Este estudio cuenta con el apoyo financiero de ZonMw como parte del programa LSH 2Treat (436001003) y la Fundación Holandesa del Riñón (14a2d507). N.A.K. agradece el apoyo del Consejo Europeo de Investigación (851960). Agradecemos el Programa de Gravitación "Regeneración Impulsada por Materiales", financiado por la Organización de los Países Bajos para la Investigación Científica (024.003.013).

Los autores no tienen nada que revelar.

El biorreactor aquí descrito permite la evaluación sistemática de las contribuciones de los efectos individuales y combinados del esfuerzo cizallador y el estiramiento cíclico sobre la inflamación y la regeneración tisular en andamios tubulares reabsorbibles. Este enfoque también permite realizar una gran variedad de análisis sobre constructos vasculares, como se ejemplifica en la sección de resultados representativos. Estos resultados muestran el impacto distintivo de los diferentes regímenes de carga hemodin?...

La ingeniería de tejidos cardiovasculares (TE) se está llevando a cabo como una opción de tratamiento alternativa a las prótesis cardiovasculares permanentes utilizadas actualmente (por ejemplo, injertos vasculares, reemplazos de válvulas cardíacas), que son subóptimas para grandes cohortes de pacientes1,2,3,4. Las aplicaciones muy buscadas incluyen injertos vasculares de ingeniería tisular (TEVG)5,6 y válvulas cardíacas (TEHV)7,8. La mayoría de las veces, las metodologías cardiovasculares de TE hacen uso de biomateriales reabsorbibles (ya sean naturales o sintéticos) que sirven como un andamio instructivo para que se forme el nuevo tejido. La formación de nuevo tejido puede ser diseñada completamente in vitro, sesementando el andamio con células y culturando en un biorreactor antes de la implantación (TE in vitro)9,10,11,o directamente in situ, en el que el andamio sintético se implanta sin precultivo para inducir la formación de nuevo tejido directamente en el cuerpo (TE in situ)12,13,14. Para los enfoques de TE cardiovascular in vitro e in situ, la regeneración funcional exitosa depende predominantemente tanto de la respuesta inmune del huésped a la construcción implantada como de la carga biomecánica adecuada.
La importancia de la carga biomecánica para la TE cardiovascular es bien reconocida15. En el caso de los implantes cardiovasculares, las células que pueblan el andamio están expuestas a estiramientos cíclicos y tensiones de cizallamiento que surgen como resultado del entorno hemodinámico. Numerosos estudios han reportado el efecto estimulante del estiramiento (cíclico) en la formación de componentes de la matriz, como el colágeno16,17,18,19,glicosaminoglicanos (GAG)20y elastina21, 22,por varios tipos de células. Por ejemplo, Huang et al. demostraron que el estiramiento biaxial elevaba la deposición y organización de colágeno y elastina en TEVG in vitro mediante el uso de un biorreactor vascular23. Si bien el énfasis generalmente se encuentra en el estiramiento como la carga dominante, estos estudios a menudo hacen uso de biorreactores impulsados por flujo en los que la muestra también está expuesta al flujo de cizalla. Aunque se sabe relativamente poco sobre la influencia aislada de las tensiones de cizallamiento en la formación de tejidos y la inflamación en 3D, hay algunos datos disponibles. Por ejemplo, Hinderer et al. y Eoh et al. demostraron que el flujo de cizallamiento, además de una microestructura de andamio 3D, era importante para la formación de elastina madura por células del músculo liso vascular humano en un sistema modelo in vitro24,25. En conjunto, estos hallazgos ilustran la relevancia tanto del estiramiento cíclico como del estrés cizallamiento para la TE cardiovascular.
Otro determinante importante para el éxito o fracaso de los implantes TE es la respuesta inmune del huésped al injerto implantado26. Esto es particularmente importante para las estrategias de TE in situ impulsadas por materiales, que en realidad se basan en la respuesta inflamatoria aguda al andamio para iniciar los procesos posteriores de afluencia celular y formación y remodelación endógena de tejidos27. El macrófago es un iniciador crítico de la regeneración funcional del tejido, lo que ha sido demostrado por múltiples estudios28,29,30. Análoga a la cicatrización de heridas, la regeneración del tejido se rige por la señalización paracrina entre los macrófagos y las células productoras de tejido como los fibroblastos y los miofibroblastos31,32,33. Además de coordinar la nueva deposición de tejido, los macrófagos están involucrados en la reabsorción activa de material de andamio extraño34,35. Como tal, la respuesta in vitro de los macrófagos a un biomaterial se ha identificado como un parámetro predictivo para el éxito in vivo de los implantes36,37,38.
La respuesta de los macrófagos a un andamio implantado depende de las características de diseño del andamio, como la composición del material y la microestructura35,39,40. Además de las propiedades del andamio, la respuesta de los macrófagos a un andamio y su diafonía con miofibroblastos también se ve afectada por las cargas hemodinámicas. Por ejemplo, se demostró que el estiramiento cíclico es un importante modulador del fenotipo de macrófagos41,42,43,44 y la secreción de citoquinas43,44,45,46 en andamios electrospun 3D. Utilizando un sistema de coculto de macrófagos y células del músculo liso vascular, Battiston et al. demostraron que la presencia de macrófagos condujo a un aumento de los niveles de elastina y GAG y que los niveles moderados de estiramiento cíclico (1.07-1.10) estimularon la deposición de colágeno I y elastina47. En trabajos anteriores, hemos demostrado que el esfuerzo por cizallamiento es un determinante importante para el reclutamiento de monocitos en andamios electrospun 3D48,49,y que tanto el esfuerzo por cizallamiento como el estiramiento cíclico afectan la señalización paracrina entre los monocitos humanos y las células del estroma mesenquimal50. Fahy et al. demostraron que el flujo de cizallado aumentaba la secreción de citoquinas proinflamatorias por los monocitos humanos51.
