Cultivo in vitro con colágeno y fibroblastos humanos de un equivalente de mucosa oral de espesor total / In vitro culture with collagen and human fibroblasts of a full-thickness oral mucosa equivalent

Objetivos. El presente trabajo tiene por objetivo obtener, mediantecultivo in vitro, láminas de tejido oral en las que se pueda identificar lasestructuras de una mucosa oral completa. La aplicación clínica del presenteestudio permitiría, en determinados casos, la sustitución del empleo de injertoslibres de piel o autólogos de mucosa oral por esta técnica. Material y Método.A partir de pequeñas biopsias de mucosa oral se hicieron cultivos primariosde queratinocitos. A partir de estos cultivos primarios se realizaron cultivossecundarios sobre una submucosa artificial constituida por colágeno y fibroblastoshumanos. Se analizaron histológicamente sus características in vitro, yulteriormente se procedió a la realización de injertos en ratones atímicospara conocer su comportamiento in vivo. Resultados. Los cultivos primariosfueron confluentes en un plazo mínimo de 10 días y máximo de 12 días, periodosimilar al observado para la confluencia de los cultivos secundarios. El tiempotranscurrido desde la toma de la muestra hasta la obtención de una mucosaartificial completa osciló entre los 20 y los 22 días, mostrando las característicashistológicas de una mucosa normal. Tras 17 días de injerto en ratonesinmunoincompetentes, sin ningún tipo de contingencia clínica, la caracterizaciónhistológica e inmunohistoquímica (citoqueratinas 13 y 19, colágenoIV y laminina) confirmó la similitud de la mucosa in vitro con la mucosa oralsana. Conclusión. Es posible mediante técnicas de cultivo in vitro la obtenciónde un equivalente de mucosa oral completa con colágeno y fibroblastos. Sibien esta mucosa muestra un importante grado de retracción, su manejo clínicoes muy favorable(AU)

Objectives. The objective of this study was to obtain,by in vitro culture, sheets of oral tissue in which complete oral mucosastructures can be identified. Clinical application of the findings ofthis study will allow the replacement of free skin grafts or autologousoral mucosa grafts by this technique in certain cases.Material and Method. Primary keratinocyte cultures were preparedfrom small biopsy samples of oral mucosa. Secondary cultures wereprepared from these primary cultures on an artificial submucosaconstituted by collagen and human fibroblasts. The cell cultureswere analyzed histologically in vitro and then used for graft implantsin athymic mice to study their behavior in vivo.Results. The primary cultures were confluent within a minimumperiod of 10 days and maximum of 12 days, which is similar to theperiod that the secondary cultures required to reach confluence.The time from sampling to achieving a complete artificial mucosaranged from 20 to 22 days. The artificial mucosa showed histologiccharacteristics of a normal mucosa. After 17 days of graftimplantation in immunoincompetent mice without any clinicalcontingency, histologic and immunohistochemical characterization(cytokeratins 19 and 13, collagen IV, and laminin) confirmed thesimilarity of the mucosa in vitro to healthy oral mucosa.Conclusion. A complete oral mucosa equivalent can be preparedwith collagen and fibroblasts using in vitro culture techniques.Although this mucosa shows considerable retraction, its clinicalhandling is very favorable(AU)

[In vitro three-dimensional reconstruction of human bladder mucosa]. / Reconstrucción tridimensional in vitro de mucosa vesical humana.

OBJECTIVE: The purpose of this study is to apply the in vitro keratinocyte culture techniques and the tissue engineering principles to human urothelium, to reconstruct an in vitro three-dimensional human bladder mucosa, suitable for grafting. MATERIAL AND METHODS: Biopsy specimens of human bladder mucosa were obtained from patients undergoing suprapubic prostatectomy, in vitro cultured and finally, an immunohistochemical study was made. RESULTS: A three-dimensional in vitro tissue was obtained, composed of a bio-artificial submucosa (fibrin gel and fibroblast) where the uroepithelial cells were seeding. We used a biodegradable polyglycolic acid mesh to facilitate the tissue manipulation and implantation. An immature epithelium was obtained with a weak immunostaining to cytokeratins. The immunohistochemical study could not demonstrate the development of basement membrane. CONCLUSIONS: In vitro keratinocyte culture techniques could be applied to other epithelial tissues like the urothelium. We obtained a three-dimensional in vitro tissue suitable for grafting in a relatively short time, which needs the matrix interactions in order to mature.

