Los autores agradecen el apoyo de los Premios Innovadores Nuevos NIH Director (1DP2 OD006499-01 a AA y 1DP2 OD006460-01 a AJE) y la Ciudad Rey Abdulaziz para la Ciencia y la Tecnología (Universidad de California en San Diego Centro de Excelencia en Nanomedicina). Nos gustaría dar las gracias a la Sra. Jessica Moore por sus comentarios críticos sobre el manuscrito.

1. Síntesis de la etiqueta fluorescente acriloil-PEG-RGDS <ol><li> Reaccionar el péptido RGDS con acriloil-PEG-succinimidil éster de carboximetil (APEG-SCM, PEG MW: 3400 g / mol) y diisopropiletilamina NN (DIPEA) en 1.2:1:2 relaciones molares en sulfóxido de dimetilo (DMSO) en atmósfera de argón a temperatura ambiente durante la noche. </li><li> Confirmar conjugación por láser asistida por matriz de desorción / ionización tiempo de vuelo (MALDI-TOF) espectrometría de masas. Añadir 1 ml de solución de reacción APEG-RGDS a un punto de la muestra sobre la diana MALDI y seco. Preparar una solución saturada de Universal MALDI Matrix en tetrahidrofurano (THF) y agitar durante 1 min. Añadir ~ 1 l de esta solución para el punto de la muestra misma. Repita el procedimiento para APEG-SCM para la comparación. Cargar y analizar. El peso molecular de APEG-RGDS debe ser mayor que APEG-SCM (Figura 2). </li><li> Para conjugar el fluoróforo, añadir una cantidad equimolar de Alexa Fluor 350 de ácido carboxílico (succinimydyl éster), disueltoen un volumen mínimo de DMSO, a la solución de reacción APEG-RGDS de 1,1 y reaccionar en atmósfera de argón a temperatura ambiente durante la noche. </li><li> Purificar APEG-350-RGDS por diálisis (PM 3500 Da) contra DI-H 2 O a 4 º C durante 48 hr en relación volumétrica 1,000:1, el intercambio de dializado al menos dos veces por día. </li><li> Se liofiliza la purifica-RGDS APEG-350 en un Labconco Freezone Plus o el sistema de congelación en seco equivalente y se almacena a -20 ° C. </li></ol> 2. La preparación de un molde 2D y fabricación de un gel 2D con PEG-350 Rgds alterna capas <ol><li> Preparar portaobjetos de vidrio hidrófobos. Coloque portaobjetos de vidrio limpias en un plato de vidrio en un horno de vacío. Calentar a 80 ° C durante 30 min para secar completamente las superficies. Lugar plato con diapositivas en una campana de extracción y añadir 250 l Sigmacote para cada diapositiva, agitando suavemente durante 30 segundos a la superficie de capa entera. Enjuague bien los portaobjetos recubiertos con 100% de metanol, seguido de lavado en agua destilada, empapando dos veces durante 5 min en al LEAt 10 ml. </li><li> Cortar separadores de silicona (0,8 mm de espesor) con 10 mm sacabocados. </li><li> Autoclave los separadores de silicona y Sigmacote tratados con portaobjetos de vidrio. </li><li> Formular soluciones para cada capa respectiva en tubos de microcentrífuga individuales mediante la mezcla de diacrilato de PEG (PEGDA) precursor (concentración final 15% w / v) con diferentes cantidades de iodixanol (60% solución madre en agua) para dar concentraciones finales que varían (por ejemplo, 40%, 30%, 20% y% 10), se completa el volumen restante con tampón fosfato salino (PBS) para obtener soluciones de densidades graduadas. De una manera similar, para capas alternas, mezcla APEG-RGDS-350 (concentración final 8 mM) con iodixanol y PBS para dar concentraciones diferentes (por ejemplo, 35%, 25%, y% 15). </li><li> Añadir fotoiniciador (2-hidroxi-4&#39;-(2-hidroxietoxi)-2-metilpropiofenona, stock 333mg/ml en N-vinil pirrolidona) a cada solución para las diversas capas (10 l de solución madre por ml de cada solución de la capa). Fotoiniciador is