En conjunto, la evidencia anterior muestra que una comprensión adecuada y el control sobre las cargas hemodinámicas es crucial para la TE cardiovascular, y que es importante considerar la respuesta inflamatoria para lograrlo. Se han descrito previamente numerosos biorreactores para el cultivo in vitro52,53,54,55,56,57,58 o ex vivo59,60,61 de tejidos cardiovasculares. Sin embargo, todos estos sistemas están diseñados para imitar las condiciones fisiológicas de carga hemodinámica tanto como sea posible. Si bien esto es muy valioso para el propósito de crear tejidos cardiovasculares in vitro o mantener cultivos ex vivo, tales sistemas no permiten estudios sistemáticos sobre los efectos individuales de las señales individuales. Esto se debe a que la aplicación tanto del estiramiento cíclico como del esfuerzo de cizallamiento en estos biorreactores es impulsada por el mismo flujo presurizado, que los une intrínsecamente. Si bien se han descrito microsistemas que permiten una manipulación mecánica multi-cue precisa para sustratos 2D62 o configuraciones de hidrogel3D 63,64, tales configuraciones no permiten la incorporación de andamios elastoméricos de biomateriales 3D.
Aquí, presentamos la aplicación de un sistema de biorreactor tubular que permite de manera única el desacoplamiento del esfuerzo por cizallamiento y el estiramiento cíclico y ayuda a investigar mecánicamente sus efectos individuales y combinados. Este sistema permite probar una amplia variedad de injertos vasculares de ingeniería tisular (por ejemplo, de origen sintético o natural, diferentes microarquicturas, varias porosidades). Para desacoplar eficazmente la aplicación de la tensión de cizallamiento y el estiramiento, los conceptos clave que utiliza el biorreactor son (1) la separación del control de la tensión de cizallamiento y el estiramiento utilizando distintos sistemas de bomba y (2) la estimulación de los andamios de una manera "de adentro hacia afuera" con dimensiones impulsadas computacionalmente. El flujo se aplica en la superficie exterior del andamio tubular mediante el uso de una bomba de flujo, mientras que el estiramiento circunferencial del andamio se induce mediante la expansión de un tubo de silicona en el que se monta el andamio mediante el uso de una bomba de deformación separada. Las dimensiones del tubo de silicona y el tubo de vidrio que contiene la construcción se eligen y validan cuidadosamente utilizando simulaciones computacionales de dinámica de fluidos, para garantizar que la tensión de cizallamiento en el andamio (debido al flujo) y el estiramiento circunferencial (debido a la expansión del tubo) no se afecten significativamente entre sí. Este diseño de adentro hacia afuera tiene varias razones prácticas. Si el estiramiento se aplica por la presión del fluido luminal (similar a la carga fisiológica), inherentemente requiere que el diseño de la muestra esté libre de fugas. Además, la presión requerida para estirar la muestra estaría completamente determinada por la rigidez de la muestra, que puede variar entre muestras y dentro de una muestra a lo largo del tiempo, lo que dificulta el control del estiramiento. Este biorreactor monta el injerto de ingeniería tisular alrededor de un tubo de silicona y permite la aplicación de tensión de cizallamiento de pared (WSS) en la pared externa del injerto y presuriza el injerto desde el interior. De esta manera, se pueden garantizar condiciones de carga iguales entre muestras y dentro de las muestras a lo largo del tiempo, y además, se permite que las muestras estén permeables, como es común para los andamios vasculares porosos19. Este biorreactor de adentro hacia afuera está específicamente diseñado para estudios sistemáticos sobre los efectos del cizallamiento y / o estiramiento, en lugar de la ingeniería de un vaso sanguíneo nativo in vitro, para el cual las configuraciones tradicionales de biorreactores vasculares son más adecuadas. Consulte la Figura 1A-B para los dibujos de diseño del biorreactor, y su correspondiente Tabla 1 para una descripción funcional y una justificación detrás de los principales componentes del biorreactor.
El uso del biorreactor está demostrado sobre la base de una serie de estudios recientes de nuestro grupo en los que investigamos las influencias individuales y combinadas del esfuerzo cizallador y el estiramiento cíclico sobre la inflamación y la formación de tejido en andamios electrospúdicos reabsorbibles para tejido cardiovascular in situ19,43,44. Con ese fin, utilizamos macrófagos humanos y miofibroblastos, ya sea en monocultivo o en cocultivo para simular las diversas fases de la cascada regenerativa in situ. Hemos demostrado que la secreción de citoquinas por parte de los macrófagos humanos se ve claramente afectada tanto por el estiramiento cíclico como por el esfuerzo de cizallamiento, afectando la deposición de la matriz y la organización por miofibroblastos humanos en estos andamios, tanto a través de la señalización paracrina como del contacto directo19,43,44. En particular, estos estudios revelaron que en el caso de la aplicación combinada de esfuerzo por cizallamiento y estiramiento, los efectos sobre la formación de tejidos y la inflamación están dominados por una de las dos cargas, o hay efectos sinérgicos de ambas cargas. Estos hallazgos ilustran la relevancia de desacoplamiento de ambas cargas para obtener una mejor comprensión de la contribución del entorno mecánico en los procesos de TE. Esta comprensión se puede aplicar para optimizar sistemáticamente los parámetros de diseño de andamios en regímenes de carga hemodinámicos relevantes. Además, los datos mecanicistas de tales entornos bien controlados pueden servir como entrada para los modelos numéricos que se están desarrollando para predecir el curso de la remodelación de tejidos in situ, como se informó recientemente para teVGs65 o TEHVs66,para mejorar aún más la capacidad predictiva.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >advanced Dulbecco&#8217;s modified EagleMedium (aDMEM)</td>
