Integra plantilla de regeneración dérmica fue proporcionado amablemente por Integra LifeSciences Corporation. Fuentes de fondos de apoyo a la obra: Este trabajo fue parcialmente financiado por el Premio CIRM-BMBF Early Traslacional II y el Centro FONDAP de regulación del genoma tanto a JTE (Nr 15090007.).

1 Preparación de andamios <ol><li> Generar muestras de los andamios utilizando 12 mm punzones biopsia. </li><li> Para introducir moléculas bioactivas o células en el andamiaje, la fuga de los andamios, apretando suavemente con una gasa estéril. Entonces rehidratar los andamios mediante la adición de 160 l de una solución que contiene las moléculas bioactivas o células de interés. Compruebe el éxito de bioactivación con las células a través de ensayos metabólicos como MTT ensayos. </li><li> Si es necesario, fijar los compuestos o células a ensayar en el interior del andamio mediante la entrega de ellos, por ejemplo en una solución de fibrina-trombina o un hidrogel, de nuevo a través de la rehidratación como se describe en el paso 1.2. NOTA: Este paso puede ser de utilidad cuando los compuestos o células no se adhieren mecánicamente al material. También se puede controlar la dinámica de liberación de los compuestos .. </li><li> Preparar las mallas titanizado, que serán colocados en cada andamio en cada grupo experimental y el control, por cutting trozos de forma redonda, de aproximadamente 14 mm de diámetro. NOTA: La malla titanizado funciona como una frontera física necesaria para delimitar el tejido regenerado del lecho de la herida. </li></ol> 2. Animales <ol><li> Antes de la implementación del modelo presentado, consulte las leyes de protección animal correspondientes y obtener el permiso de las autoridades locales. En este trabajo, todos los experimentos se realizaron con ratones, de acuerdo con la actual ley de bienestar animal alemán y aprobados por el Gobierno del Distrito de la Alta Baviera (Regierung von Oberbayern). </li><li> Elija cuidadosamente el animal y la tensión. Aunque el modelo se estableció para los ratones, que se puede traducir a otros animales comunes, tales como ratas, conejos o cerdos. </li><li> Si estás trabajando con animales de pelo, afeitarse la parte posterior de los animales antes de la implantación de los andamios. NOTA: Este trabajo utiliza ratones de 6 a 8 semanas de edad con un peso corporal entre 20 gy 25 g, sin embargo diferentes edades y bpesos ody también se pueden utilizar. </li></ol> 3. Anestesia <ol><li> Llevar a cabo la operación en condiciones estériles. Con el fin de mantener la temperatura normal del cuerpo del animal, utilizar una estera de calentamiento. </li><li> Antes de la inhalación de anestesia a los animales reciben buprenorfina a una dosis de 0,05-0,1 mg / kg de peso corporal, por vía subcutánea cada 8 horas. </li><li> Colocar el animal bajo anestesia por inhalación (isoflurano) o aplicar procedimientos estándar equivalentes. Confirme la anestesia, ya sea apretando suavemente los dedos del pie del animal oa través de un pliegue cutáneo. </li><li> Para evitar exsiccosis, inyectar 0,5 ml por vía subcutánea solución salina fisiológica al comienzo de la operación. </li></ol> 4. La escisión de la piel <ol><li> Colocar el animal en posición prona y marcar la línea media de la espalda del ratón con un bolígrafo de punta fina permanente (Figura 1A). </li><li> Defina el área de la escisión. Coloca los defectos en el área mostrada en Figure 1C. Tenga en cuenta que si los defectos se colocan demasiado caudal, los animales son más propensos a retirar los apósitos y los andamios. </li><li> Crear ronda defectos bilaterales usando un punzón de biopsia de 10 mm (Figura 1B). El punzón debe ser cuidadosamente forzado contra la piel y no debe ser utilizado para cortar completamente a través de la piel, pero para delinear el área de la escisión (Figuras 1C y 1E). </li><li> Levantar suavemente la piel marcada con una pinza y una incisión a lo largo del círculo marcado con unas tijeras quirúrgicas finas (Figura 1F). En caso de hemorragia, comprimir cuidadosamente con una gasa estéril. Una descripción paso a paso de la escisión se muestra en la Figura 1. NOTA: El defecto se crea con un punzón más pequeño que el de la generación de los andamios, para compensar la ampliación del defecto debido a la elasticidad de la piel. </li></ol> 5. Andamios Implantación <ol><li> Con el fin de hacer espacio para la titazada malla, extender la separación de la piel y el tejido subyacente en las fronteras de la herida de 2-4 mm (Figura 2A). </li><li> Coloque el titanizado de malla en el defecto directamente en el lecho de la herida y bajo los bordes de la herida (Figuras 2A y B). </li><li> Lugar andamios directamente sobre la malla. </li><li> Suturar los andamios a los