGF-ß). CONCLUSION: Our data indicated that f-spondin inhibits the differentiation of DF cells into periodontal ligament cells by inhibiting TGF-ß. These data suggested that f-spondin negatively regulates PDL differentiation which may play an important role in the immature phenotype of DF.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Saco Dental/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Adenoviridae/genética , Animales , Animales Modificados Genéticamente , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Saco Dental/citología , Saco Dental/metabolismo , Proteínas de la Matriz Extracelular/genética , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Hibridación in Situ , Células Madre Mesenquimatosas/citología , Ratones Endogámicos C57BL , Ligamento Periodontal/citología , Ligamento Periodontal/efectos de los fármacos , Ligamento Periodontal/crecimiento & desarrollo , Ligamento Periodontal/metabolismo , Proteínas Recombinantes , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Germen Dentario/metabolismo , Raíz del Diente/crecimiento & desarrollo , Raíz del Diente/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Mutations in MSX1 cause craniofacial developmental defects, including tooth agenesis, in humans and mice. Previous studies suggest that Msx1 activates Bmp4 expression in the developing tooth mesenchyme to drive early tooth organogenesis. Whereas Msx1-/- mice exhibit developmental arrest of all tooth germs at the bud stage, mice with neural crest-specific inactivation of Bmp4 (Bmp4ncko/ncko), which lack Bmp4 expression in the developing tooth mesenchyme, showed developmental arrest of only mandibular molars. We recently demonstrated that deletion of Osr2, which encodes a zinc finger transcription factor expressed in a lingual-to-buccal gradient in the developing tooth bud mesenchyme, rescued molar tooth morphogenesis in both Msx1-/- and Bmp4ncko/ncko mice. In this study, through RNA-seq analyses of the developing tooth mesenchyme in mutant and wildtype embryos, we found that Msx1 and Osr2 have opposite effects on expression of several secreted Wnt antagonists in the tooth bud mesenchyme. Remarkably, both Dkk2 and Sfrp2 exhibit Osr2-dependent preferential expression on the lingual side of the tooth bud mesenchyme and expression of both genes was up-regulated and expanded into the tooth bud mesenchyme in Msx1-/- and Bmp4ncko/ncko mutant embryos. We show that pharmacological activation of canonical Wnt signaling by either lithium chloride (LiCl) treatment or by inhibition of DKKs in utero was sufficient to rescue mandibular molar tooth morphogenesis in Bmp4ncko/ncko mice. Furthermore, whereas inhibition of DKKs or inactivation of Sfrp2 alone was insufficient to rescue tooth morphogenesis in Msx1-/- mice, pharmacological inhibition of DKKs in combination with genetic inactivation of Sfrp2 and Sfrp3 rescued maxillary molar morphogenesis in Msx1-/- mice. Together, these data reveal a novel mechanism that the Bmp4-Msx1 pathway and Osr2 control tooth organogenesis through antagonistic regulation of expression of secreted Wnt antagonists.