añadió el pasado para prevenir la polimerización antes de la estratificación de los geles en el molde, ya que es sensible a la luz. </li><li> Los pasos posteriores de este protocolo se realiza en una cabina de bioseguridad para asegurar la esterilidad. </li><li> Filtrar a esterilizar cada solución utilizando una jeringa estéril de 1 ml y 0,2 micras de filtro. Montar la configuración de molde intercalando el espaciador entre dos portaobjetos de vidrio tratados con Sigmacore y asegurar con abrazaderas de colocación como se representa en la figura 1. </li><li> Reparto de los geles de capas mediante la adición de la solución más densa (por ejemplo PEGDA con 40% iodixanol) primero, seguido por una solución menos denso (por ejemplo APEG-RGDS-350 con 35% iodixanol). Repita la estratificación alternativo para lograr varias capas de composición deseada y densidades, como se muestra en la figura 1. </li><li> Irradiar el molde con luz de 365 nm durante 3 min usando un portátil UVR-9000 lámpara. Permitir que los geles polimerizados curar durante 5 min. Retire las abrazaderas, y luego levante suavemente la tapa de cristaldiapositiva y el molde, los geles dGMP estratificados se mantendrá en las diapositivas. Usando una espátula estéril, se coloca cuidadosamente los geles en un tubo de 50 ml que contenía PBS estéril o medio de cultivo para el lavado. </li><li> Lavar los geles polimerizados en PBS a 1,000:1 relación volumétrica, el intercambio de tampón por lo menos dos veces al día para eliminar el modificador de la densidad, fotoiniciador, y polímero sin reaccionar. Alternativamente, el PBS se puede intercambiar con medio de crecimiento celular. La tienda de los geles dGMP en PBS o medio de crecimiento para el experimento de cultivo de células se describe en el paso 3. </li><li> Para visualizar capas alternas, organizar gel DGMP (estos geles no se podrá utilizar para el cultivo celular) a lo largo de una regla en la bandeja de muestras de una unidad documental VersaDoc gel. Exponer los geles en 350 nm, el tiempo de exposición variará dependiendo de la concentración del fluoróforo. Alternancia de bandas oscuras y azul en el hidrogel DGMP demostrar la formación de capas discretas de composición química distinta (Figura 3). </li></ol><ol><li> Para geles incorporan péptido RGDS, utiliza células de adhesión dependientes, como C2C12 mioblastos. </li><li> Suavemente inserte geles dGMP (almacenados en PBS) en los pocillos de placas de 48 pocillos de cultivo de células utilizando un raspador de células estéril en una cabina de bioseguridad. </li><li> Pre-caliente crecimiento medio (medio Eagle modificado de Dulbecco o medio DMEM suplementado con v / v 10% de suero fetal bovino y 1% v / v solución 100x penicilina-estreptomicina) y PBS en un baño de agua a 37 ° C. </li><li> Lavar una placa de 60% confluentes (10 mm) de C2C12 células tres veces con PBS. Aspirar fuera de PBS y las células de la cosecha mediante la adición de 1 ml de 0,25% de tripsina-EDTA y se incubaron a 37 ° C durante 2 min. Resuspender las células en medio de crecimiento y células de recuento. Semilla del DGMP gel que contiene el cultivo de células con mioblastos C2C12 bien (20.000 células / cm 2). Se incuban las células a 37 ° C en 5% de CO 2/95% de humedad relativa. Suavemente cambio medio después de 4 horas, cuidando de no volver amover ligeramente las células adheridas. </li><li> Después de 24 h, la unión de mioblastos C2C12 sobre las capas que contienen RGDS de geles dGMP puede ser confirmada por epifluorescencia y la microscopía de contraste de fase (Zeiss Axiovert 200). </li></ol>