bordes de la herida adyacentes con 4 a 6 nudos individuales, dejando los bordes ligeramente en el andamio (Figura 2C). NOTA: Evite el uso de suturas biodegradables. </li><li> Suturar una herida apósito transparente por encima de los defectos para proteger el andamio al tiempo que permite el seguimiento de la zona de la herida (Figura 2D). </li></ol> 6. Cuidado Postoperatorio <ol><li> Mantenga un ratón por jaula para evitar la manipulación mutua del vestidor y andamios. </li><li> Evaluar el estado general del animal sobre una base diaria por la observación de la actividad del motor, el peso corporal, signos de dolor, la toleranciaal vestidor y auto-mutilación. También supervisar el área de la herida para el sangrado, local y signos sistémicos de infección y la posición del apósito. </li><li> Con el fin de minimizar el dolor del animal, inyectar buprenorfina diaria en una dosis de 0,05-0,1 mg / kg de peso corporal o la medicación analgésica equivalente. </li><li> Si el ratón elimina o daña el vendaje de la herida, reemplazar o volver a instalarlo bajo anestesia. </li></ol> 7. Eutanasia y explantación del andamio <ol><li> En los puntos de tiempo deseados, sacrificar los animales por sobredosis de pentobarbital (150 mg / kg). </li><li> Marque el sitio de escisión de la piel con un marcador permanente, que debe incluir los andamios y la mayor cantidad de ratón de nuevo la piel como sea posible (Figura 3A). </li><li> Incisión en la piel a lo largo de las líneas marcadas con un par de tijeras o un bisturí. Separar la totalidad de la piel, incluyendo los andamios y la malla titanizado, desde el tejido subyacente a través de la preparación romo (Figura 3B). </li><li> Coloque el tejido explantado tendido en una placa de Petri (Figura 3C y D) </li></ol> 8. visualización y cuantificación de la Red Vascular <ol><li> Transiluminación: <ol><li> Configurar un dispositivo de transiluminación, mediante el montaje de una placa de Petri encima de una fuente intensa de luz blanca (100 vatios bombilla estándar). </li><li> Fijar una cámara digital por encima del transiluminador para obtener imágenes digitales. </li><li> Colocar las muestras en posición invertida sobre la placa de Petri sobre el dispositivo de transiluminación. </li><li> Tome fotos de los andamios completos en modo macro, así como de las áreas de igual tamaño de la piel normal. Guarde las imágenes en un formato TIFF para su posterior análisis digital. </li><li> Guarde el tejido para su posterior análisis bioquímicos o histológicos. </li></ol></li><li> Segmentación digital y cuantificación: <ol><li> Descargue e instale el software VesSeg-Herramienta, que se puede obtener free de forma gratuita en: <a href="http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255">http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255</a> . </li><li> Abra la imagen con el software VesSeg-Herramienta. </li><li> Seleccione el área de la imagen cubierta por el andamio para el análisis digital (imagen → Select). </li><li> Para aumentar el contraste de los vasos utilizar la opción &quot;umbral de histéresis&quot; (Imagen → mejora Vessel filtro → Histéresis de umbral → Top-Hat transformación → mejora buque Calcular). </li><li> Proceda con la segmentación del mapa buque (las fronteras de imagen → mejora Vessel filtro → → umbral Histéresis de umbral). Seleccione el primer umbral (&quot;cobertura de buques&quot;), por lo que cada píxel, que es ni remotamente buque similar, será etiquetado como recipiente. Seleccione el segundo umbral (&quot;cobertura de fondo&quot;), por lo que sólo aquellos píxeles que son particularmente buque similar será etiquetado como vasos. </li><li> Compruebe la propuesta automática segmentación manualmente añadir buques no capturados y eliminar estructuras falsos positivos comunes, que generalmente son largas y delgadas estructuras, como los vasos tales como pelos o suturas. </li><li> Calcular la longitud de los vasos y el área total cubierta por los buques (estadísticas de imagen → Imagen → Estadísticas imagen binaria). NOTA: Para el cálculo de las longitudes de los vasos, vasos en el mapa buque resultante se adelgazan a las líneas con un ancho de un pixel 20. </li></ol></li><li> Analizar el área de la piel nativa bajo los mismos parámetros que los andamios, la asignación de un valor de 100% para el tejido nativo y se refieren al cadalso a ese valor. Por ejemplo, si el porcentaje de píxeles blancos es 60% en el tejido normal y 30% en el andamio, representaría una vascularización 50% del andamio. Nota: La cobertura vasos blanco se asigna al área de un cuadrado alrededor del andamio. Ajuste el número de píxeles blancos consi coberturaDering que el área del círculo es significativamente menor (π x R 2) que el cuadrado (4 x R 2). </li></ol>

