Este trabajo cuenta con el apoyo financiero de NIH (Becas 1R01AR072027-01, 1R03AR069383-01), NSF Career Award (1653702) y Universidad de Connecticut.

1. Síntesis de JBNT
NOTA: El monómero JBNT se preparó como se publicó anteriormente11.
<ol><li>Síntesis del compuesto A1<ol>
<li>Preparar una solución que contenga 8,50 g de ácido cianoacético de 2 y 9,80 g de etilcarbamato en 25 ml de tolueno y 2,5 ml de N, N-dimetilformamida. Añadir 4,90 ml de cloruro de fósforo en sentido dropwise. A continuación, caliente la mezcla a 70 °C y siga revolviendo durante 1,5 h.</li>
		<li>Enfríe la mezcla de reacción a temperatura ambiente y vierta 100 g de agua helada. Extraiga la capa acuosa con acetato de etilo (3 x 250 ml) y lave con 100 ml de salmuera. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore volátiles bajo vacío para obtener un sólido de color amarillo pálido.</li>
		<li>Someta la cromatografía de columna flash de gel amarillo a sílice (3% metanol/diclorometano) para producir 15,61 g del compuesto A1 como polvo blanco.</li>
	</ol></li>
	<li>Síntesis del compuesto A2<ol>
<li>Mezclar 3,12 g del compuesto A1 y 2,75 g de carbonato de potasio en 50 ml de N, N-dimetilformamida. Revuelva a temperatura ambiente durante 2 h.</li>
		<li>Añadir 2,6 ml de disulfuro de carbono a la suspensión de reacción. Sigue revolviendo durante 4 h.</li>
		<li>Añadir etanol absoluto a la mezcla de reacción a 0 °C. Filtre el precipitado amarillo hacia fuera y lave con éter de dietil. Secar bajo vacío durante la noche para producir 6,18 g del compuesto A2 como sólido de color amarillo pálido.</li>
	</ol></li>
	<li>Síntesis del compuesto A3<ol>
<li>Añadir 2,7 ml de yoduro de metilo en 15 ml de acetonitrilo. Además, preparar por separado una solución del compuesto A2 añadiendo 6,18 g de A2 a 80 ml de agua/acetonitrilo (relación 7:3). Mezcle las dos soluciones y revuelva a temperatura ambiente durante 30 minutos.</li>
		<li>Caliente la mezcla de reacción a 95 °C y siga revolviendo durante 3 h.</li>
		<li>Enfríe la mezcla de reacción a temperatura ambiente y luego evapore los volátiles bajo vacío.</li>
		<li>Extraiga el residuo 3x con 100 ml de acetato de etilo y lávelo con salmuera. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore los volátiles bajo vacío para obtener un sólido amarillo.</li>
		<li>Someta la cromatografía de columna flash de gel amarillo a sílice (30% acetato etílico/hexanes) para producir 4,52 g del compuesto A3 como sólido amarillo claro.</li>
	</ol></li>
	<li>Síntesis del compuesto A4<ol>
<li>Preparar solución de allylamina mediante la adición de 925 μL de allylamina en 20 ml de etanol. Añada esta solución gota a gota a la solución del compuesto A3, preparado añadiendo 3,14 g de A3 en 75 ml de etanol absoluto, más de 30 min. A continuación, caliente la mezcla de reacción para reflujo durante 16 h.</li>
		<li>Enfríe la mezcla de reacción a temperatura ambiente y evapore los volátiles para dar un sólido amarillo.</li>