<ol>
	<li>Place, E. S., Evans, N. D., Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+in+biomaterials+for+tissue+engineering.">Complexity in biomaterials for tissue engineering.</a> Nat. Mater. 8, 457-470 (2009).</li><li>Liu, V. A., Jastromb, W. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+protein+and+cell+adhesivity+using+PEO-terminated+triblock+polymers.">Engineering protein and cell adhesivity using PEO-terminated triblock polymers.</a> J. Biomed. Mater. Res. 60, 126-134 (2002).</li><li>Sharma, B., 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=Designing+zonal+organization+into+tissue-engineered+cartilage.">Designing zonal organization into tissue-engineered cartilage.</a> Tissue Eng. 13, 405-414 (2007).</li><li>Hahn, M. 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=Photolithographic+patterning+of+polyethylene+glycol+hydrogels.">Photolithographic patterning of polyethylene glycol hydrogels.</a> Biomaterials. 27, 2519-2524 (2006).</li><li>Kizilel, S., Sawardecker, E., Teymour, F., Perez-Luna, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+formation+of+covalently+bonded+hydrogel+multilayers+through+surface+initiated+photopolymerization.">Sequential formation of covalently bonded hydrogel multilayers through surface initiated photopolymerization.</a> Biomaterials. 27, 1209-1215 (2006).</li><li>Harley, B. 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=Design+of+a+multiphase+osteochondral+scaffold.+II.+Fabrication+of+a+mineralized+collagen-glycosaminoglycan+scaffold.">Design of a multiphase osteochondral scaffold. II. Fabrication of a mineralized collagen-glycosaminoglycan scaffold.</a> J. Biomed. Mater. Res. A. 92, 1066-1077 (2010).</li><li>Cuchiara, M. P., Allen, A. C., Chen, T. M., Miller, J. S., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+microfluidic+PEGDA+hydrogels.">Multilayer microfluidic PEGDA hydrogels.</a> Biomaterials. 31, 5491-5497 (2010).</li><li>Roam, J. L., Xu, H., Nguyen, P. K., Elbert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+protein+concentration+gradients+mediated+by+density+differences+of+poly(ethylene+glycol)+microspheres.">The formation of protein concentration gradients mediated by density differences of poly(ethylene glycol) microspheres.</a> Biomaterials. 31, 8642-8650 (2010).</li><li>Gleghorn, J. P., Lee, C. S., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+properties+of+laminated+alginate+gels+for+tissue+engineering+of+layered+structures.">Adhesive properties of laminated alginate gels for tissue engineering of layered structures.</a> J. Biomed. Mater. Res. A. 85, 611-618 (2008).</li><li>Karpiak, J. V., Ner, Y., Almutairi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+gradient+multilayer+polymerization+for+creating+complex+tissue.">Density gradient multilayer polymerization for creating complex tissue.</a> Adv. Mater. 24, 1466-1470 (2012).</li><li>Pierschbacher, M. D., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+attachment+activity+of+fibronectin+can+be+duplicated+by+small+synthetic+fragments+of+the+molecule.">Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule.</a> Nature. 309, 30-33 (1984).</li><li>Kaushik, G., Fuhrmann, A., Cammarato, A., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Situ+Mechanical+Analysis+of+Myofibrillar+Perturbation+and+Aging+on+Soft,+Bilayered+Drosophila+Myocardium."> In Situ Mechanical Analysis of Myofibrillar Perturbation and Aging on Soft, Bilayered Drosophila Myocardium.</a> Biophysical Journal. 101, 2629-2637 (2011).</li></ol>

Los autores no tienen conflictos de intereses a revelar.