<ol>
	<li>Rahaman, M. N., Mao, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+composite+tissue+constructs+for+regenerative+medicine.">Stem cell-based composite tissue constructs for regenerative medicine.</a> Biotechnology and Bioengineering. 91 (3), 261-284 (2005).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature Biotechnology. 23, 47-55 (2005).</li><li>Machens, H. G., Berger, A. C., Mailaender, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioartificial+skin.">Bioartificial skin.</a> Cells Tissues Organs. 167, 88-94 (2000).</li><li>Priya, S. G., Jungvid, H., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+tissue+engineering+for+tissue+repair+and+regeneration.">Skin tissue engineering for tissue repair and regeneration.</a> Tissue Engineering Part B: Reviews. 14, 105-118 (2008).</li><li>Papavasiliou, G., Cheng, M. H., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+vascularization+of+polymer+scaffolds.">Strategies for vascularization of polymer scaffolds.</a> Journal of Investigative Medicine. 58 (7), 838-844 (2010).</li><li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+tissue+engineering:+breathing+life+into+constructed+tissue+substitutes.">Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes.</a> Tissue Engineering. 12, 2093-2104 (2006).</li><li>Zhong, S. P., Zhang, Y. Z., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+scaffolds+for+skin+wound+healing+and+dermal+reconstruction.">Tissue scaffolds for skin wound healing and dermal reconstruction.</a> Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology. 2 (5), 510-525 (2010).</li><li>Liu, G., Zhang, Y., Liu, B., Sun, J., Li, W., Cui, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+regeneration+in+a+canine+cranial+model+using+allogeneic+adipose+derived+stem+cells+and+coral+scaffold.">Bone regeneration in a canine cranial model using allogeneic adipose derived stem cells and coral scaffold.</a> Biomaterials. 34 (11), 2655-2664 (2013).</li><li>Hansson, A., Di Francesco, T., Falson, F., Rousselle, P., Jordan, O., Borchard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+evaluation+of+nanoparticles+for+directed+tissue+engineering.">Preparation and evaluation of nanoparticles for directed tissue engineering.</a> International Journal of Pharmaceutics. 439 (1-2), 73-80 (2012).</li><li>Sarkar, S. D., Farrugia, B. L., Dargaville, T. R., Dhara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan-collagen+scaffolds+with+nano/microfibrous+architecture+for+skin+tissue+engineering.">Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering.</a> Journal of Biomedical Materials Research Part A. 18, (2013).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+knitted+mesh-reinforced+collagen-chitosan+hybrid+scaffold+in+the+one-step+repair+of+full-thickness+skin+defects+in+rats.">The roles of knitted mesh-reinforced collagen-chitosan hybrid scaffold in the one-step repair of full-thickness skin defects in rats.</a> Acta Biomaterials. 9 (8), 7822-7832 (2013).</li><li>Rizzi, S. C., Upton, Z., Bott, K., Dargaville, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+dermal+wound+healing:+biomedical+device+approaches.">Recent advances in dermal wound healing: biomedical device approaches.</a> Expert Review of Medical Devices. 1, 143-154 (2010).</li><li>des Rieux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+systems+delivering+VEGF+to+promote+angiogenesis+for+tissue+engineering.">3D systems delivering VEGF to promote angiogenesis for tissue engineering.</a> Journal of Controlled Release. 150, 272-278 (2011).</li><li>Reckhenrich, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactivation+of+dermal+scaffolds+with+a+non-viral+copolymer-protected+gene+vector.">Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector.</a> Biomaterials. 32, 1996-2003 (2011).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Key+Regulatory+Roles+of+the+PI3K/Akt+Signaling+Pathway+in+the+Functionalities+of+Mesenchymal+Stem+Cells+and+Applications+in+Tissue+Regeneration.">The Key Regulatory Roles of the PI3K/Akt Signaling Pathway in the Functionalities of Mesenchymal Stem Cells and Applications in Tissue Regeneration.</a> Tissue Engineering Part B Rev. 19, 516-528 (2013).</li><li>Fedorovich, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endothelial+progenitor+cells+in+prevascularized+bone+tissue+engineering:+development+of+heterogenous+constructs.">The role of endothelial progenitor cells in prevascularized bone tissue engineering: development of heterogenous constructs.</a> Tissue Engineering Part A. 16 (7), 2355-2367 (2010).