Sustitucion del conducto biliar mediante tubos tridimensionales de biomaterial de colágeno/agarosa. Estudio funcional experimental

Resumen

SUSTITUCIÓN DEL CONDUCTO BILIAR MEDIANTE TUBOS TRIDIMENSIONALES DE BIOMATERIAL DE COLAGENO/AGAROSA. ESTUDIO FUNCIONAL EXPERIMENTAL. Introducción En la actualidad el tratamiento de las vías biliares extrahepáticas afectadas por cáncer, estenosis, lesiones iatrogénicas y otras patologías consiste en la resección de las mismas, y una anastomosis en los casos mas graves de la placa hilar biliar al intestino delgado. esta técnica, en muchos casos tiene un transcurso dificultado por infecciones retrogradas de la vía biliar través de los patógenos que ascienden del intestino, o debido a complicaciones como la estenosis de la anastomosis, que requieren una nueva intervención para rehacer la técnica quirúrgica. en menor numero de caso, el estancamiento de las secreciones biliares y la dificultad para el flujo de la bilis, requerirán un trasplante hepático incluso en aquellos casos en los que únicamente la porción afectada es extrahepática. en aquellos receptores de injertos hepáticos, puede ocurrir en el seno de un rechazo crónico una nueva estenosis de la anastomosis biliar, requiriendo de este modo un retrasplante. Si pudiéramos obtener un tubo artificial que funcionara morfológica e histológicamente de la misma manera que la vía biliar nativa, podría utilizarse como sustituto de las vías biliares patológicas, evitando de este modo complejas técnicas quirúrgicas que dificultan el paso de la bilis y, finalmente, evitando el transplante hepático en aquellas enfermedades en las que el parénquima hepático se encuentra indemne. nuestro grupo esta desarrollando, basado en esta idea y gracias a las nuevas biotecnologías, un conducto artificial que mantiene las propiedades morfológicas, histológicas y funcionales de la vía biliar nativa. en la bibliografía actual existen pocas referencias a conductos artificiales creados para sustituir vías biliares patológicas, algunas con la ayuda de biopolimeros y otras con la de estructuras histológicas de otra índole. hasta el momento , estas o bien no presentan una biocompatibilidad suficiente para permitir el flujo de la bilis (biopolimeros), o bien no se integran histológicamente en el ambiente (intestino, vasos sanguíneos). En cuanto hemos tenido las técnicas suficientes para obtener colágeno y poder moldearlo a nuestro antojo, nuestro equipo modificó una estructura tridimensional tubular de colágeno bovino, cubierta por hidrogenes, totalmente biocompatible. Material y Método Se construyeron tubos tridimensionales de colágeno de 4 mm de diametro y 1 mm de grosor de la pared. el tamaño de los poros formados es de 200 nm. Posteriormente se recubrieron todas sus superficies con hidrogel de agarosa al 2%. Para su almacenamiento se emplearon estufas a 37ºC. Implantación del conducto del conducto artificial en los animales de experimentación: se implantó el conducto a 40 cobayas de la raza Dunkin Hartley con un peso aproximado de 1 kg +/- 200 gr. en todas ellas se sustituyó la vía biliar extrahepática en la porción comprendida entre la inserción del conducto ciático y la ampolla de Vater. Para la intervención se realizó una incisión subcostal derecha para acceder directamente a las vías biliares. realizando una inducción anestésica con 20 mg/kg de ketamina, y manteniendo una infusión continua de propofol a razón de 0,2 mg/kg/min. Una vez identificado el conducto común hepático en el interior del ligamento hepatoyeyunal, se realizó una sección circunferencias por debajo de la inserción del conducto ciático. A este nivel se interpuso la prótesis de colágeno/agarosa con la ayuda de materiales de sutura de acido polifacético de reabsorción lenta a través del microscopio quirúrgico. Los animales de experimentación se estabularon según la normativa vigente. Durante los primeros 7 días postoperatorios se administro por vía subcutánea analgesia y antibioterapia. Estudio bioquímico y funcional: Los animales fueron sacrificados a las 4 semanas, 3 meses y 6 meses tras la sustitución del conducto biliar. Tras el sacrificio se realizo estudio histológico de las biopsias hepáticas mediante tintines de hematoxilina-eosina. Del mismo modo, los sujetos de investigación fueron sometidos a análisis biométricos y bioquímicos, junto con exploraciones radiológicas de la intervención sobre la vía biliar. Todos los resultados obtenidos fueron comparados con con los especímenes y muestras obtenidos de 10 cobayas control. Resultados Todos los animales de experimentación sobrevivieron hasta el momento de su sacrificio tras la implantación del conducto artificial. Se desarrollaron fisiológicamente, ganancia de peso y tamaño, de forma similar a los animales control (1,9kg+/- 200g). En todos los grupos se realizó una medición de los niveles sanguíneos de bilirrubina y enzimas hepáticas tras el sacrificio, obteniendo niveles normales en los 40 animales intervenidos. Macroscópicamente todos los conductos artificiales implantados parecían completamente degradados a partir de las 4 semanas, aunque persistían adherencias y ligera inflamación de los tejidos adyacentes. Los especímenes extraídos a partir de los 3 meses se encontraban completamente reabsorbidos, y el aspecto era comparable al de la vía biliar nativa, persistiendo ligeras adherencias de los tejidos circundantes. El aspecto macroscópico del hígado era completamente normal, sin observarse signos de cirrosis. Discusión El conducto tridimensional implantado se integra biológica y fisiológicamente dentro de la vía biliar, manteniendo las funciones de conducción de la bilis y evitando técnicas con morbimortalidad elevada, así como costes