{"id":104969,"date":"2018-03-11T10:28:18","date_gmt":"2018-03-11T10:28:18","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/terapia-celular-para-la-reparacion-osea-de-la-cavidad-bucal-modelo-experimental-de-defecto-mandibular\/"},"modified":"2018-03-11T10:28:18","modified_gmt":"2018-03-11T10:28:18","slug":"terapia-celular-para-la-reparacion-osea-de-la-cavidad-bucal-modelo-experimental-de-defecto-mandibular","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/estomatologia-y-ortodoncia\/terapia-celular-para-la-reparacion-osea-de-la-cavidad-bucal-modelo-experimental-de-defecto-mandibular\/","title":{"rendered":"Terapia celular para la reparaci\u00f3n \u00f3sea de la cavidad bucal. modelo experimental de defecto mandibular"},"content":{"rendered":"<h2>Tesis doctoral de <strong> Cynthia Georgina Trejo Iriarte <\/strong><\/h2>\n<p>La p\u00e9rdida \u00f3sea y dental es considerada en la actualidad uno de los grandes problemas a los que se enfrenta la odontolog\u00eda reparadora, para resolverlo su ha desarrollado el uso de implantes intra\u00f3seos. Sin embargo, cuando la calidad y cantidad \u00f3sea es insuficiente, no es posible aplicar este tratamiento. Surge entonces, la necesidad de buscar terapias alternativas para mejorar la cantidad y la calidad \u00f3sea.   actualmente, en el campo de la terapia celular mandibular, no existe una extensa bibliograf\u00eda sobre este tema que pudiera servirnos como gu\u00eda de actuaci\u00f3n. nuestro inter\u00e9s en el proceso de reparaci\u00f3n\/reconstrucci\u00f3n del hueso mandibular, nos llev\u00f3 a plantearnos la posibilidad de utilizar c\u00e9lulas mesenquimales aisladas del tejido adiposo subcut\u00e1neo (mscat) como potencial fuente celular. el objetivo de la presente tesis ha sido determinar si el tejido adiposo una adecuada fuente de c\u00e9lulas mesenquimales y si podemos establecer las condiciones \u00f3ptimas para su aplicaci\u00f3n como terapia celular osteorreparadora.  As\u00ed como saber si la terapia celular empleada, puede mejor la reparaci\u00f3n \u00f3sea mandibular.  en el primer objetivo se dise\u00f1\u00f3 el protocolo para la obtenci\u00f3n de c\u00e9lulas mesenquimales a partir del tejido adiposo subcut\u00e1neo (mscats) de ratas wistar, as\u00ed como su caracterizaci\u00f3n fenot\u00edpica a corto y largo plazo, mediante t\u00e9cnicas de microscop\u00eda \u00f3ptica y electr\u00f3nica de transmisi\u00f3n y barrido, inmunohistoqu\u00edmicas y de pcr.  los resultados obtenidos han demostrado que a partir del segundo subcultivo la poblaci\u00f3n celular se encontraba compuesta por c\u00e9lulas viables hasta muy largos plazos, con una alta capacidad de proliferaci\u00f3n (ratio de 206 en 15 d\u00edas y 5 veces mayor que fibroblastos subcut\u00e1neos de rata), y con ritmo modular proliferativo alcanzando un 80% confluencia cada 72 horas durante m\u00e1s de 200 subcultivos. La identificaci\u00f3n fenot\u00edpica caracter\u00edstica de la estirpe mesenquimal fue valorada mediante la positividad en m\u00e1s de un 90% para cd90, c-kit, vimentina, y se mostraron negativas para cd11b, cd34, cd31 y cd44. Dicha poblaci\u00f3n fue positiva en un porcentaje superior al 60% para marcadores embrionarios de indiferenciaci\u00f3n oct3\/4, sox2 y nanog durante m\u00e1s de 175 subcultivos. las mscat fueron capaces de diferenciarse a distintas l\u00edneas celulares mostrando marcadores fenot\u00edpicos caracter\u00edsticos de cada diferenciaci\u00f3n: \u00f3sea (osteocalcina), adiposa (oil red), condrog\u00e9nica (safranina) y endotelial (cd31). la diferenciaci\u00f3n inducida hacia la l\u00ednea osteog\u00e9nica fue evaluada a diferentes tiempos de estudio: 48 y 72 horas; 7, 14 ,21 y 30 d\u00edas. Los mejores resultados fueron obtenidos a los 21 d\u00edas de cultivo en medio osteog\u00e9nico, donde la poblaci\u00f3n celular mostr\u00f3 un equilibrio en la m\u00e1xima expresi\u00f3n de caracter\u00edsticas morfol\u00f3gicas y fenot\u00edpicas de los marcadores de diferenciaci\u00f3n runx2, osterix, osteopontina (op), osteocalcina (ocn) y prote\u00ednas morfogen\u00e9ticas de hueso 2\/4 (bmp2\/4). Siendo estas condiciones, las elegidas como \u00f3ptimas para su uso terap\u00e9utico (a este grupo c\u00e9lulas le hemos denominado mscost).  