		<li>Someta la cromatografía de columna flash de gel amarillo a sílice (50% acetato de etilo/hexanes al 2% de metanol/diclorometano) para producir 1,80 g del compuesto A4 como cristal blanco.</li>
	</ol></li>
	<li>Síntesis del compuesto A5<ol>
<li>Añadir 2,05 g de clorhidrato de guanidinio y 7,8 ml de etóxido de sodio (21% en etanol) en 14 ml de etanol absoluto. Caliente la mezcla a 45 °C durante 15 min.</li>
		<li>Filtre el material insoluble y agregue el filtrado directamente a la solución del compuesto A4, preparado añadiendo 2,70 g de A4 en etanol absoluto de 40 ml. Caliente la mezcla de reacción para reflujo durante 16 h.</li>
		<li>Filtre el precipitado hacia fuera para obtener 2,65 g del compuesto A5 como un sólido blanquecino.</li>
	</ol></li>
	<li>Síntesis del compuesto A6<ol>
<li>Añadir 24,9 ml de trietiltamina lentamente a la mezcla de purines obtenida añadiendo 2,11 g de compuesto A5 y 1,10 g de 4-Dimetilminopyridina en 120 ml de tetrahidrofurano durante 1 min. Revuelva a temperatura ambiente durante 2 minutos.</li>
		<li>Añadir 20,7 ml de di-tert-butyl dicarbonato gota a gota a la mezcla de reacción y seguir revolviendo a temperatura ambiente durante 40 h.</li>
		<li>Agregue 20 ml de agua para apagar la reacción. Evaporar los volátiles bajo vacío.</li>
		<li>Extraiga el residuo viscoso rojo con 500 ml de acetato de etilo. A continuación, lávalo con 250 ml de agua, 75 ml de ácido cítrico al 10%, 2 veces con 200 ml de agua, 200 ml de bicarbonato sódico saturado y 200 ml de salmuera. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore los volátiles bajo el vacío para dar un sólido rojo/naranja.</li>
		<li>Someta la cromatografía de columna flash de gel sólido a sílice (0-20% acetato de etilo/hexanes) para producir 3,87 g del compuesto A6 como espuma blanca.</li>
	</ol></li>
	<li>Síntesis del compuesto A7<ol>
<li>Añadir N-metilmorfina N-óxido (50 wt.% en agua, 1,64 ml) en sentido dropwise a la solución del compuesto A6 (2,93 g) en acetona / agua (8:1, 58,5 mL) y revolver a temperatura ambiente durante 5 minutos.</li>
		<li>Añadir tetraóxido de osmio (4% solución acuosa) a la mezcla durante un período de 3 minutos y seguir revolviendo a temperatura ambiente durante 24 h.</li>
		<li>Encienda la reacción con sulfito de sodio acuoso de 1,0 M hasta que la solución se vuelva incolora. Retire los volátiles bajo vacío para dar lugar a un purines blancos en agua. Extraiga el residuo con 350 ml de diclorometano y luego lave con 150 ml de agua seguido de un lavado con 150 ml de salmuera.</li>
		<li>Secar la capa orgánica sobre sulfato de sodio anhidro, filtrar y evaporar los volátiles bajo vacío para producir 2,45 g del compuesto A7 como un sólido blanco.</li>
	</ol></li>
	<li>Síntesis del compuesto A8<ol>
<li>Añadir 760 g de perioduro de sodio a la mezcla de 1,36 g del compuesto A7 en 35 ml de diclorometano/agua (6:1, 35 ml) y revuelva a temperatura ambiente durante 42 h.</li>