Asunto(s)
Proteína Morfogenética Ósea 4/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factor de Transcripción MSX1/metabolismo , Proteínas de la Membrana/metabolismo , Organogénesis , Transducción de Señal , Diente/embriología , Proteínas Wnt/antagonistas & inhibidores , Animales , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/genética , Cloruro de Litio/farmacología , Mandíbula/efectos de los fármacos , Mandíbula/embriología , Mandíbula/metabolismo , Proteínas de la Membrana/genética , Ratones Noqueados , Diente Molar/efectos de los fármacos , Diente Molar/embriología , Diente Molar/metabolismo , Morfogénesis/efectos de los fármacos , Organogénesis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Diente/efectos de los fármacos , Diente/metabolismo , Germen Dentario/efectos de los fármacos , Germen Dentario/embriología , Germen Dentario/metabolismo , Factores de Transcripción , Proteínas Wnt/metabolismoRESUMEN
OBJECTIVE: High fluoride can induce stress-mediated apoptosis and degradation of ameloblasts. Fas ligand (FasL) has been regarded as a key regulator in intracellular responses for stress-induced apoptosis in reproductive or cancerous cell lineages. The objective of this study is to explore the role of FasL in the regulation of ameloblast ultrastructure damage. DESIGN: Primary ameloblasts were isolated from the molar tooth germ of 4-day-old SD rats. The ameloblasts were incubated with 3.2mM NaF or nothing. After incubation for different time arranging from 12h to 72h, ELISA was used to detected the secretion levels of FasL in the medium. Then at 48h post treatment, the ameloblast ultrastructure was detected with Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM), and expression of apoptotic proteins and peroxidative enzymes/products were examined. Finally, a specific FasL inhibitor was applied to co-treat the ameloblasts with NaF, and the ameloblast ultrastructure was detected with TEM and SEM. RESULTS: The secretion of FasL was notably increased by 3.2mM NaF treatment, and the increase reached to the peak after incubation for 48h. High fluoride incubation damaged the ameloblast untrastructure manifesting a series of intracelluar stress responsing cell organelle destruction, and a marked increase in expression of apoptotic genes and oxidative stress. The FasL inhibitor treatment partially mitigated the untrastructure damage caused by high dose NaF. CONLUSION: High-fluoride leads to damage of the ameloblast ultrastructure through paritially acitivating the FasL signalling pathway.
Asunto(s)
Ameloblastos/efectos de los fármacos , Ameloblastos/ultraestructura , Proteína Ligando Fas/metabolismo , Transducción de Señal/efectos de los fármacos , Fluoruro de Sodio/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Proteína Ligando Fas/antagonistas & inhibidores , Microscopía Electrónica , Diente Molar , Estrés Oxidativo , Ratas , Germen Dentario/efectos de los fármacosRESUMEN
Although several heat shock proteins have been investigated in relation to tooth development, no available information is available about the spatial and temporal expression pattern of heat shock protein 60 (Hsp 60). To characterize Hsp 60 expression in the structures of the developing tooth germ, we used Western blotting, immunohistochemistry and in situ hybridization. Hsp 60 was present in high amounts in the inner and outer enamel epithelia, enamel knot (EK) and stratum intermedium (SI). Hsp 60 also appeared in odontoblasts beginning in the bell stage. To obtain data on the possible effect of Hsp 60 on isolated lower incisors from mice, we performed in vitro culturing. To investigate the effect of exogenous Hsp 60 on the cell cycle during culturing, we used the 5-bromo-2-deoxyuridine (BrdU) incorporation test on dental cells. Exogenously administered Hsp 60 caused bluntness at the apical part of the 16.5-day-old tooth germs, but it did not influence the proliferation rate of dental cells. We identified the expression of Hsp 60 in the developing tooth germ, which was present in high concentrations in the inner and outer enamel epithelia, EK, SI and odontoblasts. High concentration of exogenous Hsp 60 can cause abnormal morphology of the tooth germ, but it did not influence the proliferation rate of the dental cells. Our results suggest that increased levels of Hsp 60 may cause abnormalities in the morphological development of the tooth germ and support the data on the significance of Hsp during the developmental processes.
Asunto(s)
Chaperonina 60/farmacología , Odontogénesis/efectos de los fármacos , Animales , Western Blotting , Inmunohistoquímica , Hibridación in Situ , Incisivo , Ratones , Germen Dentario/efectos de los fármacosRESUMEN
OBJECTIVE: The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development. MATERIALS AND METHODS: TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR. RESULTS: Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration. CONCLUSION: These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.