			<td >Gibco</td>
			<td >12491-015</td>
			<td >cell culture medium for fibroblasts</td>
		</tr>
		<tr >
			<td >Aqua Stabil</td>
			<td >Julabo</td>
			<td >8940012</td>
			<td >prevent microorganism growth in bioreactor-hydraulic reservoir</td>
		</tr>
		<tr >
			<td >Bovine fibrinogen</td>
			<td >Sigma</td>
			<td >F8630</td>
			<td >to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Bovine thrombin</td>
			<td >Sigma</td>
			<td >T4648</td>
			<td >to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Eppendorf</td>
			<td >5804</td>
			<td >to spin down cells and conditioned medium</td>
		</tr>
		<tr >
			<td >Clamp scissor - &#34;kelly forceps&#34;</td>
			<td >Almedic</td>
			<td >P-422</td>
			<td >clamp the silicone tubing and apply pre-stretch to the scaffold so the scaffold can be sutured into the engraved groove (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >CO2 cell culture incubators</td>
			<td >Sanyo</td>
			<td >MCO-170AIC-PE</td>
			<td >for cell culturing</td>
		</tr>
		<tr >
			<td >Compressed air reservoir</td>
			<td >Festo</td>
			<td >CRVZS-5</td>
			<td >smoothing air pressure fluctuations and create time delays for pressure build-up</td>
		</tr>
		<tr >
			<td >Custom Matlab script to calculate the maximum stretches</td>
			<td >Matlab</td>
			<td >R2017. The Mathworks, Natick, MA</td>
			<td >calculate the minimum and maximum outer diameter of the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Data acquisition board</td>
			<td >National Instruments</td>
			<td >BNC-2090</td>
			<td >data processing in between amplifier system and computer</td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td >VWR</td>
			<td >VWRK4096-9005</td>
			<td >to keep sterile working conditions</td>
		</tr>
		<tr >
			<td >Fetal bovine calf serum (FBS)</td>
			<td >Greiner</td>
			<td >758087</td>
			<td >cell culture medium supplement; serum-supplement</td>
		</tr>
		<tr >
			<td >Flow culture chamber compartments, consisting of a pressure conduit with engraved grooves and small holes to apply pressure on silicone tubing, a screw thread, nose cone, top compartment with flow inlet and bottom compartment flow outlet, adapter bushing</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >flow culture chamber compartments (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Glass Pasteur pipet</td>
			<td >Assistant</td>
			<td >HE40567002</td>
			<td >apply vacuum on electrospun scaffold (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Glass tubes of the flow culture chamber</td>
			<td >Custon made, Equipment &#38; Prototype Center, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)</td>
		</tr>
		<tr >
			<td >GlutaMax</td>
			<td >Gibco</td>
			<td >35050061</td>
			<td >cell culture medium amino acid supplement, minimizes ammonia build-up</td>
		</tr>
		<tr >
			<td >High speed camera</td>
			<td >MotionScope</td>
			<td >M-5</td>
			<td >to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td>
		</tr>
		<tr >
			<td >High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lens</td>
			<td >Nikon</td>
			<td >JAA616AB</td>
			<td >to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td>
		</tr>
		<tr >
			<td >Hose clip</td>
			<td >ibidi GmbH</td>
			<td >10821</td>
			<td >block medium flow (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Hydraulic reservoir with 8 screw threads for 8 flow culture chambers</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >to apply pressure to the silicone mounted constructs (clean outside with a paper tissue with 70% ethanol, rinse reservoir with 70% ethanol followed by demi water, at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads</td>
			<td >ibidi GmbH</td>
			<td >10902</td>
			<td >set up used to control the flow in the flow culture chambers. Note 1: the ibidi pumps were modified by the manufacturer to enable 200 mbar capacity. Note 2: can be replaced by pump system of other manufacturer, as long as same flow regimes can be applied.</td>
		</tr>
		<tr >
			<td >Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)</td>
			<td >Swann Morton</td>
			<td >0301; 0933</td>
			<td >to cut the silicone tubing in the correct size for the scaffold and to cut the suture material</td>
		</tr>
		<tr >
			<td >LabVIEW Software</td>
			<td >National Instruments</td>
			<td >version 2018</td>
			<td >to control the stretch applied to the scaffolds</td>
		</tr>
		<tr >
			<td >Laminar flow biosafety cabinet with UV light</td>
			<td >Labconco</td>
			<td >302310001</td>
			<td >to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving</td>
		</tr>
		<tr >
			<td >Large and small petri dishes</td>
			<td >Greiner</td>
			<td >664-160</td>
			<td >for sterile working conditions</td>
		</tr>
		<tr >
			<td >L-ascorbic acid 2-phosphate (vitamin C)</td>
			<td >Sigma</td>
			<td >A8960</td>
			<td >cell culture medium supplement, important for collagen production</td>
		</tr>
		<tr >
			<td >LED light cold source KL2500</td>
			<td >Zeiss</td>
			<td >Schott AG</td>
			<td >to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch</td>
		</tr>
		<tr >
