Genetic evidence for the vital function of osterix in cementogenesis

To date, attempts to regenerate a complete tooth, including the critical periodontal tissues associated with the tooth root, have not been successful. Controversy still exists regarding the origin of the cell source for cellular cementum (epithelial or mesenchymal). This disagreement may be partially due to a lack of understanding of the events leading to the initiation and development of the tooth roots and supportive tissues, such as the cementum. Osterix (OSX) is a transcriptional factor essential for osteogenesis, but its role in cementogenesis has not been addressed. In the present study, we first documented a close relationship between the temporal- and spatial-expression pattern of OSX and the formation of cellular cementum. We then generated 3.6 Col 1-OSX transgenic mice, which displayed accelerated cementum formation vs. WT controls. Importantly, the conditional deletion of OSX in the mesenchymal cells with two different Cre systems (the 2.3 kb Col 1 and an inducible CAG-CreER) led to a sharp reduction in cellular cementum formation (including the cementum mass and mineral deposition rate) and gene expression of dentin matrix protein 1 (DMP1) by cementocytes. However, the deletion of the OSX gene after cellular cementum formed did not alter the properties of the mature cementum as evaluated by backscattered SEM and resin-cast SEM. Transient transfection of Osx in the cementoblasts in vitro significantly inhibited cell proliferation and increased cell differentiation and mineralization. Taken together, these data support 1) the mesenchymal origin of cellular cementum (from PDL progenitor cells); 2) the vital role of OSX in controlling the formation of cellular cementum; and 3) the limited remodeling of cellular cementum in adult mice.

Effects of vitamin K deficiency and its mechanisms in vertebrate's early development: zebrafish as a model

Disertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

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Implantación de Geoportales con soporte técnico profesionalizado en software libre

La creciente dinamización de las IDE's genera una demanda de la construcción de Geoportales y por ende la demanda de herramientas que además de facilitar su construcción, configuración e implementación, ofrezcan la posibilidad de contratar un soporte técnico profesionalizado. OpenGeo Suite, paquete de software libre profesional e integrado, que permite desde el almacenamiento de datos geográficos, hasta su publicación utilizando estándares OGC e implementación de soluciones web GIS con librerías de código abierto Javascript. OpenGeo Suite permite un despliegue multiplataforma (Linux, Windows y OSX), con cuatro componentes de software libre fuertemente integrados basados en el uso de estándares OGC. Los componentes del lado del servidor están orientados al almacenamiento, configuración y publicación de datos por parte de usuarios técnicos en SIG: PostgreSQL+ la extensión espacial PostGIS que se encarga del almacenamiento de la información geográfica dando soporte a funciones de análisis espacial. pgAdmin como sistema de gestión de base de datos, facilitando la importación y actualización de datos. Geoserver se encarga de la publicación de la información geográfica proveniente de diferentes orígenes de datos: PostGIS, SHP, Oracle Spatial, GeoTIFF, etc. soportando la mayoría de estándares OGC de publicación de información geográfica WMS, WFS, WCS y de formatos GML, KML, GeoJSON, SLD. Además, ofrece soporte a cacheado de teselas a través de Geowebcache. OpenGeo Suite ofrece dos aplicaciones: GeoExplorer y GeoEditor, que permiten al técnico construir un Geoportal con capacidades de edición de geometrías.OpenGeo Suite ofrece una consola de administración (Dashboard) que facilita la configuración de los componentes de administración. Del lado del cliente, los componentes son librerías de desarrollo JavaScript orientadas a desarrolladores de aplicaciones Web SIG. OpenLayers con soporte para capas raster, vectoriales, estilos, proyecciones, teselado, herramientas de edición, etc. Por último, GeoExt para la construcción del front-end de Geoportales, basada en ExtJS y fuertemente acoplada a OpenLayers

Caracterização de osteoblastos diferenciados de células-tronco mesenquimais da medula óssea de ratos espontâneamente hipertensos (SHR)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering

A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)calcium phosphate (PLGACaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470590, 590850 and 8501200 mu m. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470590 mu m. These results show that PLGACaP is a promising biomaterial for bone engineering. However, ideally, combinations of larger (similar to 1000 mu m) and smaller (similar to 500 mu m) pores in a single scaffold would optimize cellular and tissue responses during bone healing. Copyright (C) 2011 John Wiley & Sons, Ltd.