Asunto(s)
Transducción de Señal/fisiología , Receptor Toll-Like 4/fisiología , Calcificación de Dientes/fisiología , Germen Dentario/fisiología , Ameloblastos/efectos de los fármacos , Animales , Colágeno Tipo X/análisis , Colágeno Tipo X/efectos de los fármacos , Esmalte Dental/efectos de los fármacos , Esmalte Dental/metabolismo , Proteínas del Esmalte Dental/análisis , Proteínas del Esmalte Dental/efectos de los fármacos , Dentina/efectos de los fármacos , Dentina/metabolismo , Órgano del Esmalte/efectos de los fármacos , Órgano del Esmalte/metabolismo , Proteínas I-kappa B/análisis , Proteínas I-kappa B/efectos de los fármacos , Lipopolisacáridos/farmacología , Ratones , Odontoblastos/efectos de los fármacos , Odontoblastos/metabolismo , Odontogénesis/efectos de los fármacos , Odontogénesis/fisiología , Técnicas de Cultivo de Órganos , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/efectos de los fármacos , Calcificación de Dientes/efectos de los fármacos , Germen Dentario/efectos de los fármacosRESUMEN
Dithiocarbamate propinebs are organometal fungicides that are widely used for the control of diseases in plants. In this study, pregnant female rats received 400 ppm propineb concentrations in 5 ml distilled water for 16 days of gestation, and then infant rats were obtained by cesarean section. In the histological analysis on the frontal sections, the use of propineb was found effective on odontoblast cell hyperplasia, cell infiltration in the dental papilla, and degeneration in the mesenchymal cells of the outer enamel. The expression of MMP-2 (Matrix Metalloproteinase-2) and VEGF (Endothelial cell growth factor) in the connective tissue was evaluated by immunohistochemistry. The drinking water given to the mothers in propineb tooth bud, enamel and dentin, resulted in morphological changes suggestive of a delay in formation, which cross the placental barrier and possibly affect the tooth development.
Los ditiocarbamatos (Propineb) son fungicidas organometálicos que son ampliamente utilizados para el control de enfermedades en las plantas. En este estudio, ratas hembras preñadas recibieron concentraciones de 4000 ppm de propineb en 5 ml de agua destilada durante 16 días de su gestación. Luego, las crías de las ratas fueron obtenidas mediante cesárea para su estudio estudio histológico. En el análisis histológico de las secciones frontales, el uso de propineb fue positivo para la hiperplasia de las células odontoblástica, infiltración de células en la papila dental, y la degeneración en las células mesenquimales del epitelio externo del esmalte. La expresión de MMP-2 (metaloproteinasa de la matriz 2) y VEGF (factor de crecimiento de células endoteliales) en el tejido conectivo se evaluó por inmunohistoquímica. El agua potable con propineb dada a las madres actuó sobre el brote dentario, esmalte y dentina; se tradujo en cambios morfológicos indicativos de un retraso en la formación. Por tanto, el propineb atraviesa la barrera placentaria y posiblemente afecten el desarrollo de los dientes.
Asunto(s)
Animales , Masculino , Femenino , Embarazo , Ratas , Fungicidas Industriales/toxicidad , Odontogénesis/efectos de los fármacos , Zineb/análogos & derivados , Esmalte Dental/efectos de los fármacos , Dentina/efectos de los fármacos , Inmunohistoquímica , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Germen Dentario/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Zineb/toxicidadRESUMEN
BMP and Wnt signaling pathways play a crucial role in organogenesis, including tooth development. Despite extensive studies, the exact functions, as well as if and how these two pathways act coordinately in regulating early tooth development, remain elusive. In this study, we dissected regulatory functions of BMP and Wnt pathways in early tooth development using a transgenic noggin (Nog) overexpression model (K14Cre;pNog). It exhibits early arrested tooth development, accompanied by reduced cell proliferation and loss of odontogenic fate marker Pitx2 expression in the dental epithelium. We demonstrated that overexpression of Nog disrupted BMP non-canonical activity, which led to a dramatic reduction of cell proliferation rate but did not affect Pitx2 expression. We further identified a novel function of Nog by inhibiting Wnt/ß-catenin signaling, causing loss of Pitx2 expression. Co-immunoprecipitation and TOPflash assays revealed direct binding of Nog to Wnts to functionally prevent Wnt/ß-catenin signaling. In situ PLA and immunohistochemistry on Nog mutants confirmed in vivo interaction between endogenous Nog and Wnts and modulation of Wnt signaling by Nog in tooth germs. Genetic rescue experiments presented evidence that both BMP and Wnt signaling pathways contribute to cell proliferation regulation in the dental epithelium, with Wnt signaling also controlling the odontogenic fate. Reactivation of both BMP and Wnt signaling pathways, but not of only one of them, rescued tooth developmental defects in K14Cre;pNog mice, in which Wnt signaling can be substituted by transgenic activation of Pitx2. Our results reveal the orchestration of non-canonical BMP and Wnt/ß-catenin signaling pathways in the regulation of early tooth development.