el segundo objetivo ha sido la aplicaci\u00f3n de las l\u00edneas celulares mscat y mscost como terapia celular aut\u00f3loga en un modelo de reparaci\u00f3n mandibular en rata wistar. Se realizaron defectos \u00f3seos cavitarios en el cuerpo de la mand\u00edbula. Se establecieron 3 grupos de estudio:  grupo mscat, el defecto cavitario fue tratado con 10&#215;106 mscat aut\u00f3logas previamente cultivadas en amniomax, durante 21 d\u00edas. grupo mscost, cuando el defecto cavitario fue tratado con 10&#215;106 mscost aut\u00f3logas.  grupo control, se realiz\u00f3 un defecto cavitario en el lado izquierdo a todos los especimenes, el cual no fue tratado.  la poblaci\u00f3n celular implantada en el defecto \u00f3seo fue marcada con el fluor\u00f3 cromo pkh26 para su seguimiento dentro del defecto. de los tres grupos evaluados, en el grupo mscots la presencia de matriz osteoide en el lugar del defecto fue constatada a los 14 d\u00edas, para el grupo mscat a los 30 d\u00edas y se observ\u00f3 ausente en el grupo control. La neoformaci\u00f3n \u00f3sea que rellenaba el defecto cavitario a los 30 d\u00edas del transplante celular en el grupo mscost alcanz\u00f3 porcentajes superiores al 70%, versus el 30% observado en el grupo mscat. En el grupo control solo se pudo observar una peque\u00f1a remodelaci\u00f3n en los bordes del defecto. en el an\u00e1lisis de los marcadores oste\u00f3genicos pudimos observar que: porcentaje de expresi\u00f3n de runx2 mostr\u00f3 un incremento estad\u00edsticamente significativo (p<0.01) en el grupo mscost respecto a los grupos mscat y control, esto fue observado en todos los tiempos de estudio. El patron de expresi\u00f3n de ocn mostr\u00f3 diferencias significativas (p<0.05) entre los grupos mscost, mscat y control en los d\u00edas 7, 14 y 21, siendo el grupo mscost el que present\u00f3 una mayor expresi\u00f3n. Se observ\u00f3 un aumento de expresi\u00f3n en el grupo mscat a los 21 d\u00edas para ocn, que result\u00f3 significativo (p>0.05) con repecto al grupo control.  destaca la ausencia de c\u00e9lulas de estirpe monocitario (macr\u00f3fago\/osteoclasto) valoradas mediante ed1 y trap. los resultados obtenidos nos permiten formular las siguientes conclusiones:   la poblaci\u00f3n aislada a partir de tejido adiposo subcut\u00e1neo de rata en las condiciones espec\u00edficas utilizadas en nuestro modelo (medio de cultivo amniomax) posee las propiedades inherentes a las c\u00e9lulas mesenquimales y permiten que dichas caracter\u00edsticas puedan ser conservadas y mantenidas en el tiempo. esta poblaci\u00f3n mscat es susceptible de ser diferenciada a la l\u00ednea osteog\u00e9nica; se defini\u00f3 un protocolo \u00f3ptimo para obtenci\u00f3n de las mscost (inducci\u00f3n de la diferenciaci\u00f3n en c\u00e9lulas en segundo subcultivo utilizando el medio con suplemento osteog\u00e9nico durante 21 d\u00edas).   los resultados obtenidos tras llevar a cabo nuestro modelo in vivo demostraron que en los defectos \u00f3seos sometidos a terapia celular mscost se observ\u00f3 una neoformaci\u00f3n \u00f3sea m\u00e1s r\u00e1pida y superior con respecto a los otros grupos de estudio. La m\u00e1xima eficacia se obtiene tras una diferenciaci\u00f3n hacia la l\u00ednea osteog\u00e9nica in vitro durante 3 semanas previas al implante in vivo.  las conclusiones anteriores, nos permiten elevar nuestras hip\u00f3tesis a categor\u00eda de tesis y afirmar que: el tejido adiposo es una fuente accesible de celulas mesenquimales para fines terapeuticos. Las mscat aisladas por nosotros, cumplen los requisitos exigidos para su uso como terapia celular, as\u00ed como su capacidad de diferenciaci\u00f3n a mscost, cuya eficacia ha sido probada, mostrando una aceleraci\u00f3n en el proceso de remodelaci\u00f3n y consolidaci\u00f3n \u00f3sea del defecto cavitario en la mand\u00edbula de rata.   cell therapy for bone repair of the oral cavity: an experimental model of mandibular defect.  