		<li>Filtre el material insoluble y concentre el filtrado bajo vacío. Extraiga el residuo resultante con 250 ml de diclorometano, y luego lave con 100 ml de agua y 100 ml de salmuera. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore los volátiles bajo vacío para dar el producto crudo.</li>
		<li>Someta el producto crudo a cromatografía de columna flash de gel de sílice (5-40% acetato de etilo/hexanes, luego 5% metanol/diclorometano) para producir 1,08 g del compuesto A8.</li>
	</ol></li>
	<li>Síntesis del compuesto A9<ol>
<li>Preparar una solución de 830 mg del compuesto A8 en 15 ml de 1,2-dicloroetano. Añadir la solución de gota a gota a la mezcla de 6-benzyloxycarbonylamino-2-L-amino-ácido hexanoico trimetilsilyl éster etílico (649.5 mg) y N,N-diisopropylethylamine (591 μL) en 24 ml de 1,2-dicloroetano durante 3 min y revuelve a temperatura ambiente durante 15 min.</li>
		<li>Añadir 360,3 mg de triacetoxyborohidrato sódico a la solución y seguir revolviendo a temperatura ambiente durante 24 h.</li>
		<li>Saciar la mezcla de reacción con 6 ml de agua. Extraer la mezcla de reacción 3x con 200 ml de diclorometano y lavar con 200 ml de ácido cítrico al 10% en agua, 2x con 200 ml de agua, 200 ml de bicarbonato sódico saturado y 200 ml de salmuera en ese orden. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore los volátiles bajo vacío para obtener el producto crudo.</li>
		<li>Someta el producto crudo a cromatografía de columna flash de gel de sílice (5-30% acetato de etilo/hexanes) para producir 885 mg compuesto A9.</li>
	</ol></li>
	<li>Síntesis del monómero JBNT<ol>
<li>Disolver 0,54 g del compuesto A9 (0,54 g) en 10 ml de solución de ácido trifluoroacético/tioanisole al 94% y agitar a temperatura ambiente durante 72 h.</li>
		<li>Agregue el éter de dietil (80 ml) a la mezcla de reacción y centrífuga el precipitado hacia abajo. Vierta el ácido trifluoroacético claro que contiene sobrenadante y lave el precipitado blanco con éter de dietil y metanol para producir 168,6 mg de monómero crudo JBNT(Archivo Suplementario 1).</li>
		<li>Purifique el monómero JBNT crudo mediante una cromatografía líquida de alto rendimiento (HPLC) con una columna de fase inversa. El programa de aislamiento se muestra a continuación: Disolvente A: 100% agua; disolvente B: 100% acetonitrilo; disolvente C: solución de agua pH=1 HCl; caudal: 3 mL/min (véase la Tabla 1).</li>
		<li>Recoge el pico más grande a unos 7,2 minutos.</li>
	</ol></li>
</ol>2. Fabricación para JBNT/FN
<ol><li>Añadir 80 μL de solución acuosa JBNT de 1 mg/ml a 40 μL de solución acuosa FN de 1 mg/ml y pipeta varias veces.</li>
	<li>Compruebe la presencia de suspensión sólida blanca después de la tubería durante unos 10 s. NOTA: Se tomó un video para capturar el proceso de autoensamblaje de NM (Archivo suplementario 2).</li>
</ol>3. Observación de especímenes liofilizados
NOTA: Este paso se realiza para mostrar la estructura de andamios de NM hecha de JBNT y FN.
<ol><li>Prepare soluciones FN, JBNT y FN/JBNT NM para la observación de materiales liofilizados. Diluir 10 μL de 100 μg/ml de FN con 40 μL de agua destilada para hacer una solución de 20 μg/ml de FN.</li>