sanitarios altos. Las integración tisular que se produce a partir de las 4 semanas lleva a pensar que el conducto sirve de estructura para la regeneración a conducto biliar nativo. Podríamos deducir múltiples aplicaciones que pueden desarrollarse en la clínica actual. Ante la presencia de estenosis o afecciones cancerígenas propias de la vía biliar extrahepática, estos segmentos pueden ser extirpados u sustituidos por el conducto artificial mediante anastomosis término-terminales. De este modo podemos preservar la papila de Vater, manteniendo las funciones biológicas de esta válvula, evitando infecciones retrógradas que ocurren en las anastomosis biliodigestivas. Si nuestro proyecto llega a ser factible, la disponibilidad en todo momento de un neoconducto biliar puede evitar derivaciones con grandes riesgos y ser utilizadas en multitud de técnicas que necesiten la extirpación de segmentos de conductos biliares, evitado las complicaciones derivadas, y finalmente, el trasplante. Podemos pensar que nuestra prótesis se ha integrado correctamente si no aparecen complicaciones tras su implantación. No debemos observar signos o síntomas derivados de la obstrucción al flujo de la bilis producidos por una estenosis del conducto, así como tampoco observar signos que nos hagan pensar en la presencia de una fuga de bilis intraabdominal, debido a dehiscencias o puntos de fuga en las anastomosis. Ninguna de estas complicaciones estuvo presente en nuestros casos. La función favorable que hemos observado en os neoconductos formados en los periodos estudiados nos sugiere que estos injertos podrían ser aplicables a la práctica clínica. Algunos autores han publicado recientemente el exitoso resultado en la formación de órganos artificiales como los vasos sanguíneos y el intestino delgado, mediante cultivos celulares. Este principio ha sido utilizado por algunos autores como Rosen et al. para facilitar la reconstrucción de partes de la circunferencia del conducto biliar mediante el uso de submucosa intestinal, a partir de la cual se producía la reepitelización. El examen macroscópico mostró que a partir de las 4 semanas, nuestro conducto biocompatible era completamente similar morfológicamente a la vía biliar nativa. Nuestro tubo tridimensional de colágeno implantado en los animales de experimentación adquirió una función y forma similares a la de la vía biliar nativa a partir de las 4 semanas tras el injerto. La creación de un neoconducto biliar nos abre nuevas vías terapéuticas para el tratamiento de afecciones confinadas a la vía biliar extrahepática, evitando complejas técnicas quirúrgicas con elevados costes socioeconómicos y alta morbimortalidad. Si en estudios futuros se demuestra la viabilidad a largo plazo de esta prótesis, nuestro conducto tendrá indudablemente una gran aplicación clínica. Visto Bueno Director de la Tesis Doctorando PABLO JUAN TORNE POYATOS ALEJANDRO J. PEREZ ALONSO REPAIRMENT OF EXTRAHEPATIC BILE DUCTS USING THREE DIMENSIONAL COLLAGEN/AGAROSE TUBES. EXPERIMENTAL FUNCTIONAL STUDY. Introduction ¿ Currently the surgical treatment of extrahepatic bile ducts affected by different conditions such as cancer, stenosis, iatrogenic injuries and other diseases is based in the excision, and in severe cases, the anastomosis of biliary hiliar plate to the small intestine is necessary. This technique may have a difficult course due to the retrograde infections of the gallbladder through the intestinal pathogens, or due to complications such as stenosis of the anastomotic area requiring reoperation. In other cases, the block of bile secretions, or the presence of a difficult flow of bile will require liver transplantation even in those cases where the affected portion is only extrahepatic. Patients receiving liver grafts may suffer a stenosis of the bladder anastomosis within the context of a chronic rejection, requiring retransplantation. The design of an artificial tube morphologically and histologically similar to the native bile duct would be interesting to be used as a substitute for pathologic biliary tract, thus avoiding complex surgical techniques that may block the flow of bile, and hipothetically avoiding liver transplantation in those diseases in which the liver parenchyma is undamaged. Based on this idea and supported by new biotechnologies, our research group is developing a tube graft that maintains the morphological, and functional characteristics of native bile duct. ¿ In the current literature few works about artificial canals to replace pathologic bile ducts, some of them based on biopolymers, and others based on other histological structures have been published. Issues associated with the artificial canals developed included the presence of insufficient biocompatibility to permit the flow of bile (biopolymers) and the inadequate histological integration in the environment (intestine, blood vessels). The current work presents the results of a modified tubular three-dimensional structure of bovine collagen, covered by biocompatible hydrogels, designed by our team. These new constructs were transplanted into experimental animals (guinea pigs) replacing the native bile duct. Biochemical and physiological functionalities were assessed. Material and methods ¿Substitute biliary tubes: Three-dimensional collagen tubes were constructed measured 4 mm in diameter and 1 mm wall thickness. The size of the pores was 200 nm. Subsequently all surfaces were coated with 2% agarose hydrogel, in order to avoid bile leaks through the pores. Heaters at 37 ° C were used for storage. Transplantation in experimental animals: the construct was transplanted to 40 guinea pigs weighing approximately 1 kg + / - 200 gr. In all of the experimental