the main problems faced by odontology in daily practice are bone and tooth loss, actually has now developed intraosseous implants are use. However, these implants can only be used when there is a sufficient amount and quality of preexisting bone such that there is a need for other therapeutic strategies that will improve bone quantity and quality. in the field of cell therapy of the jaw, existing literature is too scarce to serve as a research guide. our interest in the jaw repair\/reconstruction process prompted our idea of using adult mesenchymal stem cells derived from subcutaneous adipose tissue (mscat) as a potential cell source. the fist objective in this thesis was to establish if the adipose tissue is a good source of mesenchymal stem cells and is it possible to establish the optimal conditions for their use in cell therapy procedures in bone tissue repair. Something does cell therapy improve the bone repair process in the jaw.   for the first objective, we designed a protocol to harvest mscat from the subcutaneous tissue of wistar rats, which have been grown in amniomax medium. The cells were characterized in short- and long-term using light microscopy and scanning and transmission electron microscopy, immunohistochemical, pcr techniques and flow cytometry proliferation assay.   obtained results showed that after the 2nd passage cell population was composed by viable cells to long periods with highly proliferative (206 ratio in 15 days and five times higher than subcutaneous rat fibroblasts), and modular proliferative rate, reaching 80% confluency every 72 hours for more than 200 passages. the characteristic phenotype identity of mesenchymal cell population was assessed by positive expression higher than 90% for cd90, c-kit., Vimentin and negativity for cd11b, cd34, cd31 and cd44. The named population was positive in more than 60% for undifferentiated embryonic markers oct3\/4, sox2 and nanog for more than 175 subcultures.   mscat were able to differentiate to the same cells lineages showing characteristic phenotypic markers of each line: osteogenic (osteocalcin), adipogenic (oil red), chondrogenic (safranin) and endothelial (cd31).  the differentiation to osteogenic lineage was assessed in several study times: 48 and 72 hours; 7, 14, 21 and 30 days. The best results were obtained at 21 days in osteogenic culture medium, where the cell population showed an optimal balance of osteogenic characteristics and phenotype markers runx2, osterix, osteopontin (op), osteocalcin (ocn) and bone morphogenetic proteins 2\/4 (bmp 2\/4) . Therefore, these conditions were chosen for their therapeutic use, being these cells denominated mscost.  after second passage, the cell population was mainly comprised of mesenchymal cells with a high capacity of proliferation, adhesion, migration and differentiation under conditioned stimuli.  the second objective was to apply the cell lines mscat and mscost as autologous cell therapy in the wistar rat model of jaw repair. Cavitary bone defects in the mandible body. mscat group: created defect was filled with 106 autologous mscat cultured for 21 days in amniomaxmedium. mscost group: created defect was filled with 106 autologous mscost cultured for 21 days in amniomax medium with osteog\u00e9nic cocktail.  control group: in all animals, a jaw defect was created on the left side that was not filled.  