Unveiling novel genes upregulated by both rhBMP2 and rhBMP7 during early osteoblastic transdifferentiation of C2C12 cells

Abstract Findings We set out to analyse the gene expression profile of pre-osteoblastic C2C12 cells during osteodifferentiation induced by both rhBMP2 and rhBMP7 using DNA microarrays. Induced and repressed genes were intercepted, resulting in 1,318 induced genes and 704 repressed genes by both rhBMP2 and rhBMP7. We selected and validated, by RT-qPCR, 24 genes which were upregulated by rhBMP2 and rhBMP7; of these, 13 are related to transcription (Runx2, Dlx1, Dlx2, Dlx5, Id1, Id2, Id3, Fkhr1, Osx, Hoxc8, Glis1, Glis3 and Cfdp1), four are associated with cell signalling pathways (Lrp6, Dvl1, Ecsit and PKCδ) and seven are associated with the extracellular matrix (Ltbp2, Grn, Postn, Plod1, BMP1, Htra1 and IGFBP-rP10). The novel identified genes include: Hoxc8, Glis1, Glis3, Ecsit, PKCδ, LrP6, Dvl1, Grn, BMP1, Ltbp2, Plod1, Htra1 and IGFBP-rP10. Background BMPs (bone morphogenetic proteins) are members of the TGFβ (transforming growth factor-β) super-family of proteins, which regulate growth and differentiation of different cell types in various tissues, and play a critical role in the differentiation of mesenchymal cells into osteoblasts. In particular, rhBMP2 and rhBMP7 promote osteoinduction in vitro and in vivo, and both proteins are therapeutically applied in orthopaedics and dentistry. Conclusion Using DNA microarrays and RT-qPCR, we identified both previously known and novel genes which are upregulated by rhBMP2 and rhBMP7 during the onset of osteoblastic transdifferentiation of pre-myoblastic C2C12 cells. Subsequent studies of these genes in C2C12 and mesenchymal or pre-osteoblastic cells should reveal more details about their role during this type of cellular differentiation induced by BMP2 or BMP7. These studies are relevant to better understanding the molecular mechanisms underlying osteoblastic differentiation and bone repair.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

Osterix inhibits bone destruction in osteosarcoma through transcriptional repression of Interleukin-1alpha expression

Osteosarcoma, a malignant bone tumor, rapidly destroys the cortical bone. We demonstrated that mouse K7M2 osteosarcoma cells were deficient in osterix (osx), a zinc finger-containing transcription factor required for osteoblasts differentiation and bone formation. These cells formed lytic tumors when injected into the tibia. The destruction of bone is mediated by osteoclasts in osteosarcoma. The less expression of osterix with osteolytic phenotype was also observed in more tumor cell lines. Replacement of osterix in K7M2 cells suppressed lytic bone destruction, inhibited tumor growth in vitro and in vivo, and suppressed lung metastasis in vivo and the migration of K7M2 to lung conditioned medium in vitro. By contrast, inhibiting osterix by vector-based small interfering RNA (siRNA) in two cell lines (Dunn and DLM8) that expressed high levels of osterix converted osteoblastic phenotype to lytic. Recognizing and binding of Receptor Activator of NF-κB (RANK) on osteoclast precursors by its ligand RANKL is the key osteoclastogenic event. Increased RANKL results in more osteoclast activity. We investigated whether K7M2-mediated bone destruction was secondary to an effect on RANKL. The conditioned medium from K7M2 could upregulate RANKL in normal osteoblast MC3T3, which might lead to more osteoclast formation. By contrast, the conditioned medium from K7M2 cells transfected with osx-expressing plasmid did not upregulate RANKL. Furthermore, Interleukin-1alpha (IL-1α) was significantly suppressed following osx transfection. IL-1α increased RANKL expression in MC3T3 cells, suggesting that osx may control RANKL via a mechanism involving IL-1α. Using a luciferase reporter assay, we demonstrated that osx downregulated IL-1α through a transcription-mediated mechanism. Following suppression of osterix in Dunn and DLM8 cells led to enhanced IL-1α promoter activity and protein production. Site-directed mutagenesis and Chromatin immunoprecipitation (ChIP) indicated that osterix downregulated IL-1α through a Sp1-binding site on the IL-1α promoter. These data suggest that osterix is involved in the lytic phenotype of osteosarcoma and that this is mediated via transcriptional repression of IL-1α. ^