DGMP es una estrategia sencilla para la preparación de geles de capas múltiples que no se basan en instrumentos costosos. Este protocolo se puede adaptar para crear andamios utilizando otros materiales biocompatibles, tales como colágeno y ácido hialurónico. Pequeñas moléculas bioactivas, por ejemplo de células que promueve la adhesión péptido RGDS, puede estar amarrado a la matriz de polímero para evitar la mezcla de las señales entre las capas. Las proteínas se pueden encapsular en capas distintas, sin necesidad de conjugación química, ya que, dependiendo del tamaño de la malla de la matriz, son menos propensos a difundirse a través de hidrogeles 10. Aquí hemos utilizado iodixanol (Nycoprep), un modificador de la densidad inerte, que se ha utilizado anteriormente para las aplicaciones de células viables. Otros modificadores de la densidad tales como sacarosa y dextrosa también se puede utilizar. Al variar el tiempo de establecimiento (t s), se puede ajustar con precisión las interfaces entre las dos capas para producir una transición suave o fuerte, según sea necesario (tiempo de asentamiento más aporta mayor suavidad a las transiciones) &lt;sup&gt; 10. Por ejemplo, transiciones más suaves entre las capas de gel dGMP podría ser utilizado para generar un gradiente continuo de una señal biológica para estudiar procesos celulares, tales como la quimiotaxis. El efecto modificador de la densidad sobre la rigidez de gel se muestra en la Figura 5 para un 15% de gel de aPEGda; una caracterización más completa de la rigidez y de la porosidad como una función de las concentraciones de iodixanol PEGDA y se está evaluando actualmente. Mientras que la concentración de PEGDA en este ejemplo es relativamente alta, se observó un módulo de 60% mayor elástica en geles con 30% iodixanol comparación con los geles sin. El cambio en la rigidez del gel se puede ajustar mediante la modulación de la concentración de macrómero o la densidad de reticulación. También hemos aplicado la técnica DGMP para crear 3D geles de poliacrilamida utilizando múltiples capas y los precursores de PEG 10. La variación de la concentración o el grado de reticulación del prepolímero permite variación estructural en elandamios, que pueden ser utilizados para explorar el comportamiento celular tales como el crecimiento y la migración polarizada en 3D. En resumen, DGMP es una técnica adaptable que se puede aplicar para fabricar andamios 2D y 3D a partir de una variedad de materiales biocompatibles para una amplia gama de aplicaciones de investigación biomédica y de base.

<table border="1"><tbody><tr><td> Reactivo o Instrumento </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Polietilenglicol succinimydyl carboximetil (a-PEG-SCM) </td><td> Laysan </td><td> 120-64 </td></tr><tr><td> Polyethelyene glicol diacrilato (PEGDA) </td><td> Dajac Labs </td><td> 9359 </td></tr><tr><td> Arginina-glicina-ácido aspártico-serina (RGDS) </td><td> American Peptide </td><td> 49-01-4 </td></tr><tr><td> N, N - diisopropiletilamina (DIPEA) </td><td> Sigma </td><td> D125806 </td></tr><tr><td> Sulfóxido de dimetilo (DMSO) </td><td> Sigma </td><td> D2438 </td></tr><tr><td> N, N-dimetilformamida (DMF) </td><td> Pescador </td><td> D119-4 </td></tr><tr><td> El tetrahidrofurano (THF) </td><td> Pescador </td><td> T397 </td></tr><tr><td> Casete de diálisis (3500Da) </td><td> Thermo Scientific </td><td> 66330 </td></tr><tr><td> Alexa Fluor 350 carboxílico succinimydyl </td><td> Life Technologies </td><td> A-10168 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> Separadores de silicona </td><td> Grainger </td><td> 1MWA4 </td></tr><tr><td> Biopsia golpes </td><td> Acuderm </td><td> P1025 (10 mm) P850 (8 mm) </td></tr><tr><td> Fosfato de Dulbecco tamponada con solución salina (DPBS) </td><td> Hyclone </td><td> SH30028 </td></tr><tr><td> Iodixanol (NycoPrep) </td><td> Pescador </td><td> NC9388846 </td></tr><tr><td> 2-hidroxi-4&#39;-(2-hidroxietoxi)-2-metilpropiofenona </td><td> Sigma </td><td> 410896 </td></tr><tr><td> Dulbecco modificado a medio Eagle (DMEM) </td><td> Life Technologies </td><td> 11054 </td></tr><tr><td> El suero fetal bovino </td><td> Life Technologies </td><td> 10082 </td></tr><tr><td> Penicilina-estreptomicina </td><td> Life Technologies </td><td> 15140 </td></tr><tr><td> C2C12 mioblastos </td><td> ATCC </td><td> CRL-1772 </td></tr><tr><td> MALDI </td><td> Bruker </td><td> N / A </td></tr><tr><td> UVR-9000 </td><td> Bayco </td><td> UVR-9000 </td></tr><tr><td> VersaDoc </td><td> Bio-Rad </td><td> N / A </td></tr></tbody></table>