			<td >Luer (female and male) locks and connectors, white luer caps</td>
			<td >ibidi GmbH</td>
			<td >various, see (https://ibidi.com/26-flow-accessories)</td>
			<td >to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Measuring amplifier (PICAS)</td>
			<td >PEEKEL instruments B.V.</td>
			<td >n.a.</td>
			<td >to amplify the signal from the pressure sensor and feedback to LabView</td>
		</tr>
		<tr >
			<td >Medium reservoir (large syringes 60 mL) and reservoir holders</td>
			<td >ibidi GmbH</td>
			<td >10974</td>
			<td >medium reservoir (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter</td>
			<td >Rubber BV</td>
			<td >1805</td>
			<td >to allow for a larger flow rate, the ibidi medium tubing with larger diameter is used. Note: the part of medium tubing guided through the fluidic unit valves are the same as the default ibidi medium tubing</td>
		</tr>
		<tr >
			<td >Motion Studio Software</td>
			<td >Idtvision</td>
			<td >2.15.00</td>
			<td >to make the high speed time lapse images for stretch monitoring</td>
		</tr>
		<tr >
			<td >Needle (19G)</td>
			<td >BD Microlance</td>
			<td >301700</td>
			<td >together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles</td>
		</tr>
		<tr >
			<td >Needle driver</td>
			<td >Adson</td>
			<td >2429218</td>
			<td >to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Paper tissues</td>
			<td >Kleenex</td>
			<td >38044001</td>
			<td >for cleaning of the equipment with 70% ethanol</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td >Sigma</td>
			<td >P7793-1EA</td>
			<td >quick fix if leakage occurs</td>
		</tr>
		<tr >
			<td >Penicillin/streptomycin (P/S)</td>
			<td >Lonza</td>
			<td >DE17-602E</td>
			<td >cell culture medium supplement; prevent bacterial contamination</td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline (PBS)</td>
			<td >Sigma</td>
			<td >P4417-100TAB</td>
			<td >for storage and washing steps (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Plastic containers (60 mL) with red screw caps</td>
			<td >Greiner</td>
			<td >206202</td>
			<td >to prepare the fibrinogen solution</td>
		</tr>
		<tr >
			<td >Pneumatic cylinder</td>
			<td >Festo</td>
			<td >AEVC-20-10-I-P</td>
			<td >to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 &#181;m wall thickness, 20 mm length)</td>
			<td >SyMO-Chem, Eindhoven, The Netherlands</td>
			<td >n.a.</td>
			<td >produced using electrospinning from 15% (w/w) chloroform (Sigma; 372978) polymer solutions. See Van Haaften et al Tissue Engineering Part C (2018) for more details</td>
		</tr>
		<tr >
			<td >Pressure conduit without holes (for static control)</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Pressure sensor and transducer</td>
			<td >BD</td>
			<td >TC-XX and P 10 EZ</td>
			<td >the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure</td>
		</tr>
		<tr >
			<td >Proportional air pressure control valve and pressure sensor</td>
			<td >Festo</td>
			<td >MPPES-3-1/8-2-010, 159596</td>
			<td >provides compressed air to the pneumatic actuated pump</td>
		</tr>
		<tr >
			<td >Roswell Park Memorial Institute 1640 (RPMI-1640)</td>
			<td >Gibco</td>
			<td >A1049101</td>
			<td >cell culture medium for monocyte/macrophage</td>
		</tr>
		<tr >
			<td >Safe lock Eppendorf tubes (1.5 mL)</td>
			<td >Eppendorf</td>
			<td >30120086</td>
			<td >multiple applications (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Sodium dodecyl sulfate solution 20%</td>
			<td >Sigma</td>
			<td >5030</td>
			<td >Used to clean materials, at a concentration of 0.1%.&#160;&#160;</td>
		</tr>
		<tr >
			<td >Silicone O-rings</td>
			<td >Technirub</td>
			<td >1250S</td>
			<td >to prevent leakage (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)</td>
			<td >Rubber BV</td>
			<td >1805</td>
			<td >to mount the electrospun tubes on the pressure conduits (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Sterile tube (15 mL)</td>
			<td >Falcon</td>
			<td >352095</td>
			<td >multiple applications</td>
		</tr>
		<tr >
			<td >Suture, 5-0 prolene with pre-attached taper point needle</td>
			<td >Ethicon, Johnson&#38;Johnson</td>
			<td >EH7404H</td>
			<td >Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)</td>
		</tr>
		<tr >
			<td >Syringe (24 mL)</td>
			<td >B. Braun Melsungen AG</td>
			<td >2057932</td>
			<td >to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber</td>
		</tr>
		<tr >
			<td >Syringe filter (0.2 &#181;m)</td>
			<td >Satorius</td>
			<td >17597-K</td>
			<td >to filter the fibrinogen solution</td>
		</tr>
		<tr >
			<td >T150 cell culture flask with filter cap</td>
			<td >Nunc</td>
			<td >178983</td>
			<td >to degas culture medium</td>
		</tr>
		<tr >
			<td >T75 Cell culture flask with filter cap</td>
			<td >Nunc</td>
			<td >156499</td>
			<td >to culture static control samples</td>
		</tr>
		<tr >
			<td >Teflon bellow</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Tray (stainless steel)</td>
			<td >PolarWare</td>
			<td >15-248</td>
			<td >for easy transport of the fluidic culture chambers and the bioreactor from incubator to laminar flow cabinet and back (clean with a paper tissue with 70% ethanol before and after use)</td>
		</tr>
		<tr >
			<td >Tweezers</td>
			<td >Wironit</td>
			<td >4910</td>
			<td >sterile handling of individual parts (autoclave at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Ultrapure water</td>
			<td >Stakpure</td>
			<td >Omniapure UV 18200002</td>
			<td >to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)</td>
		</tr>
		<tr >
			<td >UV light</td>
			<td >Philips</td>
			<td >TUV 30W/G30 T8</td>
			<td >for decontamination of grafts and bioreactor parts before seeding</td>
		</tr>
	</tbody>
</table>