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+and+angiogenesis+of+tissue-engineered+bone+constructed+by+prevascularized+&#946;-tricalcium+phosphate+scaffold+and+mesenchymal+stem+cells.">Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized &#946;-tricalcium phosphate scaffold and mesenchymal stem cells.</a> Biomaterials. 36, 9452-9461 (2010).</li><li>Cuadra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+results+of+burned+hands+treated+with+Integra.">Functional results of burned hands treated with Integra.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 65 (2), 228-234 (2012).</li><li>Wilcke, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF(165)+and+bFGF+protein-based+therapy+in+a+slow+release+system+to+improve+angiogenesis+in+a+bioartificial+dermal+substitute+in+vitro+and+in+vivo.">VEGF(165) and bFGF protein-based therapy in a slow release system to improve angiogenesis in a bioartificial dermal substitute in vitro and in vivo.</a> Langenbecks Arch Surg. 392 (3), 305-314 (2007).</li><li>Condurache, A., Aach, T., Grzybowsky, S., Machens, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+segmentation+and+analysis+in+laboratory+skin+transplant+micro-angiograms.">Vessel segmentation and analysis in laboratory skin transplant micro-angiograms.</a> Proceedings of the Eighteenth IEEE Symposium on Computer-Based Medical Systems. , 21-26 (2005).</li><li>Danner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+human+sweat+gland-derived+stem+cells+for+enhancing+vascularization+during+dermal+regeneration.">The use of human sweat gland-derived stem cells for enhancing vascularization during dermal regeneration.</a> Journal of Investigative Dermatology. 132 (6), 1707-1716 (2012).</li><li>Shaterian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+Time+Analysis+of+the+Kinetics+of+Angiogenesis+and+Vascular+Permeability+in+an+Animal+Model+of+Wound+Healing.">Real Time Analysis of the Kinetics of Angiogenesis and Vascular Permeability in an Animal Model of Wound Healing.</a> Burns. 35 (6), 811-817 (2009).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nature Medicine. 9 (6), 713-725 (2003).</li><li>Bergeron, L., Tang, M., Morris, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+vascular+injection+techniques+for+the+study+of+perforator+flaps.">A review of vascular injection techniques for the study of perforator flaps.</a> Plastic and Reconstructive Surgery. 117, 2050-2057 (2006).</li><li>Schlatter, P., K&#246;nig, M. F., Karlsson, L. M., Burri, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+intussusceptive+capillary+growth+in+the+chorioallantoic+membrane+(CAM)+of+the+chicken+embryo.">Quantitative study of intussusceptive capillary growth in the chorioallantoic membrane (CAM) of the chicken embryo.</a> Microvascular Research. 54 (1), 65-73 (1997).</li><li>Lehr, H. A., Leunig, M., Menger, M. D., Nolte, D., Messmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+skinfold+chamber+technique+for+intravital+microscopy+in+nude+mice.">Dorsal skinfold chamber technique for intravital microscopy in nude mice.</a> American Journal of Pathology. 143 (4), 1055-1062 (1993).</li><li>Menger, M. D., J&#228;ger, S., Walter, P., Hammersen, F., Messmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+studies+on+the+microvasculature+of+transplanted+islets+of+Langerhans+in+vivo.">A novel technique for studies on the microvasculature of transplanted islets of Langerhans in vivo.</a> International journal of microcirculation, clinical and experimental. 9 (1), 103-117 (1990).</li><li>Laschke, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+spheroids+of+adipose-derived+mesenchymal+stem+cells+are+potent+initiators+of+blood+vessel+formation+in+porous+polyurethane+scaffolds.">Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds.</a> Acta Biomaterials. 9 (6), 6876-6884 (2013).</li><li>Ega&#241;a, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+human+mesenchymal+cells+to+improve+vascularization+in+a+mouse+model+for+scaffold-based+dermal+regeneration.">Use of human mesenchymal cells to improve vascularization in a mouse model for scaffold-based dermal regeneration.</a> Tissue Eng Part A. 15 (5), 1191-1200 (2009).</li><li>Condurache, A., Aach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+segmentation+in+angiograms+using+hysteresis+thresholding.">Vessel segmentation in angiograms using hysteresis thresholding.</a> Proceedings of the Ninth IAPR Conference on Machine Vision Applications. , 269-272 (2005).</li><li>Ega&#241;a, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+method+to+visualize+and+quantify+vascular+networks+in+native+and+tissue+engineered+skin.">Ex vivo method to visualize and quantify vascular networks in native and tissue engineered skin.</a> Langenbecks Archives of Surgery. 394, 349-356 (2009).</li></ol>