Reconstrucción tridimensional in vitro de mucosa vesical humana / In vitro three-dimensional reconstruction of human bladder mucosa

Objetivo: Aplicar las técnicas de cultivo in vitro de queratinocitos así como los principios de la ingeniería tisular al epitelio urinario humano, con el fin de reconstruir tridimensionalmente una mucosa vesical humana in vitro, apta para trasplantar. Material y Métodos: Se obtuvieron muestras de mucosa vesical de pacientes programados para cirugía abierta de próstata, previo consentimiento de los mismos, las cuales fueron cultivadas in vitro, para proceder posteriormente al estudio histomorfológico de los tejidos obtenidos. Resultados: Se obtuvo un tejido tridimensional compuesto por una submucosa bioartificial a base de un gel de fibrina y fibroblastos, sobre la que descansan las células uroepiteliales, pudiendo utilizar una malla de ácido poliglicólico, que facilite la manipulación de la mucosa y el posterior injerto de la misma. El tejido obtenido tenía el aspecto de un epitelio inmaduro con muy escasa reacción a citoqueratinas, sin poderse demostrar inmunohistoquímicamente el desarrollo de una membrana basal. Conclusiones: Las técnicas de cultivo in vitro de queratinocitos son aplicables a otros epitelios, entre ellos el urotelio humano. En un periodo de tiempo relativamente corto se puede obtener un tejido in vitro tridimensional apto para trasplantar, precisando posiblemente de las interacciones con el lecho receptor para poder madurar

Objetive: The purpose of this study is to apply the in vitro keratinocyte culture techniques and the tissue engineering principles to human urothelium, to reconstruct an in vitro three-dimensional human bladder mucosa, suitable for grafting. Material and Methods: Biopsy specimens of human bladder mucosa were obtained from patients undergoing suprapubic prostatectomy, in vitro cultured and finally, an immunohistochemical study was made. Results: A three-dimensional in vitro tissue was obtained, composed of a bio-artificial submucosa (fibrin gel and fibroblast) where the uroepithelial cells were seeding. We used a biodegradable polyglycolic acid mesh to facilitate the tissue manipulation and implantation. An immature epithelium was obtained with a weak immunostaining to cytokeratins. The immunohistochemical study could not demonstrate the development of basement membrane. Conclusions: In vitro keratinocyte culture techniques could be applied to other epithelial tissues like the urothelium. We obtained a three-dimensional in vitro tissue suitable for grafting in a relatively short time, which needs the matrix interactions in order to mature

Estudio experimental sobre la viabilidad del injerto libre de epitelio urinario autólogo cultivado in vitro / Experimental study about viability of autologous free graft in vitro cultivated urinary epithelium

OBJETIVO: Con el presente trabajo pretendimos aplicar las técnicas de cultivo in vitro de queratinocitos así como los principios de la ingeniería tisular al epitelio urinario, con el fin de conseguir un tejido autólogo tridimensional apto para trasplantar, y confirmar a su vez la viabilidad del injerto libre del mismo en un modelo experimental. MATERIAL Y MÉTODOS: Se procedió a un diseño experimental en el animal de laboratorio donde se aplicaron las técnicas del cultivo celular y de la ingeniería de tejidos. Se obtuvieron muestras de mucosa vesical de conejo, las cuales fueron cultivadas in vitro, implantándose posteriormente el tejido obtenido en cada animal, estableciéndose 3 grupos, con diferentes periodos de seguimiento (7,14 y 30 días), para proceder al estudio histomorfológico de la viabilidad de dichos implantes. RESULTADOS: Se obtuvo un tejido tridimensional compuesto por una submucosa bioartificial a base de un gel de fibrina y fibroblastos, sobre la que descansan las células uroepiteliales, utilizando una malla de ácido poliglicólico, la cual facilitó la manipulación del tejido y el posterior injerto del mismo. Todos los implantes resultaron viables y se pudo comprobar como los injertos con mayor periodo de seguimiento (30 días) se encontraban mejor conformados, con múltiples capas celulares. CONCLUSIONES: Las técnicas de cultivo in vitro de queratinocitos son aplicables a otros epitelios, entre ellos el urinario. En un periodo de tiempo relativamente corto se puede obtener un tejido in vitro tridimensional apto para trasplantar. El estudio histológico puso de manifiesto que el injerto libre de epitelio urinario autólogo cultivado es totalmente viable, apuntando futuras aplicaciones clínicas