a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch

El uso de biomateriales reabsorbibles para inducir la regeneración directamente en el cuerpo es una estrategia atractiva desde una perspectiva traslacional. Tales materiales inducen una respuesta inflamatoria tras la implantación, que es el impulsor de la resorción posterior del material y la regeneración de nuevos tejidos. Esta estrategia, también conocida como ingeniería de tejidos in situ, se persigue para obtener reemplazos cardiovasculares como injertos vasculares de ingeniería tisular. Tanto los procesos inflamatorios como los regenerativos están determinados por las señales biomecánicas locales en el andamio (es decir, estiramiento y esfuerzo por cizallamiento). Aquí, describimos en detalle el uso de un biorreactor desarrollado a medida que permite de manera única el desacoplamiento de la tensión de estiramiento y cizallamiento en un andamio tubular. Esto permite la evaluación sistemática y estandarizada de la capacidad inflamatoria y regenerativa de los andamios tubulares bajo la influencia de cargas mecánicas bien controladas, que demostramos sobre la base de un experimento de cocultivo dinámico utilizando macrófagos humanos y miofibroblastos. Los pasos prácticos clave en este enfoque: la construcción y configuración del biorreactor, la preparación de los andamios y la siembra de células, la aplicación y el mantenimiento del flujo de estiramiento y cizallamiento, y la recolección de muestras para el análisis, se discuten en detalle.