	<li>Preparar 100 mg/ml de solución de JBNT diluyendo 5 μL de 1 mg/ml de JBNTs en 45 μL de agua di.</li>
	<li>Mezclar 10 μL de 100 μg/mL FN y 5 μL de 1 mg/ml de JBNT en 35 μL de agua DI para formar la solución NM.</li>
	<li>Cubra tres pozos de microplacas inferiores redondas transparentes de 96 pozos con el FN, la solución JBNT y la solución NM, respectivamente. Cada pozo recibe 50 μL de la solución.</li>
	<li>Realizar la liofilización, como se describe a continuación, para visualizar la estructura del andamio del NM.<ol>
<li>Congele la placa a -80 °C durante la noche.</li>
		<li>Encienda el instrumento liofilizado y la bomba de vacío.</li>
		<li>Pulse el botón de enfriamiento para reducir la temperatura del sistema a -50 °C.</li>
		<li>Coloque la placa congelada en el instrumento liofilizado y cierre todas las aberturas de los instrumentos.</li>
		<li>Haga clic en el botón de inicio para reducir la presión del sistema a 0,9 mbar.</li>
		<li>Después de las 4 h, pulse el botón de aireación y abra la válvula para aumentar la presión del sistema a la presión atmosférica.</li>
		<li>Saque el plato del instrumento liofilizado.</li>
		<li>Utilice un microscopio para observar el FN, los JBNT y el NM autoensamblado resultante. Tome varias fotografías con una cámara para los materiales.</li>
	</ol>
</li>
</ol>4. Medición de espectros de absorción
NOTA: Utilice el cambio de espectros para FN y JBNT para presentar el JBNT/FN NM12autoensamblado.
<ol><li>Mezclar 30 μL de 100 μg/mL FN con 15 μL de agua destilada y 5 μL de 1 mg/ml de JBNT para preparar la solución JBNT/FN NM. La suspensión sólida blanca se puede ver después de canalizar la solución varias veces. NOTA: Las concentraciones finales de JBNT y FN en la solución son 100 μg/mL y 60 μg/mL, respectivamente. Las soluciones JBNT y FN a la misma concentración a la utilizada en la solución NM también se prepararon como soluciones de control.</li>
	<li>Diluir 5 μL de 1 mg/ml de JBNT con 45 μL de agua destilada para preparar la solución de control JBNT.</li>
	<li>Diluir 30 μL de 100 μg/mL FN con 20 μL de agua destilada para preparar la solución de control FN.</li>
	<li>Mida los espectros de absorción UV-vis de las soluciones FN, JBNT y JBNT/FN NM con el espectrofotómetro (ver Tabla de materiales)para caracterizar el ensamblaje de NM.<ol>
<li>Haga clic en el botón UV-Vis. Limpie la superficie de ensayo del espectrofotómetro y deje caer 2 μL de agua destilada sobre él. Mida como en blanco de 190-850 nm.</li>
		<li>Limpie la superficie de prueba del espectrofotómetro y suelte 2 μL de solución FN en él. Mida los espectros de absorción UV-Vis de la solución FN de 190 nm a 850 nm.</li>
		<li>Limpie la superficie de prueba del espectrofotómetro y suelte 2 μL de solución JBNT sobre ella. Mida los espectros de absorción de la solución JBNT de 190 nm a 850 nm.</li>