animals, the common bile duct was replaced in the portion between the insertion of the cystic duct and ampulla of Vater. For the intervention a right subcostal incision was performed for direct access to the biliary tract, with an anesthetic induction (20 mg / kg of ketamine) and a maintenance phase (continuous infusion of propofol at 0.2 mg / kg / min). After identifying the common hepatic duct inside the hepatoduodenal ligament, a circumferential section was performed below of the insertion of the cystic duct. At this level the collagen prosthesis was interposed using a slow-resorption poly-lactic acid suture. All the experimental animals survived up to their sacrifices at 7 days (Group 1, 13 guinea pigs), 15 days (Group 2, 13 guinea pigs) or 45 days (Group 3, 14 guinea pigs) after transplantation. Results¿¿ After implantation of the artificial canal all experimental animals survived until the time of sacrifice (7 days, 15 days, 45 days). All the experimental animals were similar to control animals regarding physiological development, weight gain and size. A measurement of blood levels of bilirubin and liver enzymes were performed after sacrifice and normal levels were obtained in the 40 animals and in the 10 controls. ¿Macroscopic study of the neo-bile duct: In 4 weeks, all implanted artificial constructs appeared completely degraded, but persistent adhesions and slight inflammation of adjacent tissues were observed. At 3 months, specimens were completely resorbed, and the appearance was comparable to the native bile duct, persisting slight adhesions of the surrounding tissues. The gross appearance of the liver was completely normal, without signs of cirrhosis. H & E microscopic study: In all cases, the liver tissue did not suffer any alteration, with no presence of stasis or biliary cirrhosis. Discussion¿¿ Our transplanted three-dimensional construct integrates into the bile duct morphologically and physiologically, maintaining the functions of bile and avoiding high morbidity techniques as well as high health costs. The tissue integration that occurs after 4 weeks, suggests that the construct serves as a structure for regeneration from distal and proximal native bladder tissues. Multiple applications can be developed using these constructs: In the presence of stenosis or extrahepatic bile duct cancers, these segments can be removed and replaced by this artificial duct through anastomotic sutures. Thus, the papilla of Vater may be preserved, keeping the biological functions of this valve and preventing retrograde infections which are common in biliodigestive anastomosis. If our project proves to be feasible, availability of a neo-conduct may avoid biliary derivations with high risks and morbidity. Furthermore it may be used in multiple techniques in which multiple removals of bile duct segments are required, avoiding complications and finally transplantation. We think that our prosthesis is integrated properly as long as no complications after implantation are observed. Signs or symptoms resulting from obstruction of bile flow produced by a stenosis, signs that suggest the presence of intra-abdominal bile leak due to dehiscence or leaks in the anastomosis may indicate that the technique has not been performed properly. None of these complications was present in our cases. The positive outcomes that we observed in the artificial duct during the study period, suggest that these grafts could be applicable to clinical practice. Some authors have recently published the successful results in the formation of artificial organs such as blood vessels and small intestine, using cell cultures. This principle has been used by some authors such as Rosen et al. to facilitate reconstruction of parts of the circumference of the bile duct through the use of intestinal submucosa, from which the reepithelialization is produced. In our cases the re-epithelialization occurs from the ends of artificial duct, extending all the way along through the cell migration which it is allowed by the pores of the three dimensional structure forming collagen. The rate of re-epithelialization was similar in both ends. This fact allows their use in a greater number of clinical indications and makes the technique easier. Broadly speaking, we could say that the presence of bile pluripotent cells are viable and they may differentiate into epithelial cells uniformly, migrating through the implanted construct. However, more histological studies are warranted to confirm our results. Gross examination showed that after 4 weeks, our construct was completely biocompatible and morphologically similar to the native bile duct. The histological test and immunohistochemical techniques showed that the new tissue was histologically similar to the native one, with a similar antigen expression. In the new created environment, the bile flowed through into the duodenum with no blocking. Through our research we were not able to determine what factors stimulate cell differentiation and reepithelialization, and whether these factors are from components in the bile or they reach the area by chemotaxis. ¿ Our three-dimensional collagen tube transplanted into experimental animals developed a similar structure and functions to the native bile duct from the 4 weeks after grafting. The creation of a biliary neo-conduct opens new therapeutic avenues for the treatment of disease confined to the extrahepatic bile duct, avoiding complex surgical techniques with high socioeconomic costs and mortality. If future studies confirm the long-term viability of this prosthesis, our construct will certainly be of great clinical application. 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