Este biorreactor fue desarrollado para estudiar los efectos individuales y combinados del esfuerzo de cizallamiento y el estiramiento cíclico en el crecimiento y remodelación del tejido vascular en andamios de biomateriales 3D. El diseño del biorreactor permite cultivar hasta ocho construcciones vasculares bajo diversas condiciones de carga(Figura 1A). Los constructos vasculares se colocan en una cámara de cultivo de flujo(Figura 1B)en la que tanto el estira...

Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

El objetivo de este protocolo es ejecutar un cocultivo dinámico de macrófagos humanos y miofibroblastos en andamios electrospun tubulares para investigar la regeneración de tejidos impulsada por materiales, utilizando un biorreactor que permite el desacoplamiento del esfuerzo de cizallamiento y el estiramiento cíclico.

Un biorreactor multipíca para evaluar la capacidad inflamatoria y regenerativa de los biomateriales bajo flujo y estiramiento

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

a-multi-cue-bioreactor-to-evaluate-inflammatory-regenerative-capacity

Research

JoVE Journal

Engineering

5.4K vistas.  Eindhoven University of Technology. El objetivo de este protocolo es ejecutar un cocultivo dinámico de macrófagos humanos y miofibroblastos en andamios electrospun tubulares para investigar la regeneración de tejidos impulsada por materiales, utilizando un biorreactor que permite el desacoplamiento del esfuerzo de cizallamiento y el estiramiento cíclico.

Video: A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.

A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.

Medición material Microestructura Bajo Flujo de 1-2 Plano de Flujo de ángulo pequeño dispersión de neutrones

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is ...

Investigación

Ingeniería

A Testing Platform for Durability Studies of Polymers and Fiber-reinforced Polymer Composites under Concurrent Hygrothermo-mechanical Stimuli

The durability of polymers and fiber-reinforced polymer composites under service condition is a critical aspect to be addressed for their robust designs and condition-based maintenance. These materials are adopted in a wide range of engineering applications, from aircraft and ship structures, to bridges, wind turbine blades, biomaterials and biomedical implants. Polymers are viscoelastic materials, and their response may be highly nonlinear and thus make it challenging to predict and monitor their in-service performance. The laboratory-scale testing platform presented herein assists the investigation of the influence of concurrent mechanical loadings and environmental conditions on these materials. The platform was designed to be low-cost and user-friendly. Its chemically resistant materials make the platform adaptable to studies of chemical degradation due to in-service exposure to fluids. An example of experiment was conducted at RT on closed-cell polyurethane foam samples loaded with a weight corresponding to ~50% of their ultimate static and dry load. Results show that the testing apparatus is appropriate for these studies. Results also highlight the larger vulnerability of the polymer under concurrent loading, based on the higher mid-point displacements and lower residual failure loads. Recommendations are made for additional improvements to the testing apparatus.

The durability of polymers and fiber-reinforced polymer composites in service is a critical aspect for their designs and condition-based maintenance. We present a novel low-cost laboratory testing platform for the investigation of the influence of concurrent mechanical and environmental loadings, and may help design more efficient yet safer composite structures.

Una plataforma de pruebas de Estudios de durabilidad de Polímeros y reforzados con fibra de polímero composites bajo estímulos Concurrente Hygrothermo-mecánica

The durability of polymers and fiber-reinforced polymer composites under service condition is a critical aspect to be addressed for their robust designs ...

Experimental Protocol to Investigate Particle Aerosolization of a Product Under Abrasion and Under Environmental Weathering

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering, which is a fundamental element to the approach of nanosafety-by-design of nanostructured products for their durable development. This approach is basically a preemptive one in which the focus is put on minimizing the emission of engineered nanomaterials' aerosols during the usage phase of the product's life cycle. This can be attained by altering its material properties during its design phase without compromising with any of its added benefits. In this article, an experimental protocol is presented to investigate the nanosafety-by-design of three commercial nanostructured products with respect to their mechanical solicitation and environmental weathering. The means chosen for applying the mechanical solicitation is an abrasion process and for the environmental weathering, it is an accelerated UV exposure in the presence of humidity and heat. The eventual emission of engineered nanomaterials is studied in terms of their number concentration, size distribution, morphology and chemical composition. The purpose of the protocol is to study the emission for test samples and experimental conditions which are corresponding to real life situations. It was found that the application of the mechanical stresses alone emits the engineered nanomaterials' aerosols in which the engineered nanomaterial is always embedded inside the product matrix, thus, a representative product element. In such a case, the emitted aerosols comprise of both nanoparticles as well as microparticles. But if the mechanical stresses are coupled with the environmental weathering, the experimental protocol reveals then the eventual deterioration of the product, after a certain weathering duration, may lead to the emission of the free engineered nanomaterial aerosols too.