		<li>Limpie la superficie de prueba del espectrofotómetro y suelte 2 μL de solución JBNT/FN NM en él. Mida los espectros de absorción de la solución JBNT/FN NM de 190 nm a 850 nm.</li>
	</ol>
</li>
</ol>5. Preparación de JBNT/FN NM para microscopía electrónica de transmisión (TEM)
<ol><li>Limpie varias rejillas antes de manchar negativamente con un limpiador de plasma básico (consulte Tabla de materiales).</li>
	<li>Realice la tinción negativa de las muestras siguiendo dos procesos diferentes.<ol>
<li>Gota 3 μL de 1 mg/ml de solución acuosa JBNTs y 3 μL de solución JBNT/FN NM en rejillas separadas y déjela durante 2 minutos. A continuación, enjuague cada rejilla con 100 μL de solución de acetato de urilo (UA) del 0,5%. Escurrir el exceso de solución con papel filtrante y secar el aire de las rejillas.</li>
		<li>Mezcle 9 μL de solución JBNT/FN NM con 3 μL de solución 2.0% UA y pipeta varias veces. Pipetear la solución mixta en las rejillas y mantenerla en las rejillas durante 2 minutos. Utilice un papel de filtro para eliminar la solución mixta de las rejillas y secar las rejillas a temperatura ambiente.</li>
	</ol>
</li>
	<li>Por último, caracterizar la muestra de JBNT, JBNT/FN NM manchada con el paso 5.2.1, y JBNT/FN NM manchada con el paso 5.2.2 con el TEM.</li>
</ol>6. Ensayo de función biológica in vitro
<ol><li>Añada 200 μL de controles negativos (NC, PBS), JBNT, FN y las soluciones JBNT/FN NM en un pozo de una diapositiva de 8 pozos, respectivamente.</li>
	<li>Secar el reloj de cubierta de 8 pozos con el instrumento liofilizado para recubrir materiales en la parte inferior de los pozos. El protocolo para el secado por congelación es el mismo que el paso 3.5.</li>
	<li>A continuación, añadir 10.000 células/pozos hMSCs (Pasaje 3) en los pozos e incubarlos durante 24 h a 37 °C con un 5% de CO2.</li>
	<li>Después de la incubación, pipetear el medio de cultivo celular de los pozos y enjuagar el cultivo con solución PBS 1x.</li>
	<li>Añadir 100 μL de la solución fija a cada pozo con células y dejar durante 5 min. Enjuague las células suavemente con 1x PBS dos veces.</li>
	<li>Diluir 1% Tritón-X 100 a 0.1%. Añadir 100 μL de 0.1% Triton-X 100 a cada pozo con células y dejar por 10 min. Enjuague las células suavemente con 1x PBS dos veces.</li>
	<li>Diluir la ródamina fhalloidina a 1,65 μM. Añadir 100 μL de ródamina faloides a cada pozo con células e incubar el reloj de cubierta de cámara manteniendo la luz a temperatura ambiente durante 30 minutos. Enjuague las células suavemente con 1x PBS dos veces.</li>
	<li>Capture las imágenes celulares con un microscopio fluorescente.</li>
	<li>Cuente los números de celda en imágenes fluorescentes para cada grupo. Calcule la densidad de adherencia celular promediando tres áreas aleatorias en cada pozo.</li>
	<li>Presente todos los datos como media ± desviación estándar (SD). Realice análisis estadísticos utilizando una prueba T de una cola, seguida de un análisis de la varianza (ANOVA) con p<0.05, que se considera estadísticamente significativa.</li>
</ol>