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Diente/embriología , Diente/metabolismo , Vía de Señalización Wnt , Animales , Proteínas Portadoras/metabolismo , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Integrasas/metabolismo , Factor de Transcripción MSX1/metabolismo , Mesodermo/embriología , Ratones Transgénicos , Modelos Biológicos , Odontogénesis/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Smad/metabolismo , Diente/citología , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Germen Dentario/embriología , Germen Dentario/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
The dentition in zebrafish is extremely and richly vascularized, but the function of the vasculature, in view of the continuous replacement of the teeth, remains elusive. Through application of SU5416, a vascular endothelial growth factor receptor inhibitor, we studied the role of the blood vessels in the dentition of the zebrafish. We were unable to show an effect on the development of first-generation teeth as well as first tooth replacement. However, in juvenile fish, a delay was observed in the developmental state of the replacement tooth compared with what was expected based on the maturation state of the functional tooth. Furthermore, we observed a difference between treated and nontreated fish in the distance between blood vessels and developing replacement teeth. In conclusion, our results provide support for a nutritive, rather than an inductive, function of the vasculature in the process of tooth development and replacement.
Asunto(s)
Odontogénesis/efectos de los fármacos , Diente/crecimiento & desarrollo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Proteínas de Pez Cebra/antagonistas & inhibidores , Pez Cebra/crecimiento & desarrollo , Fosfatasa Alcalina/análisis , Inhibidores de la Angiogénesis/farmacología , Animales , Vasos Sanguíneos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Indoles/farmacología , Larva/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Pirroles/farmacología , Diente/irrigación sanguínea , Diente/efectos de los fármacos , Germen Dentario/efectos de los fármacosRESUMEN
Our previous research has shown that the extracellular matrix metalloproteinase inducer (EMMPRIN) is expressed during and may function in the early development of tooth germs. In the present study, we observed the specific expression of EMMPRIN in ameloblasts and odontoblasts during the middle and late stages of tooth germ development using immunohistochemistry. Furthermore, to extend our understanding of the function of EMMPRIN in odontogenesis, we used an anti-EMMPRIN function-blocking antibody to remove EMMPRIN activity in tooth germ culture in vitro. Both the formation and mineralisation of dental hard tissues were suppressed in the tooth germ culture after the abrogation of EMMPRIN. Meanwhile, significant reductions in VEGF, MMP-9, ALPL, ameloblastin, amelogenin and enamelin expression were observed in antibody-treated tooth germ explants compared to control and normal serum-treated explants. The current results illustrate that EMMPRIN may play a critical role in the processing and maturation of the dental matrix.
Asunto(s)
Basigina/genética , Calcificación Fisiológica/genética , Diente Molar/metabolismo , Ameloblastos/citología , Ameloblastos/enzimología , Ameloblastos/metabolismo , Animales , Anticuerpos Neutralizantes/farmacología , Basigina/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Expresión Génica , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Odontogénesis/genética , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Germen Dentario/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