OBJETIVE: The purpose of this study is to apply the in vitro keratinocyte culture techniques and the tissue engineering principles to urothelium, to obtain a three-dimensional autologous tissue suitable for grafting. We also showed the viability of free graft cultured urothelium in an experimental model. MATERIAL AND METHODS: An animal experimental model was designed to apply the techniques of cellular culture and tissue engineering. Biopsy specimens of bladder mucosa were obtained, in vitro cultured and posteriorly implanted in each animal. We established three groups based on different follow-up periods (7, 14 and 30 days), and made a final histomorphological study to demonstrate the viability of the graft at the end of its respective follow-up period. RESULTS: A three-dimensional in vitro tissue was obtained, composed of a bio-artificial submucosa (fibrin gel and fibroblast) where the uroepithelial cells were seeding; a biodegradable polyglycolic acid mesh was used to facilitate the tissue manipulation and implantation. In the morphological study all the implants appeared viable, but the grafts with longer implantations periods were better conformed, showing a tisular structure with multiple cellular layers. CONCLUSIONS: In vitro keratinocyte culture techniques could be applied to other epithelial tissues as the urothelium. We obtained a three-dimensional in vitro tissue suitable for grafting in a relatively short time. The histological study demonstrated that free autologous urothelial graft is totally viable, opening future clinics applications

Estado actual de la ingeniería de tejidos en urología. Revisión de la literatura / Current state of the tissue engineering in urology. Revision of the literature

En la década de los 80 surge un nuevo campo de la medicina que aplica los principios del cultivo celular a polímeros sintéticos biodegradables de soporte con el fin de crear sustitutos biológicos autólogos que puedan mejorar, mantener o restaurar la función de órganos o tejidos dañados. La Ingeniería Tisular constituye una nueva disciplina en plena fase de desarrollo, especialmente en USA, con múltiples potenciales aplicaciones en las diferentes especialidades médicas. Nuestra especialidad no puede permanecer ajena al interés y esperanzador futuro suscitado por esta nueva ciencia. En el presente trabajo realizamos una amplia revisión bibliográfica en Medline con objeto de conocer los antecedentes, estado actual y las posibles aplicaciones futuras de la Ingeniería de Tejidos en Urología

In the eighties a new field of the medicine appears wich applies the principles of cellular cultivation to synthetic biodegradable polymers scaffolds with the purpose of creating autologous biological substitutes that could improve, maintain or restore the function of organs or damaged tissues. The Tissue Engineering constitutes a new discipline in full phase of development especially in USA, with multiple potential applications in several medical specialities. Our speciality can’t remain indifferent to interest and encouraging future originated by this new science. In this work we have made a wide bibliographical revision in the Medline to know the antecedents, current state and the possible future applications of Tissue Engineering in Urology

[Experimental study about viability of autologous free graft in vitro cultivated urinary epithelium]. / Estudio experimental sobre la viabilidad del injerto libre de epitelio urinario autólogo cultivado in vitro.

OBJECTIVE: The purpose of this study is to apply the in vitro keratinocyte culture techniques and the tissue engineering principles to urothelium, to obtain a three-dimensional autologous tissue suitable for grafting. We also showed the viability of free graft cultured urothelium in an experimental model. MATERIAL AND METHODS: An animal experimental model was designed to apply the techniques of cellular culture and tissue engineering. Biopsy specimens of bladder mucosa were obtained, in vitro cultured and posteriorly implanted in each animal. We established three groups based on different follow-up periods (7, 14 and 30 days), and made a final histomorphological study to demonstrate the viability of the graft at the end of its respective follow-up period. RESULTS: A three-dimensional in vitro tissue was obtained, composed of a bio-artificial submucosa (fibrin gel and fibroblast) where the uroepithelial cells were seeding; a biodegradable polyglycolic acid mesh was used to facilitate the tissue manipulation and implantation. In the morphological study all the implants appeared viable, but the grafts with longer implantations periods were better conformed, showing a tisular structure with multiple cellular layers. CONCLUSIONS: In vitro keratinocyte culture techniques could be applied to other epithelial tissues as the urothelium. We obtained a three-dimensional in vitro tissue suitable for grafting in a relatively short time. The histological study demonstrated that free autologous urothelial graft is totally viable, opening future clinics applications.