Análisis de reflectores en MatLab

Sabor, Software de Análisis de BOcinas y Reflectores, es una herramienta didáctica la cual es utilizada en los laboratorios de la escuela para realizar prácticas de la asignatura Antenas y Compatibilidad Electromagnética, esta herramienta da a los alumnos una visión gráfica de lo que se enseña en clase de teoría de lo que son los campos en las aperturas de los reflectores. El proyector pretende sustituir al primer Sabor , ya que se queda obsoleto debido al sistema operativo, ya que funciona solo para Windows XP y con ordenadores de 32 bits, y también realizar mejoras y corregir errores de la versión anterior. El proyecto se ha desarrollado en Matlab que es un software matemático con grandes ventajas en cuanto a cálculo, desarrollo gráfico, y a la creación de nuevos algoritmos en su propio lenguaje y además está disponible para las plataformas Unix, Windows, Mac OSX y GNU/Linux. El objetivo del proyecto ha sido implementar, al igual que las versiones anteriores, cinco tipos de reflectores, como son: Parabólico, Offset, Cassegrain y los dos Dobles Offset, Cassegrain y Gregorian, y han sido analizados con un alimentador ideal ,cos-q, y por último los resultados obtenidos se han comparado con las versiones anteriores de Sabor, como son Sabor 3.0 y el primer Sabor. El proyecto consta de partes muy bien diferencias como son :  La interpretación correctas de las formulas que se han utilizado para la realización de este proyecto ,dichas formulas han sido las dadas por el proyecto fin de carrera titulado Sabor3.0 de Francisco Egea Castejón.  GUIDE, the graphical user interface development environment, con el que se creó: GUI, graphical user interface, que es la parte de Matlab dedicada a crear interfaces de usuario , herramienta utilizada para crear nuestras distintas ventanas dedicadas para la obtención de datos para analizar los distintos reflectores y para mostrar por pantalla los distintos resultados.  Programación Orientada a Objetos de Matlab y sus distintas propiedades como son la herencia lo cual es muy útil para ocupar menos memoria ya que con un único método podemos realizar distintos cálculos con los distintos reflectores, objetos, solo cambiando las propiedades de cada objeto  Y por último ha sido la realización de validación de los resultados con la ayuda de las versiones anteriores de Sabor, que están detallados en el capítulo 5 y la unión con bocinas del proyecto fin de carrera Análisis de Bocinas en Matlab de Javier Montero. Por otra parte tenemos las mejoras realizadas a las antiguas versiones como son: realización de registros que el usuario puede guardar y cargar con las distintas variables, también se ha realizado un fichero .txt en el que consta la amplitud del campo con su respectiva theta para que el usuario pueda visualizarlo en cualquier plataforma gráfica de datos como por ejemplo exel. ABSTRACT. Sabor, Software de Análisis de BOcinas y Reflectores, is a teaching tool, which is used to do laboratory practice in the subject of Antennas y Compatibilidad Electromagnética, this tool gives students a graphic view of the knowledge that are given in theory class in regard to aperture field of reflectors. This project intend to replace the first Sabor, because it is outdated, due to the operating system, because Sabor works only with Widows XP and computer with 32 bits, and to make improves and correct errors that were detected in the last version of Sabor too. This project has been carried out in Matlab, which is a mathematical software with high-level language for numerical computation, visualization and application development, and furthermore it is available to different platforms such as Unix, Windows ,Mac OSX and GNU/Linux This project has focused on implementing, the same as last versions, five kind of reflectors, such as : Parabolic, Offset, Cassegrain and two offset dual reflector Cassegrain y Gregorian ,and these were analysed with a cos-q ideal feed, and finally the results were checked with the versions of Sabor, as well as Sabor 3.0 and the first Sabor. This project consist of four parts:  The correct interpretation of the formulas , which were used to do this project, from the final project Sabor3.0 by Francisco Egea Castejón.  GUIDE, the graphical user interface development environment, tool that was used to create : GUI, graphical user interface, part of Matlab dedicated to create user interface.  Object Oriented Programming of Matlab and different properties like inheritance, that is very useful for saving memory space because with only one method we can analyse different kind of reflectors, object, only change the properties of the object.  At finally, the results were contrasted with the results from the previous versions and the link reflectors with horns from the final project Análisis de Bocinas en Matlab by Javier Montero. On the other hand, we have the improvements such as: registers and .txt file. The registers are used by user to save and load different variables and .txt file is useful because it allows to the user plotting in different platforms for example exel.