Declaración de conflicto de intereses: Todos los autores: Ninguno Divulgación de información financiera: Ninguno de los autores tiene un interés financiero en cualquiera de los productos, dispositivos o medicamentos mencionados en este manuscrito.

Hay una necesidad de establecer enfoques eficaces en la mejora de la perfusión sanguínea en construcciones de ingeniería tisular, lo que exige el desarrollo de nuevos métodos fiables para estudiar los procesos de vascularización dentro de los biomateriales. Los métodos comunes para hacer visible la vascularización andamio ex vivo incluyen el uso de la microscopía, que proporciona una herramienta de alta resolución. En la mayoría de los casos, sin embargo, este método está limitado al análisis de pe...

En las últimas décadas, la ingeniería de tejidos ha abierto una nueva opción terapéutica para reemplazar defectos de tejidos con células del propio cuerpo 1. Con el fin de apoyar el proceso fisiológico de la regeneración de tejidos, andamios están diseñados como una estructura biodegradable, que proporciona un escenario en el que las células de la lecho de la herida pueden crecer y restaurar el defecto de 2,3. Vascularización insuficiente se considera que es el principal obstáculo, que retiene el avance clínico de andamios bioartificiales 4. Con el crecimiento hacia el interior de las células, la demanda de nutrientes y oxígeno aumenta y vascularización del material se convierte en esencial. Por lo tanto, la vascularización insuficiente o tardía puede llevar a necrosis central de productos de ingeniería tisular 5. Además, los vasos sanguíneos proporcionan células inmunocompetentes y eliminar los residuos metabólicos en la zona de regeneración. Las tasas de infección altas y bajas de regeneración son sóloalgunas de las consecuencias de la perfusión insuficiente de sangre observados en la ingeniería de tejidos, que tienen por objeto que se estén produciendo por el aumento de la vascularización de los andamios de 6,7. Varias estrategias encaminadas a mejorar la vascularización foco en el papel clave de la propia biomaterial y la microestructura del andamio. Hay esfuerzos de investigación intensiva para desarrollar nuevos enfoques en el cambio del proceso de curación de la reparación a la regeneración, por lo tanto (re) generar un tejido con las propiedades fisiológicas más cercanas a la de ser restaurados 8,9. Biomateriales que fueron estudiados y evaluados en cuanto a su potencial de regeneración incluyen colágeno, fibrina, quitosano y alginato 10,11. Estos biomateriales se pueden utilizar y combinar como columna vertebral para la construcción de nuevos andamios utilizando diferentes estrategias como descelularización tejido, auto-ensamblaje, prototipado rápido y electrospinning 12. Con el fin de ENHAnce propia capacidad de regeneración del cuerpo, los andamios se pueden bioactivados. La incorporación de factores de crecimiento angiogénico recombinante o gen 13 vectores que codifican para tales factores 14 ha demostrado mejorar la vascularización del andamio. El uso de células madre se ha demostrado ampliamente ser una estrategia prometedora para mejorar la vascularización, donde las células estromales mesenquimales y células progenitoras endoteliales han ganado la mayor atención 15,16. Otros enfoques intentan construir construcciones que contienen redes de vasos prefabricados antes del trasplante 17. A pesar de los intensos esfuerzos en el diseño de andamio y su bio-activación, ninguna estrategia ha mejorado la vascularización en un nivel clínicamente significativo y, con la excepción de los reemplazos dérmicos en las lesiones por quemaduras masivas, la