[Current status of tissue engineering in urology. Review of the literature]. / Estado actual de la ingeniería de tejidos en urología. Revisión de la literatura.

In the eighties a new field of the medicine appears wich applies the principles of cellular cultivation to synthetic biodegradable polymers scaffolds with the purpose of creating autologous biological substitutes that could improve, maintain or restore the function of organs or damaged tissues. The Tissue Engineering constitutes a new discipline in full phase of development especially in USA, with multiple potential applications in several medical specialities. Our speciality can't remain indifferent to interest and encouraging future originated by this new science. In this work we have made a wide bibliographical revision in the Medline to know the antecedents, current state and the possible future applications of Tissue Engineering in Urology.

Tratamiento de úlceras vasculares crónicas con equivalentes cutáneos obtenidos mediante ingeniería tisular / Treatment of chronic vascular ulcers with skin equivalents obtained by tissue engineering

Objetivo. Valorar la efectividad de un equivalente cutáneo diseñado en un banco de tejidos, en el tratamiento de úlceras vasculares crónicas. Pacientes y métodos. Entre septiembre de 1999 y diciembre de 2001 se incluyó a 25 pacientes con úlceras vasculares de evolución tórpida (>4 meses) en quienes, tras ingreso hospitalario y tratamiento estándar, se objetivó un estancamiento en su cicatrización. Se constataron nueve úlceras venosas, dos arteriales, siete hipertensivas y siete mixtas (componente flebostático y arterial). Descartada la infección clínica y bacteriológica de la úlcera, los injertos se colocaron una vez a la semana hasta la cicatrización o suspensión del tratamiento por no respuesta. Se realizaron controles fotográficos digitales semanales para medir el área de las lesiones. A partir del segundo injerto, el tratamiento se realizó ambulatoriamente. El seguimiento medio fue de 18 meses (intervalo: 6-30 meses). Se valoró la ausencia de rechazo, la tasa de cicatrización, el tiempo de reducción del área al 50 y 75%, y la recidiva ulcerosa. Resultados. No se detectaron signos de rechazo. Tasa global de cicatrización del 80% (20/25); en hipertensivas y mixtas, tasa del 100% (14/14). Tiempo medio de cierre: 5,3 semanas (intervalo: 3-12 semanas). Número medio de injertos utilizados: 5,8 (intervalo: 3-13 injertos). La recidiva ulcerosa fue del 25% (cuatro venosas y una hipertensiva). De las cinco úlceras que no cicatrizaron (cuatro venosas y una arterial), ninguna empeoró: en tres no hubo respuesta y en dos se redujo el área más de un 85%. Conclusiones. Los equivalentes cutáneos podrían constituir una buena alternativa en el tratamiento de úlceras vasculares crónicas (AU)

Aims. To assess the effectiveness of a skin equivalent designed in a tissue bank in the treatment of chronic vascular ulcers. Patients and methods. Between September 1999 and December 2001, 25 patients with vascular ulcers that progressed in a torpid fashion (>4 months) were admitted to hospital and administered standard therapy, following which sluggish healing processes were observed. Nine venous, two arterial, seven hypertensive and seven mixed ulcers (the latter with a phlebostatic and arterial component) were found. After ruling out the possibility of the ulcer being affected by clinical and bacteriological infections, the grafts were placed once a week until healing had been achieved or the treatment was suspended due to lack of response. Weekly digital photographic monitoring was carried out to measure the extension of the lesions. From the second graft onwards, patients were treated in the outpatients department. Average follow-up time was 18 months (interval: 6-30 months). Values measured included the absence of rejection, healing rate, the time required to reduce the extension to 50 and 75%, and recurrence of ulcers. Results. No signs of rejection were detected. Overall healing rate was 80% (20/25); in hypertensive and mixed cases, the rate was 100% (14/14). Average closing time: 5.3 weeks (interval: 3-12 weeks). Average number of grafts used: 5.8 (interval: 3-13 grafts). Recurrence of ulcers was 25% (four venous and one hypertensive). Of the five ulcers that did not heal (four venous and one arterial), none of them got worse: there was no response in three cases and two of them showed a reduction of the extension that reached 85%. Conclusions. Skin equivalents could be a good alternative in the treatment of chronic vascular ulcers (AU)