density gradient multilayered polymerization (dgmp): a novel technique for creating multi-compartment, customizable scaffolds for tissue engineering

Complejas matrices de cultivo de tejidos, en el que los tipos y concentraciones de los estímulos biológicos (por ejemplo, factores de crecimiento, inhibidores, o pequeñas moléculas) o la estructura de la matriz (por ejemplo, composición, concentración, o la rigidez de la matriz) varían en el espacio, que permiten una amplia gama de investigaciones sobre cómo estas variables afectan la diferenciación celular, la migración y otros fenómenos. El principal reto en la creación de matrices de capas es el mantenimiento de la integridad estructural de las interfaces de capa sin difusión de los componentes individuales de cada capa 1. Las metodologías actuales para ello es photopatterning 2-3, litografía 4, functionalization5 secuencial, liofilización 6 de microfluidos 7, 8 o centrifugación, muchos de los cuales requieren instrumentos sofisticados y habilidades técnicas. Otros confían en unión secuencial de las capas individuales, que pueden llevar a la deslaminación de las capas 9 </sup&gt;. DGMP supera estos problemas mediante el uso de un modificador de la densidad inerte tal como iodixanol para crear capas de densidades variables 10. Dado que el modificador de la densidad se puede mezclar con cualquier prepolímero o molécula bioactiva, DGMP permite que cada capa andamio para personalizar. Simplemente variando la concentración del modificador de la densidad evita que se mezclen las capas adyacentes mientras permanecen acuosa. Polimerización subsiguiente solo paso da lugar a una estructura de andamio continuo de múltiples capas, en la que cada capa tiene química distinta y propiedades mecánicas. El modificador de la densidad se puede quitar fácilmente con suficiente lavado sin perturbación de las capas individuales o sus componentes. Esta técnica es por lo tanto muy adecuado para la creación de hidrogeles de diversos tamaños, formas y materiales. Un protocolo para la fabricación de un 2D-polietilenglicol (PEG) gel, en el que las capas alternantes incorporar RGDS-350, se describe a continuación. Utilizamos PEG bomo es biocompatible e inerte. RGDS, una adhesión celular péptido 11, se utiliza para demostrar restricción espacial de una señal biológica, y la conjugación de un fluoróforo (Alexa Fluor 350) nos permite distinguir visualmente distintas capas. Este procedimiento se puede adaptar para otros materiales (por ejemplo, colágeno, ácido hialurónico, etc) y se puede ampliar para fabricar geles 3D con algunas modificaciones 10.