traducción de los materiales de bioingeniería en la rutina clínica sólo está teniendo lugar vacilante 18 . Una de las razones por las que la vascularizaciónde construcciones de tejidos artificiales sigue siendo un problema sin resolver, es la dificultad para evaluar el éxito de las nuevas tecnologías en los enfoques in vivo. Aunque los experimentos in vitro pueden proporcionar importantes conocimientos sobre el potencial de la vascularización de los andamios, se requieren modelos animales apropiados para estudiar los parámetros clave tales como la biocompatibilidad del material, la seguridad y eficacia del tratamiento y, de particular importancia, la vascularización del tejido construir. Por lo tanto, herramientas fiables para visualizar y cuantificar redes de vasos sanguíneos in vivo son esenciales. En este estudio se presenta un método simple y fiable que permite la visualización y cuantificación de la red vascular en el interior andamios explantados. Este método se basa en la transiluminación del tejido y la segmentación digital. Dado que este método no es invasivo, permite nuevos análisis moleculares e histológicas del material objetivo.

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name of Material/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:431px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Ethilon P-3 13 mm 3/8 circle 5-0</td>
			<td style="width:223px;">Ethicon, Norderstedt, Germany</td>
			<td style="width:119px;">698G</td>
			<td>Ethilon polyamid-6 precision point-reverse cutting suture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (10 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (12 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital camera&#160;</td>
			<td style="width:223px;">Ricoh, Hannover, Germany</td>
			<td style="width:119px;">Cx1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gazin Mullkompresse&#160;</td>
			<td style="width:223px;">Lohmann und Rauscher, Neuwied, Germany</td>
			<td style="width:119px;">13622</td>
			<td>Sterile gauze (10 cm x 10 cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Double-layer collagen-based scaffold (8 &#39;x 10&#39;)</td>
			<td style="width:223px;">Integra Life Science Corporation, Plainsboro, NJ, USA</td>
			<td style="width:119px;">88101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, liquid-gas for inhalative anesthesia&#160;</td>
			<td style="width:223px;">Baxter, Unterschleissheim, Germany</td>
			<td style="width:119px;">100196040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pentobarbital, 16 g / 100 ml</td>
			<td style="width:223px;">Fa. Merial, Hallbergmoos</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu</td>
			<td style="width:223px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:119px;">Strain code 088</td>
			<td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Buprenorphine (0.3 mg/ml)</td>
			<td style="width:223px;">Essex Pharma GmbH, Munich, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Titanized mesh (15 cm x 15 cm), extralight</td>
			<td style="width:223px;">PFM Medical AG, K&#246;ln, Germany</td>
			<td style="width:119px;">6000029</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Tissucol Duo S Immuno 2 ml</td>
			<td style="width:223px;">Baxter Germany GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;">B1332020110614</td>
			<td>Fibrin-thrombin solution&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Transparent adhesove drape (30.5 cm x 26 cm)</td>
			<td style="width:223px;">KCI Medical Products, Wimborne Dorset, UK</td>
			<td style="width:119px;">M6275009/10</td>
			<td></td>
		</tr>
	</tbody>
</table>