MicroRNAs (miRNAs) are known to regulate a variety of gene functions in many tissues and organs, but their expression and function in tooth development are not well-understood. A specific miRNA, miR-135a, was determined to be highly expressed at the bud stage. Interestingly, after the cap stage, miR-135a was expressed in the epithelium and mesenchyme but not in the inner enamel epithelium. To identify the relationship between miR-135a and its putative target genes, Bmpr-Ia and Bmpr-Ib, in early tooth development, miR-135a was ectopically overexpressed with a lentivirus. This overexpression resulted in the repression of Bmpr-Ia and Bmpr-Ib. Furthermore, miR-135a inhibited both Bmpr-Ia and Bmpr-Ib transcription. BMP2 proteins were expressed ectopically in tooth germs during the cap stage to determine the relationship between miR-135a and BMP signaling in early tooth development. When miR-135a was ectopically expressed, no tooth formation was observed after 4 wk of incubation in the kidney capsule. This study suggested that Bmp signaling, specifically Bmpr-Ia and Bmpr-Ib, regulates tooth formation via miR-135a.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Regulación de la Expresión Génica/genética , MicroARNs/genética , Odontogénesis/genética , Transducción de Señal/genética , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/farmacología , Epitelio/metabolismo , Vectores Genéticos/genética , Lentivirus/genética , Masculino , Mesodermo/metabolismo , Ratones , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Germen Dentario/efectos de los fármacos , Germen Dentario/metabolismo , Germen Dentario/trasplante , Transcripción Genética/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Periodontal ligament stem cells (PDLSCs) have recently been proposed as a novel option in periodontal regenerative therapy. However, one of the issues is the difficulty of stably generating PDLSCs because of the variation of stem cell potential between donors. Here, we show that Semaphorin 3A (Sema3A) can induce mesenchymal-stem-like properties in human periodontal ligament (PDL) cells. Sema3A expression was specifically observed in the dental follicle during tooth development and in parts of mature PDL tissue in rodent tooth and periodontal tissue. Sema3A expression levels were found to be higher in multipotential human PDL cell clones compared with low-differentiation potential clones. Sema3A-overexpressing PDL cells exhibited an enhanced capacity to differentiate into both functional osteoblasts and adipocytes. Moreover, PDL cells treated with Sema3A only at the initiation of culture stimulated osteogenesis, while Sema3A treatment throughout the culture had no effect on osteogenic differentiation. Finally, Sema3A-overexpressing PDL cells upregulated the expression of embryonic stem cell markers (NANOG, OCT4, and E-cadherin) and mesenchymal stem cell markers (CD73, CD90, CD105, CD146, and CD166), and Sema3A promoted cell division activity of PDL cells. These results suggest that Sema3A may possess the function to convert PDL cells into mesenchymal-stem-like cells.
Asunto(s)
Células Madre Mesenquimatosas/citología , Ligamento Periodontal/citología , Semaforina-3A/farmacología , Animales , Biomarcadores/metabolismo , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos BALB C , Osteogénesis/efectos de los fármacos , Ligamento Periodontal/efectos de los fármacos , Ligamento Periodontal/embriología , Ligamento Periodontal/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/farmacología , Germen Dentario/efectos de los fármacos , Germen Dentario/embriología , Germen Dentario/metabolismoRESUMEN
Vascularization is essential for organ and tissue development. Teeth develop through interactions between epithelium and mesenchyme. The developing capillaries in the enamel organ, the dental epithelial structure, occur simultaneously by mechanisms of vasculogenesis and angiogenesis at the onset of dentinogenesis. The vascular neoformation in the dental mesenchyme has been reported to start from the cap stage. However, the mechanisms of vascularization in the dental mesenchyme remain unknown. In the hope of understanding the mechanisms of the formation of dental mesenchymal vasculature, mouse lower molar germs from embryonic day (E) 13.5 to E16.5 were processed for immunostaining of CD31 and CD34, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and transmission electron microscopy (TEM). In addition, the role of apoptosis for the vascularization in dental mesenchyme was examined by in vitro culture of E14.0 lower molars in the presence of the apoptosis inhibitor (z-VAD-fmk) and a subsequent subrenal culture. Our results showed that CD31- and CD34-positive cells progressively entered the central part of the dental papilla from the peridental mesenchyme. For TEM, angioblasts, young capillaries with thick endothelium and endothelial cells containing vacuoles were observed in peripheral dental mesenchyme, suggesting vasculogenesis was taking place. The presence of lateral sprouting, cytoplasmic filopodia and transluminal bridges in the dental papilla suggested angiogenesis was also occurring. Inhibition of apoptosis delayed the angiogenic vascularization of the dental papilla. Therefore, these data demonstrated that molar mesenchyme is progressively vascularized by mechanisms of both vasculogenesis and angiogenesis and apoptosis partially contributes to the vascularization of the dental papilla.