the cells implanted in the bone defects were labelled with pkh26 fluorochrome so that they could be followed at the defect site. This is how we first demonstrated objectively in this area, that new bone formation was guided by transplanted cells.  from the three groups evaluated, in mscost group at 14 days the presence of osteoid matriz in the defect site was checked, this was observed at 21 days in mscat group and in control group could not be observed.   observed bone neoformation filling the defect at 30 days was over 70% in mscost, group, versus 30% in mscat group. In control group only a small remodeling was observed at the edges of the bone defect area.  in the immunohistochemical analysis we found that: runx2 marker suffered an statistically significant increase (p<0.01) in mscost group, versus mscat and control groups observed at all times of study. The ocn pattern of expression showed statistically significant differences (p<0.05) between groups mscost, mscat and control at 7, 14, and 21 days, being mscost the group that presented the greatest expression. It was also observed a significant increase (p<0.05) in the expression ocn marker in mscost group of 21 days versus control group. resulted interesting the absence of monocyte\/macrophage lineage cells in the defect area, a fact that was assessed by ed-1 and trap staining.  based on these findings, the following conclusions may be established:  the cell population isolated from subcutaneous adipose tissue of rat, in the specifics conditions used in our model (amniomax media culture), shows inherent properties of mesenchymal cells. Used conditions allow both preservation and maintainace of these characteristics over time.  mscat population is susceptible to differentiation to osteogenic lineage; we have defined an optimal protocol for obtaining mscost (osteogenic induction in the second subculture using media with osteogenic cocktail for 21 days).  the results obtained after carrying out our in vivo model showed that in the bone defects undergoing mscost cell therapy was observed a faster healing, higher new bone formation and superior quality of mineralization compared with the other study groups.  the best efficiency was obtained with 3 weeks of in vitro osteogenic differentiation in vitro 3 weeks previous to in vivo transplant.   according to these conclusions, our starting hypotheses can be elevated to the category of thesis and we can state that:  the adipose tissue is an accessible source of mesenchymal cells for therapeutic use. The mscat isolated by us satisfy all the requisites for their use as cell therapy, and its ability to differentiate to mscost, whose effectiveness has been tested, showing acceleration in the remodelling and bone healing process, in the cavitary defect in the rat mandible.\n\n\n\n&nbsp;\n\n\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Terapia celular para la reparaci\u00f3n \u00f3sea de la cavidad bucal. modelo experimental de defecto mandibular<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Terapia celular para la reparaci\u00f3n \u00f3sea de la cavidad bucal. modelo experimental de defecto mandibular <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Cynthia Georgina Trejo Iriarte <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Alcal\u00e1<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 18\/11\/2010<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Julia Bujan Varela<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: eliseo Carrascal marino <\/li>\n<li>colette Lacabanne (vocal)<\/li>\n<li>Francisco Jos\u00e9 Manso platero (vocal)<\/li>\n<li>Juan  Manuel Bell\u00f3n caneiro (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Cynthia Georgina Trejo Iriarte La p\u00e9rdida \u00f3sea y dental es considerada en la actualidad uno de los 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