In this article, an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering is presented. Results on the emission of engineered nanomaterials, in the form of aerosols are presented. The specific experimental set-up is described in detail.

Protocolo experimental para investigar las partículas aerosolización del producto objeto de la abrasión y Bajo El desgaste del Medio Ambiente

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental ...

Design and Use of a Full Flow Sampling System (FFS) for the Quantification of Methane Emissions

The use of natural gas continues to grow with increased discovery and production of unconventional shale resources. At the same time, the natural gas industry faces continued scrutiny for methane emissions from across the supply chain, due to methane&#39;s relatively high global warming potential (25-84x that of carbon dioxide, according to the Energy Information Administration). Currently, a variety of techniques of varied uncertainties exists to measure or estimate methane emissions from components or facilities. Currently, only one commercial system is available for quantification of component level emissions and recent reports have highlighted its weaknesses.
In order to improve accuracy and increase measurement flexibility, we have designed, developed, and implemented a novel full flow sampling system (FFS) for quantification of methane emissions and greenhouse gases based on transportation emissions measurement principles. The FFS is a modular system that consists of an explosive-proof blower(s), mass airflow sensor(s) (MAF), thermocouple, sample probe, constant volume sampling pump, laser based greenhouse gas sensor, data acquisition device, and analysis software. Dependent upon the blower and hose configuration employed, the current FFS is able to achieve a flow rate ranging from 40 to 1,500 standard cubic feet per minute (SCFM). Utilization of laser-based sensors mitigates interference from higher hydrocarbons (C2+). Co-measurement of water vapor allows for humidity correction. The system is portable, with multiple configurations for a variety of applications ranging from being carried by a person to being mounted in a hand drawn cart, on-road vehicle bed, or from the bed of utility terrain vehicles (UTVs). The FFS is able to quantify methane emission rates with a relative uncertainty of &#177; 4.4%. The FFS has proven, real world operation for the quantification of methane emissions occurring in conventional and remote facilities.

Design and Use of a Full Flow Sampling System FFS for the Quantification of Methane Emissions

We have designed, developed, and implemented a novel full flow sampling system (FFS) for quantification of methane emissions and greenhouse gases from across the natural gas supply chain.

El diseño y uso de un sistema de muestreo de flujo completo (FFS) para la cuantificación de las emisiones de metano

The use of natural gas continues to grow with increased discovery and production of unconventional shale resources. At the same time, the natural gas ...

A Robotic Platform to Study the Foreflipper of the California Sea Lion

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most of their thrust with their large foreflippers. This protocol describes a robotic platform designed to study the hydrodynamic performance of the swimming California sea lion (Zalophus californianus). The robot is a model of the animal&#39;s foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the &#39;clap&#39;). The kinematics of the sea lion&#39;s propulsive stroke are extracted from video data of unmarked, non-research sea lions at the Smithsonian Zoological Park (SNZ). Those data form the basis of the actuation motion of the robotic flipper presented here. The geometry of the robotic flipper is based a on high-resolution laser scan of a foreflipper of an adult female sea lion, scaled to about 60% of the full-scale flipper. The articulated model has three joints, mimicking the elbow, wrist and knuckle joint of the sea lion foreflipper. The robotic platform matches dynamics properties&#8212;Reynolds number and tip speed&#8212;of the animal when accelerating from rest. The robotic flipper can be used to determine the performance (forces and torques) and resulting flowfields.

A robotic platform is described that will be used to study the hydrodynamic performance&#8212;forces and flowfields&#8212;of the swimming California sea lion. The robot is a model of the animal&#39;s foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the &#39;clap&#39;).

Una plataforma robótica para estudiar la Foreflipper del león marino de California

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most ...

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each thermoplastic of interest. Solvent bonding is a simple and versatile method that can be used to fabricate devices from a variety of plastics. An appropriate solvent is added between two device layers to be bonded, and heat and pressure are applied to the device to facilitate the bonding. By using an appropriate combination of solvent, plastic, heat, and pressure, the device can be sealed with a high quality bond, characterized as having high bond coverage, bond strength, optical clarity, durability over time, and low deformation or damage to microfeature geometry. We describe the procedure for bonding devices made from two popular thermoplastics, poly(methyl-methacrylate) (PMMA), and cyclo-olefin polymer (COP), as well as a variety of methods to characterize the quality of the resulting bonds, and strategies to troubleshoot low quality bonds. These methods can be used to develop new solvent bonding protocols for other plastic-solvent systems.