MALDI-TOF análisis confirma la conjugación del péptido RGDS para acriloil-PEG (Figura 2). Gel imagen revela alterna RGDS-350 (azul) capas después de la fotopolimerización (Figura 3A). Como se muestra en la Figura 3A, tamaño del gel 2D DGMP se puede variar en función del diámetro de los moldes de silicona (10 mm, izquierdo; 8 mm, derecha), y por lo tanto son fácilmente adaptables para su uso en múltiples ensayos - en este caso para adaptarse a una 48 así placa de cultivo de células (Figura 3B). Epifluorescencia y la microscopía de contraste de fase de C2C12 mioblastos cultivados en un gel de DGMP muestra unión selectiva en RGDS-350-que contiene capas de PEG (Figura 4), ​​lo que demuestra la compartimentación de la adhesión celular péptido (RGDS). <img src="/files/ftp_upload/50018/50018fig1.jpg" alt="Figura 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50018/50018fig1highres.jpg"/> Figura 1. Molecular Weight análisis por MALDI-TOF comparar APEG-SCM para APEG-RGDS obtenidos después de la conjugación del péptido RGDS. <img src="/files/ftp_upload/50018/50018fig2.jpg" alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/50018/50018fig2highres.jpg"/> Figura 2. Representación esquemática de DGMP fabricación de gel. Después de los gradientes se disponen en capas, pueden dejarse en reposo durante períodos variables de tiempo (t s) para crear interfaces graduadas, seguido por fotopolimerización. Estratificados geles dGMP puede ser fácilmente extraído del molde para su posterior utilización. <a href="https://www.jove.com/files/ftp_upload/50018/50018fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . <img src="/files/ftp_upload/50018/50018fig3.jpg" alt="Figura 3" /> Figura 3. A) geles 2D multicapa obtenidos después de la fotopolimerización imágenes utilizando 350 nm y blancas canales de luz de VersaDoc. gel unidad documental. La escala de grises de la imagen revela alternando capas que contienen RGDS en blanco. B) Inserción de gel DGMP en 48 pocillos de cultivo celular. <img src="/files/ftp_upload/50018/50018fig4.jpg" alt="Figura 4" /> Figura 4. Contraste de fase y de la imagen fusionada de epifluorescencia C2C12 mioblastos cultivados en DGMP geles (barra de escala 50 mm). <img src="/files/ftp_upload/50018/50018fig5.jpg" alt="Figura 5" /> Figura 5. Efecto de la elasticidad del gel iodixanol en superficie. Atómica mediciones de fuerza de microscopía de muestras estáticas de sustratos PEG reticulados utilizando métodos previamente establecidos con un activador 2 nN fuerza 12. * P &lt;0,05 y ** p &lt;0,01.

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Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Aquí se describe una estrategia única para la creación de matrices biocompatibles, capas continuas con interfaces entre capas distintas para la ingeniería de tejidos. Tal andamio podría proporcionar un entorno adaptable ideal para modular el comportamiento celular por varias señales biológico, químico o mecánico

Gradiente de densidad multicapa polimerización (DGMP): una técnica novedosa para la creación de multi-compartimiento, Andamios personalizables para Ingeniería de Tejidos

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

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Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

12.4K vistas.  University of California, San Diego. Aquí se describe una estrategia única para la creación de matrices biocompatibles, capas continuas con interfaces entre capas distintas para la ingeniería de tejidos. Tal andamio podría proporcionar un entorno adaptable ideal para modular el comportamiento celular por varias señales biológico, químico o mecánico

Video: Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomérico andamios en ingeniería de tejidos arteriales

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Investigación

Bioingeniería

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproducción de procesamiento de las Fibras electrospun de Ingeniería de Tejidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

Un biorreactor Novela de alta densidad de cultivo de diversas comunidades microbianas

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

a base de varilla de fabricación de dispositivos robóticos adaptables pinza neumática suaves para la manipulación del tejido delicado

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Los andamios neonatal cardíaca: Matrices novedosos para Estudios regenerativos

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Escritura de Electrospinning de andamios Poly(ε-caprolactone) tridimensional con morfologías controlables para aplicaciones de ingeniería de tejidos del derretimiento

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting de cartílago y piel análogos de tejido utilizando un pasivo novela mezcla de unidad técnica de Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Un protocolo para Decellularizing ratón cócleas para ingeniería de tejidos del oído interno

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Cultivo de células de mamífero en andamios tridimensionales péptido

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...