Asunto(s)
Apoptosis , Capilares/ultraestructura , Mesodermo/irrigación sanguínea , Mesodermo/embriología , Diente Molar/irrigación sanguínea , Diente Molar/embriología , Neovascularización Fisiológica , Clorometilcetonas de Aminoácidos/farmacología , Animales , Antígenos CD34/metabolismo , Apoptosis/efectos de los fármacos , Capilares/embriología , Técnica del Anticuerpo Fluorescente , Etiquetado Corte-Fin in Situ , Mesodermo/citología , Mesodermo/ultraestructura , Ratones , Ratones Endogámicos ICR , Diente Molar/citología , Diente Molar/ultraestructura , Neovascularización Fisiológica/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Transporte de Proteínas/efectos de los fármacos , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Germen Dentario/embriología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
UNLABELLED: Dental amalgam is the most common restorative material used in dentistry. It was reported that amalgam might constitute potential toxic hazards to pregnant patients and foetuses through mercury release and absorption. The present study aimed to investigate the vital tissue response in contact to dental amalgam plus determination of blood mercury levels in mother and offspring Wistar strain albino rats. Pregnant mothers were divided into two main groups each had dental amalgam implanted into either an oral mucosa incision or a bony socket following extraction. Third and fourth groups included the offspring rats of mothers from the first and second groups, respectively. The blood mercury levels and histopathology of oral tissues were analyzed in mothers at one and six months post-implantation and in offspring rats one day after birth. The blood mercury levels of mothers increased significantly at six months (P<0.01) as compared to levels at one month. However, blood mercury levels were not significant (P>0.05) when the two offspring (third and fourth) groups were compared. Histopathology results from mothers showed inflammatory response at the bottom of the socket, one month after amalgam implantation. At six months, teeth germs showed vacuolation of the abnormal odontoblasts with globular dentine formation. Degenerated periodontal fibres and thin trabeculae forming the bony sockets with large marrow spaces were evident. A fibrous connective tissue capsule surrounded the amalgam mass inside the mucosa of mothers at one month and was evident also at 6 months with a huge inflammatory cell infiltrate. Teeth germs showed elongated odontoblasts with intercellular oedema, thinner dentine and bony trabeculae with wider marrow spaces. Offspring rats showed comparable oral tissue response. CONCLUSIONS: There is a positive correlation between blood mercury levels and oral tissue response in mothers, however, the negative impact of mercury on oral tissues of offspring rats was due to high mercury levels in their mothers' blood during pregnancy. We would recommend that women should - as far as possible - postpone having dental amalgam filling placed or removed during pregnancy to avoid its harmful effect on the foetus. Further clinical studies are recommended to test our findings in man.
Asunto(s)
Amalgama Dental/química , Mercurio/efectos adversos , Mucosa Bucal/efectos de los fármacos , Germen Dentario/efectos de los fármacos , Alveolo Dental/efectos de los fármacos , Animales , Animales Recién Nacidos , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Tejido Conectivo/efectos de los fármacos , Tejido Conectivo/patología , Dentina/efectos de los fármacos , Dentina/patología , Dentinogénesis/efectos de los fármacos , Femenino , Mercurio/sangre , Mercurio/química , Mucosa Bucal/patología , Odontoblastos/efectos de los fármacos , Odontoblastos/patología , Ligamento Periodontal/efectos de los fármacos , Ligamento Periodontal/patología , Embarazo/sangre , Efectos Tardíos de la Exposición Prenatal , Ratas , Ratas Wistar , Estomatitis/inducido químicamente , Factores de Tiempo , Lengua/efectos de los fármacos , Germen Dentario/patología , Alveolo Dental/patologíaRESUMEN
We have previously demonstrated that tooth size is determined by dental mesenchymal factors. Exogenous bone morphogenetic protein (BMP)4, Noggin, fibroblast growth factor (FGF)3 and FGF10 have no effect on tooth size, despite the expressions of Bmp2, Bmp4, Fgf3, Fgf10 and Lef1 in the dental mesenchyme. Among the wingless (Wnt) genes that are differentially expressed during tooth development, only Wnt5a is expressed in the dental mesenchyme. The aims of the present study were to clarify the expression pattern of Wnt5a in developing tooth germs and the role of Wnt5a in the regulation of tooth size by treatment with exogenous WNT5A with/without an apoptosis inhibitor on in vitro tooth germs combined with transplantation into kidney capsules. Wnt5a was intensely expressed in both the dental epithelium and mesenchyme during embryonic days 14-17, overlapping partly with the expressions of both Shh and Bmp4. Moreover, WNT5A retarded the development of tooth germs by markedly inducing cell death in the non-dental epithelium and mesenchyme but not widely in the dental region, where the epithelial-mesenchymal gene interactions among Wnt5a, Fgf10, Bmp4 and Shh might partly rescue the cells from death in the WNT5A-treated tooth germ. Together, these results indicate that WNT5A-induced cell death inhibited the overall development of the tooth germ, resulting in smaller teeth with blunter cusps after tooth-germ transplantation. Thus, it is suggested that Wnt5a is involved in regulating cell death in non-dental regions, while in the dental region it acts as a regulator of other genes that rescue tooth germs from cell death.