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V., Khorsand, B., Geary, S. M., Salem, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Printing+of+scaffolds+for+tissue+regeneration+applications.">3D Printing of scaffolds for tissue regeneration applications.</a> Advanced Healthcare Materials. 4 (12), 1742-1762 (2015).</li><li>Shi, 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=Structurally+and+functionally+optimized+silk-fibroin-gelatin+scaffold+using+3D+printing+to+repair+cartilage+injury+in+vitro+and+in+vivo.">Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo.</a> Advanced Material. 29, 1701089 (2017).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. G., Fenniri, H., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+inflammatory+activation+by+self-assembling+Rosette+nanotubes+in+human+Calu-3+pulmonary+epithelial+cells.">Low inflammatory activation by self-assembling Rosette nanotubes in human Calu-3 pulmonary epithelial cells.</a> Small. 4 (6), 817-823 (2008).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. G., Fenniri, H., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosette+nanotubes+show+low+acute+pulmonary+toxicity+in+vivo.">Rosette nanotubes show low acute pulmonary toxicity in vivo.</a> International Journal of Nanomedicine. 3 (3), 373-383 (2008).</li></ol>

El Dr. Yupeng Chen es cofundador de NanoDe Therapeutics, Inc.

En este estudio, desarrollamos un NM biomimético autoensamblado, que se formó con JBNTs e FN inspirados en el ADN. Al preparar la solución JBNT, el polvo liofilizado JBNT debe disolverse en el agua en lugar de PBS porque PBS causará aglomeración de JBNT, lo que inhibe su montaje. Además, el NM también debe ensamblarse en agua si queremos observar las estructuras nano-fibril del NM, porque la sal en PBS se agrupará con fibras NM, lo que puede reducir en gran medida la resolución de las imágenes.
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Las células madre mesenquimales humanas (hMSCs) han demostrado el potencial de autorretración y auto-diferenciación a lo largo de diferentes linajes mesenquimales, lo que ayuda en la regeneración y mantenimiento de los tejidos1. Sobre la base del potencial de diferenciación, hMSCs se consideran como candidatos para lesiones de tejido mesenquimal y terapia de trastorno hematopoyético2. hMSCs han demostrado la capacidad de promover la cicatrización de heridas mediante el aumento de la reparación de tejidos, la angiogénesis, y la reducción de la inflamación3. Sin embargo, sin asistencia bioquímica o biomateriales, la eficiencia para que los hMSCs alcancen un tejido objetivo y funcionen en la ubicación deseada es baja4. Aunque se han utilizado varios andamios de ingeniería para atraer hMSCs para adherirse a las lesiones, algunos sitios como la fractura de placas de crecimiento, en medio de un hueso largo, no son fácilmente accesibles por los andamios prefabricados convencionales, que pueden no caber perfectamente en un sitio lesionado de forma irregular.
Aquí, hemos desarrollado un nanomaterial biomimético que puede autoensamblarse in situ y ser inyectado a un área objetivo de difícil acceso. El nm bio-andamio inyectable se compone de nanotubos base Janus (JBNT) y fibronectina (FN). Los JBNT, también conocidos como los Rosette Nanotubes (RNTs), se derivan de pares de bases de ADN, específicamente timina y adenina, aquí5,6,7. Como se ve en la Figura 1,los nanotubos se forman cuando seis moléculas de la base de ADN derivada se autoensamblan a través de enlaces de hidrógeno para formar un plano6. Seis moléculas se apilan unas sobre otras en un plano a través de una fuerte interacción de pi-stacking7,que puede ser de hasta 200-300 μm de longitud. Los JBNT están diseñados para imitar morfológicamente las fibras de colágeno para que el FN reaccione con ellas.
FN es una glicoproteína adhesiva de alto peso molecular, que se puede encontrar en la matriz extracelular (ECM)9. Estos pueden mediar el apego de las células madre a otros componentes del ECM, particularmente el colágeno10. Diseñamos JBNT para imitar morfológicamente las fibras de colágeno para que FN pueda reaccionar con ellas para formar NM en pocos segundos a través de la unión no covalente. Por lo tanto, NM es un prometedor bio-andamio para ser inyectado en un sitio de fractura ósea que no podría ser accesible por los andamios fabricados convencionalmente. Aquí, el NM inyectable presenta una excelente capacidad para mejorar el anclaje hMSC in vitro, exhibiendo su potencial para servir como un andamio para la regeneración de tejidos.