a full skin defect model to evaluate vascularization of biomaterials in vivo

Vascularización insuficiente se considera que es uno de los principales factores que limitan el éxito clínico de construcciones de ingeniería tisular. Con el fin de evaluar nuevas estrategias encaminadas a mejorar la vascularización, se requieren métodos confiables para hacer la en-el crecimiento de nuevos vasos sanguíneos en los andamios visibles bioartificiales y cuantificar los resultados. En el último par de años, nuestro grupo ha introducido un modelo de defecto de la piel completa que permite la visualización directa de los vasos sanguíneos mediante transiluminación y ofrece la posibilidad de cuantificación a través de la segmentación digital. En este modelo, se crea quirúrgicamente defectos completos de la piel en la parte posterior de los ratones y las sustituye por el material ensayado. Moléculas o células de interés también se pueden incorporar en tales materiales para estudiar su efecto potencial. Después de un tiempo de observación de la propia elección de uno, los materiales se explantaron para su evaluación. Heridas bilaterales ofrecen la posibilidad de hacer comparaciones internas ªa minimizar los artefactos entre los individuos, así como de reducir el número de animales necesarios para el estudio. En comparación con otros enfoques, nuestro método ofrece un análisis simple, fiable y rentable. Hemos aplicado este modelo como una herramienta rutinaria para llevar a cabo el cribado de alta resolución cuando se prueba la vascularización de diferentes biomateriales y enfoques bio-activación.

Un defecto de la piel completa bilateral fiable se puede crear en el ratón (Figura 1) donde la piel puede ser reemplazado por un biomaterial en estudio (Figura 2). Aquí, no se observan complicaciones mayores durante o después del procedimiento quirúrgico, ni signos macroscópicos de infección o reacción de cuerpo extraño. En casos raros, un andamio se pierde cuando un ratón elimina. Contracción de la herida que nunca se observó (Figura 3). Transiluminación te...

Watch this Scientific Journal Video about A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo at JoVE.com

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

La vascularización es clave para enfoques en la ingeniería de tejidos éxito. Por lo tanto, se requieren tecnologías fiables para evaluar el desarrollo de las redes vasculares en los tejidos-construcciones. Aquí presentamos un método simple y rentable para visualizar y cuantificar la vascularización in vivo.

Una piel llena de defectos modelo para evaluar la vascularización de Biomateriales<em&gt; En Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

a-full-skin-defect-model-to-evaluate-vascularization-biomaterials

Research

JoVE Journal

Bioengineering

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

12.1K vistas.  Technische Universität München.  La vascularización es clave para enfoques en la ingeniería de tejidos éxito. Por lo tanto, se requieren tecnologías fiables para evaluar el desarrollo de las redes vasculares en los tejidos-construcciones. Aquí presentamos un método simple y rentable para visualizar y cuantificar la vascularización in vivo. 

Video: A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

La piel humana tridimensional Reconstrucción del modelo: una herramienta para estudiar la piel normal y la progresión del melanoma

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Investigación

Bioingeniería

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Diseño de un biorreactor de presión cíclica de la Ex Vivo Estudio de las válvulas aórtica del corazón

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microentornos-Sandwich como para aprovechar Celulares / Materiales Interacciones

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingeniería de tejidos por intrínseca Vascularización en una<em&gt; En Vivo</em&gt; Cámara Ingeniería de Tejidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Un método para evaluar puntualidad y la exactitud de las respuestas Volitiva Motorizados para vibrotáctil Estímulos

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Un modelo de embrión de pez cebra para In Vivo visualización y análisis Intravital de Biomaterial asociados Staphylococcus aureus infección

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Un modelo confiable y Reproducible tamaño crítico Femoral defecto segmentario en ratas estabilizado con un Fixator externo personalizado

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

In Vivo 2-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Modelo Calvarial de Aumento Óseo en Conejo para Evaluación del Crecimiento Óseo y Neovascularización en Materiales de Sustitución Ósea

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...