Asunto(s)
Diente/anatomía & histología , Diente/embriología , Proteínas Wnt/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Bioensayo , Tipificación del Cuerpo/efectos de los fármacos , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Tampones (Química) , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Modelos Biológicos , Tamaño de los Órganos/efectos de los fármacos , Diente/citología , Diente/efectos de los fármacos , Germen Dentario/citología , Germen Dentario/efectos de los fármacos , Germen Dentario/embriología , Germen Dentario/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/farmacología , Proteína Wnt-5aRESUMEN
Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoA(DN)) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoA(DN) fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy. The 3 strains expressed at either low (TgEGFP-RhoA(DN)-8), intermediate (TgEGFP-RhoA(DN)-2), or high (TgEGFP-RhoA(DN)-13) levels, and the molar teeth from the 3 strains had enamel hypoplasia and surface defects. We conclude that RhoA(DN) expressed in ameloblasts interferes with normal enamel development through the pathway that is induced by sodium fluoride.
Asunto(s)
Ameloblastos/metabolismo , Esmalte Dental/metabolismo , Esmalte Dental/patología , Genes Dominantes/genética , Proteína de Unión al GTP rhoA/metabolismo , Ameloblastos/efectos de los fármacos , Ameloblastos/patología , Animales , Esmalte Dental/efectos de los fármacos , Esmalte Dental/ultraestructura , Ratones , Ratones Transgénicos , Diente Molar/efectos de los fármacos , Diente Molar/metabolismo , Diente Molar/patología , Diente Molar/ultraestructura , Inhibidores de Proteínas Quinasas/farmacología , Germen Dentario/efectos de los fármacos , Germen Dentario/patología , Transgenes/genéticaRESUMEN
INTRODUCTION: Biocompatibility of pulp capping materials is important for successful use in dentistry. These materials should be nontoxic and permissive for proliferation and induction of odontogenic differentiation of pulp cells. The aim of our study was to evaluate the effects of enamel matrix derivative (EMD), mineral trioxide aggregate (MTA), and calcium hydroxide-containing cement (DYCAL) on proliferation and odontogenic differentiation of human tooth germ stem cells (hTGSCs) in which cells belonging to both pulp tissue and dental follicle exist. METHODS: The 96-well plates, 24-well plates, and special chamber slides were coated with biomaterials for cell proliferation, differentiation, and scanning electron microscopy analysis. Odontogenic differentiation of hTGSCs was evaluated by analyzing mRNA expression of dentin sialophosphoprotein (DSPP) by real-time polymerase chain reaction expression analysis, measurement of alkaline phosphatase activity, and visualization of calcium depositions by von Kossa staining. RESULTS: Our results demonstrate that EMD is the best material in terms of inducing differentiation and proliferation of hTGSCs. DYCAL was found to be toxic to hTGSCs; however, EMD-coated DYCAL showed less toxicity. EMD-coated MTA was not efficient at inducing proliferation and differentiation. CONCLUSIONS: Pulp capping materials come in direct contact with dental pulp cells; thus, they require comprehensive evaluation of interactions between cells and biomaterials. Therefore, we cultured hTGSCs, capable of odontogenic differentiation, on pulp capping materials directly. Our results suggest that combination of capping materials with EMD would increase the quality of capping by increasing biocompatibility of capping materials.