Solvent bonding is a simple and versatile method for fabricating thermoplastic microfluidic devices with high quality bonds. We describe a protocol to achieve strong, optically clear bonds in PMMA and COP microfluidic devices that preserve microfeature details, by a judicious combination of pressure, temperature, an appropriate solvent, and device geometry.

Unión con disolvente para la fabricación de PMMA y CP dispositivos de microfluidos

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each ...

In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions

To help address production issues in refineries caused by the fouling of process units and lines, we have developed a setup as well as a method to visualize the behavior of petroleum samples under process conditions. The experimental setup relies on a custom-built micro-reactor fitted with a sapphire window at the bottom, which is placed over the objective of an inverted microscope equipped with a cross-polarizer module. Using reflection microscopy enables the visualization of opaque samples, such as petroleum vacuum residues, or asphaltenes. The combination of the sapphire window from the micro-reactor with the cross-polarizer module of the microscope on the light path allows high-contrast imaging of isotropic and anisotropic media. While observations are carried out, the micro-reactor can be heated to the temperature range of cracking reactions (up to 450 &#176;C), can be subjected to H2 pressure relevant to hydroconversion reactions (up to 16 MPa), and can stir the sample by magnetic coupling. 
Observations are typically carried out by taking snapshots of the sample under cross-polarized light at regular time intervals. Image analyses may not only provide information on the temperature, pressure, and reactive conditions yielding phase separation, but may also give an estimate of the evolution of the chemical (absorption/reflection spectra) and physical (refractive index) properties of the sample before the onset of phase separation.

In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions

This article describes a setup and method for the in situ visualization of oil samples under a variety of temperature and pressure conditions that aim to emulate refining and upgrading processes. It is primarily used for studying isotropic and anisotropic media involved in the fouling behavior of petroleum feeds.

In Situ La visualización del comportamiento de fases de muestras de aceite bajo condiciones de proceso de la refinería

To help address production issues in refineries caused by the fouling of process units and lines, we have developed a setup as well as a method to ...

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light scattering from intraocular lenses (IOLs) using goniophotometer principles. This protocol describes the SLSP platform and how it employs a 360&#176; rotational photodetector sensor that is scanned around an IOL sample while recording the intensity and location of scattered light as it passes through the IOL medium. The SLSP platform can be used to predict, non-clinically, the propensity for current and novel IOL designs and materials to induce light scatter. Non-clinical evaluation of light scattering properties of IOLs can significantly reduce the number of patient complaints related to unwanted glare, glistening, optical defects, poor image quality, and other phenomena associated with the unintended light scattering. Future studies should be conducted to correlate SLSP data with clinical results to help identify which measured light scatter is most problematic for patients that have undergone cataract surgery subsequent to IOL implantation.

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

Metodología basada en el perfilador de dispersión de luz de barrido (SLPS) para evaluar cuantitativamente la dispersión de luz hacia delante y hacia atrás desde lentes intraoculares

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

A Novel Biaxial Testing Apparatus for the Determination of Forming Limit under Hot Stamping Conditions

The hot stamping and cold die quenching process is increasingly used to form complex shaped structural components of sheet metals. Conventional experimental approaches, such as out-of-plane and in-plane tests, are not applicable to the determination of forming limits when heating and rapid cooling processes are introduced prior to forming for tests conducted under hot stamping conditions. A novel in-plane biaxial testing system was designed and used for the determination of forming limits of sheet metals at various strain paths, temperatures, and strain rates after heating and cooling processes in a resistance heating uniaxial testing machine. The core part of the biaxial testing system is a biaxial apparatus, which transfers a uniaxial force provided by the uniaxial testing machine to a biaxial force. One type of cruciform specimen was designed and verified for the formability test of aluminum alloy 6082 using the proposed biaxial testing system. The digital image correlation (DIC) system with a high-speed camera was used for taking strain measurements of a specimen during a deformation. The aim of proposing this biaxial testing system is to enable the forming limits of an alloy to be determined at various temperatures and strain rates under hot stamping conditions.

This protocol proposes a novel biaxial testing system used on a resistance heating uniaxial tensile test machine in order to determine the forming limit diagram (FLD) of sheet metals under hot stamping conditions.

Un nuevo aparato de ensayo biaxiales para la Determinación del límite de conformación bajo estampación en caliente Condiciones

The hot stamping and cold die quenching process is increasingly used to form complex shaped structural components of sheet metals. Conventional ...

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture &#8211; information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

Microfluidic Imaging Cytometría de flujo por Asimétrica de detección de tiempo de estiramiento Microscopía óptica (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...