<table><tbody><tr><td>1,2-dichloroethane</td><td>Alfa Aesar</td><td>39121</td><td></td></tr><tr><td>2-cyanoacetic acid</td><td>Sigma-Aldrich</td><td>C88505</td><td></td></tr><tr><td>4-Dimethylaminopyridine</td><td>TCI America</td><td>D1450</td><td></td></tr><tr><td>8 wells Chambered Coverglass</td><td>Thermo Fisher</td><td>155409</td><td></td></tr><tr><td>96-well plate</td><td>Corning</td><td>353072</td><td></td></tr><tr><td>absolute ethanol</td><td>Thermo Fisher</td><td>BP2818500</td><td></td></tr><tr><td>acetone</td><td>Sigma-Aldrich</td><td>179124</td><td></td></tr><tr><td>acetonitrile</td><td>Sigma-Aldrich</td><td>34851</td><td></td></tr><tr><td>allylamine</td><td>Sigma-Aldrich</td><td>145831</td><td></td></tr><tr><td>Basic Plasma Cleaner</td><td>Harrick Plasma</td><td>PDC32G</td><td></td></tr><tr><td>citric acid</td><td>Sigma-Aldrich</td><td>251275</td><td></td></tr><tr><td>concentrated hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>Deionized water</td><td>Thermo Fisher</td><td>15230147</td><td></td></tr><tr><td>dichloromethane</td><td>Sigma-Aldrich</td><td>270997</td><td></td></tr><tr><td>diethyl ether</td><td>Sigma-Aldrich</td><td>296082</td><td></td></tr><tr><td>Di-tert-butyl dicarbonate</td><td>Sigma-Aldrich</td><td>361941</td><td></td></tr><tr><td>ethyl acetate</td><td>Sigma-Aldrich</td><td>319902</td><td></td></tr><tr><td>ethylcarbamate</td><td>Sigma-Aldrich</td><td>U2500</td><td></td></tr><tr><td>Fibronectin</td><td>Thermo Fisher</td><td>PHE0023</td><td></td></tr><tr><td>Fixative Solution (4 % formaldehyde prepared in PBS)</td><td>Thermo Fisher</td><td>R37814</td><td></td></tr><tr><td>guanidinium hydrochloride</td><td>Alfa Aesar</td><td>A13543</td><td></td></tr><tr><td>hexanes</td><td>Sigma-Aldrich</td><td>227064</td><td></td></tr><tr><td>Human mesenchymal stem cells</td><td>Lonza</td><td>PT-2501</td><td></td></tr><tr><td>methanol</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>methyl iodide</td><td>Sigma-Aldrich</td><td>289566</td><td></td></tr><tr><td>N,N-Diisopropylethylamine</td><td>Alfa Aesar</td><td>A17114</td><td></td></tr><tr><td>N,N-dimethylformamide</td><td>Sigma-Aldrich</td><td>227056</td><td></td></tr><tr><td>N-Methylmorpholine N-oxide</td><td>Alfa Aesar</td><td>A19802</td><td></td></tr><tr><td>Osmium tetraoxide</td><td>Alfa Aesar</td><td>45385</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher</td><td>15140163</td><td></td></tr><tr><td>Phosphate Buffer Solution</td><td>Thermo Fisher</td><td>20012050</td><td></td></tr><tr><td>phosphoryl chloride</td><td>Sigma-Aldrich</td><td>201170</td><td></td></tr><tr><td>potassium carbonate</td><td>Sigma-Aldrich</td><td>347825</td><td></td></tr><tr><td>reverse phase column</td><td>Thermo Fisher</td><td>25305-154630</td><td></td></tr><tr><td>Rhodamine Phalloidin</td><td>Thermo Fisher</td><td>R415</td><td></td></tr><tr><td>silica gel</td><td>TCI America</td><td>S0821</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>sodium ethoxide</td><td>Alfa Aesar</td><td>L13083</td><td></td></tr><tr><td>sodium periodide</td><td>Sigma-Aldrich</td><td>71859</td><td></td></tr><tr><td>sodium sulfate</td><td>Sigma-Aldrich</td><td>239313</td><td></td></tr><tr><td>sodium sulfite</td><td>Sigma-Aldrich</td><td>S0505</td><td></td></tr><tr><td>sodium triacetoxyborohydride</td><td>Alfa Aesar</td><td>B22060</td><td></td></tr><tr><td>spectrophotometer(NanoDrop One/One&lt;sup&gt;c&lt;/sup&gt; UV-Vis)</td><td>Thermo Fisher</td><td>ND-ONE-W</td><td></td></tr><tr><td>Stem Cell Growth Medium BulletKit</td><td>Lonza</td><td>PT-3001</td><td></td></tr><tr><td>tetrahydrofuran</td><td>Sigma-Aldrich</td><td>401757</td><td></td></tr><tr><td>thioanisole</td><td>Sigma-Aldrich</td><td>T28002</td><td></td></tr><tr><td>toluene</td><td>Sigma-Aldrich</td><td>179418</td><td></td></tr><tr><td>triethylamine</td><td>Alfa Aesar</td><td>A12646</td><td></td></tr><tr><td>trifluoroacetic acid</td><td>Alfa Aesar</td><td>A12198</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Fisher</td><td>HFH10</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Thermo Fisher</td><td>25200056</td><td></td></tr></tbody></table>

fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion

Un NM biomimético fue desarrollado para servir como un andamio biológico de ingeniería de tejidos, que puede mejorar el anclaje de las células madre. El NM biomimético se forma a partir de JBNT y FN a través del autoensamblaje en una solución acuosa. Los JBNT miden 200-300 μm de longitud con canales huecos hidrofóbicos internos y superficies hidrófilos externas. Los JBNT se cobran positivamente y los FNs se cobran negativamente. Por lo tanto, cuando se inyectan en una solución acuosa neutral, se unen a través de la unión novalente para formar los paquetes NM. El proceso de autoensamblaje se completa en pocos segundos sin ningún iniciador químico, fuente de calor o luz UV. Cuando el pH de la solución NM es menor que el punto isoeléctrico de los FN (pI 5.5-6.0), los paquetes NM se auto-liberarán debido a la presencia de FN cargado positivamente.
NM es conocido por imitar la matriz extracelular (ECM) morfológicamente y por lo tanto, se puede utilizar como un andamio inyectable, que proporciona una excelente plataforma para mejorar la adhesión hMSC. El análisis de densidad celular y los experimentos de imágenes de fluorescencia indicaron que los NMs aumentaron significativamente el anclaje de los hMSC en comparación con el control negativo.

Nuestros estudios descubrieron que la formación del NM de JBNT y FN es rápida, lo que sucedió en 10 segundos. Como se muestra en la Figura 2,floccule blanco se obtuvo cuando la solución JBNT se mezcló con la solución FN y se pipeted varias veces. El proceso de formación de NM es completamente biomimético. No se necesitan estímulos externos. El proceso de fabricación es mucho más fácil que el de algunos biomateriales emergentes, que se basa en la luz ultravioleta o iniciador quím...

Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

El objetivo de este protocolo es mostrar el montaje de una nanomatrix biomimética (NM) con nanotubos base Janus (JBNT) y fibronectina (FN). Cuando se co-cultivan con células madre mesenquimales humanas (hMSCs), los NMs exhiben una excelente bioactividad en el fomento de la adhesión a los hMSCs.

Fabricación de una nano-matriz biomimética con nanotubos base Janus y fibronectina para adherencia a células madre

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

fabrication-biomimetic-nano-matrix-with-janus-base-nanotubes

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

4.0K vistas.  University of Connecticut. El objetivo de este protocolo es mostrar el montaje de una nanomatrix biomimética (NM) con nanotubos base Janus (JBNT) y fibronectina (FN). Cuando se co-cultivan con células madre mesenquimales humanas (hMSCs), los NMs exhiben una excelente bioactividad en el fomento de la adhesión a los hMSCs.

Video: Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparación de fibrina Andamios 3D para Aplicaciones madre para cultivo celular

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Investigación

Bioingeniería

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Fuerza Litografía Cardiaca de Ingeniería de Tejidos

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

Las nanofibras ECM proteínas y Uso Asamblea Superficie iniciadas Nanoestructuras Engineered

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Electrospun nanofibras andamios con gradaciones en Organización de fibra

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

La expansión de nanoestructurada Los sustratos usando la técnica de la puntada para Nanotopographical modulación del comportamiento de la célula

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

Basado en el dendrímero nanopatrones desigual para adherencia de superficie de Control local: un método para dirigir la diferenciación condrogénica

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabricación de pozos de agarosa personalizado para la siembra de células y tejido anillo autoensamblaje con moldes impresa en 3D

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

Fabricación de Nanopattern gradiente térmico Nanoimprinting técnica y proyección de la respuesta de células formadoras de colonias endoteliales humanas

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-&#946;1) via bioaffinity. The JBNm was assembled at a TGF-&#946;1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-&#946;1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-&#946;1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-&#946;1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-&#946;1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

Fabricación y caracterización de la nanomatriz base Janus capa por capa para promover la regeneración del cartílago

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...