Name: two methods for decellularization of plant tissues for tissue engineering applications
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Nos gustaría agradecer a John Wirth de los jardines de Olbrich para suministrar amablemente los especímenes utilizados en este proyecto. Este trabajo es apoyado en parte por el National Heart, Lung and Blood Institute (R01HL115282 para G.R.G.) Fundación de ciencia nacional (DGE1144804 J.R.G y G.R.G.) y el Departamento de cirugía de la Universidad de Wisconsin y el fondo de antiguos alumnos (H.D.L.). Este trabajo también fue apoyado en parte por la Agencia de protección ambiental (grant no. 83573701 de estrella), los institutos nacionales de salud (R01HL093282-01A1 y UH3TR000506) y la National Science Foundation (IGERT DGE1144804).

1. descelularización del tejido vegetal usando el enfoque de detergente
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Utilice frescas o congeladas de F. hispida, muestras foliares. Congelación de muestras frescas no utilizadas en un congelador de-20 ° C y un almacén para uso futuro (hasta un año). Nota: Utilizar tejido de tallo o de hoja de casi cualquier planta deseada. Veces de un almacenamiento prolongado pueden causar daño a los tejidos.<ol>
<li>Determinar el tamaño y la forma de muestras a procesar en base a uso de...

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En este documento, se describen dos métodos para decellularize los tejidos vegetales. Los resultados presentados aquí, junto con los resultados de25estudios anteriores sugieren que los protocolos planteados son probablemente aplicable a un amplio espectro de especies de plantas y se pueden realizar en tallos y hojas. Estos procedimientos son simples y no requieren de equipo especializado, para que planta descelularización puede llevarse a cabo en la mayoría de los laboratorios. Cabe destacar q...

Ingeniería de tejidos surgió en la década de 1980 para crear tejidos vivos sustitutos y potencialmente dirección importantes órganos y tejidos escasez1. Una de las estrategias ha utilizar andamios para estimular y guiar el cuerpo para regenerar los tejidos o los órganos. Aunque avanzados métodos de fabricación tales como impresión en 3-d han producido andamios con características físicas únicas, la capacidad para la fabricación de andamios con una amplia gama de propiedades físicas y biológicas viables sigue siendo un reto2 , 3. por otra parte, debido a la falta de una red vascular funcional, estas técnicas han sido limitadas en regeneración de tejido de 3 dimensiones. El uso de tejidos animales y humanos decellularized como andamios ha ayudado a eludir este problema4,5,6,7. Sin embargo, el alto costo, la variabilidad lote a lote y la disponibilidad limitada pueden limitar uso generalizado de animal decellularized andamios8. También hay preocupaciones sobre la potencial transmisión de enfermedades a los pacientes y reacción inmunológica a algunos tejidos mamíferos decellularized9.
Celulosa, derivada de plantas y fuentes bacterianas, se ha utilizado ampliamente para generar Biomateriales para una amplia gama de aplicaciones en medicina regenerativa. Algunos ejemplos incluyen:10,11, cartílago12,13,14 y15de cicatrización ósea. Los andamios que se componen de celulosa tienen un beneficio adicional que son durables y resistentes a ser desglosadas por células de mamífero. Esto es debido a que las células de mamífero no producen las enzimas necesarias para descomponer las moléculas de celulosa. En comparación, andamios producción con macromoléculas de la matriz extracelular, como colágeno, se descomponen fácilmente con16 y no sea idóneos para aplicaciones a largo plazo. Andamios de colágeno pueden ser estabilizados por reticulación química. Sin embargo, existe una relación inversa debido a la toxicidad inherente de los cross-linkers que afectan la biocompatibilidad de los andamios17. Por el contrario, la celulosa tiene el potencial de permanecer presentes en el lugar de implantación durante períodos prolongados de tiempo porque son impermeable a la degradación enzimática de las células mamíferas18,19,20. Esto puede modificarse mediante la regulación de la tasa de degradación a través de pretratamiento de hidrólisis y la entrega de los andamios con celulasas21. La biocompatibilidad de celulosa de origen vegetal decellularized andamios en vivo también se ha demostrado en un estudio realizado en ratones22.
A través de cientos de millones de años de evolución, las plantas han refinado su estructura y composición para aumentar la eficiencia de transporte de fluidos y la retención. Planta vasculares vasos minimizan resistencia hidráulica por ramifican en vasos más pequeños, similares a la vasculatura mamífera según23de la ley de Murray. Después de descelularización, se mantiene la red compleja de la planta de los vasos y poros interconectados. Teniendo en cuenta la gran cantidad de especies de plantas distintas disponibles, andamios de origen vegetal tienen el potencial para superar limitaciones de diseño que afecta actualmente a andamios en tejido ingeniería24,25. Por ejemplo, Modulevsky et al demostraron que la angiogénesis y migración celular ocurrió cuando apple decellularized tejido fue implantado por vía subcutánea en la parte posterior de un ratón22. Semejantemente, Gershlak et al. , demostró que las células endoteliales podrían ser crecidas dentro de la vasculatura de hojas decellularized24. En un experimento separado, Gershlak et al. también fueron capaces de demostrar que cardiomiocitos podrían cultivarse en la superficie de las hojas y pudieron contratar24.
Las plantas también incluyen organización compleja desde el celular a la escala macroscópica, que es difícil de conseguir incluso con las más avanzadas técnicas de fabricación desarrolladas hasta la fecha. El diseño jerárquico complejo de tejidos de la planta hace más fuerte que la suma de sus componentes26. Las plantas poseen una gran cantidad de propiedades mecánicas diferentes que van desde componentes rígidos y resistentes como los tallos, a unos mucho más flexibles y maleables como hojas27. Hojas varían dependiendo de la especie en términos de tamaño, forma, rompen la resistencia, el grado de vascularización y pueden llevar a diferentes grados de hidrofilicidad. En general, estas propiedades de la planta sugieren que decellularized las plantas pueden servir como dispositivos médicos únicos y altamente funcionales, como los andamios de la ingeniería del tejido.
Este protocolo se centra en dos métodos para decellularize los tejidos vegetales, como hojas y tallos, para el uso como andamios en ingeniería tisular. El primer método es una técnica basada en el detergente que utiliza una serie de baños para extraer ADN y materia celular, que ha sido adaptada de una técnica ampliamente utilizada decellularize mamíferos y vegetales tejidos6,22,25 ,28,29,30. El segundo método está libre de detergente y es una adaptación de un protocolo de "skeletonization" generalmente se utiliza para eliminar los tejidos blandos de hojas31. Trabajos previos demostraron que hirviendo las hojas en una solución de lejía y bicarbonato de sodio facilita la separación de la vasculatura de los tejidos blandos circundantes31. Esta técnica se puede citar a experimentos realizados en elth de 17 y18 siglos, tales como el trabajo de Albertus Seba32 y33de Edward Parrish . Estos experimentos de centrar dejando materia vegetal, como hojas y frutos, sumergieron en agua durante largos períodos de tiempo (semanas a meses) y permitir que los tejidos más suaves al decaimiento, naturalmente. Aquí el enfoque "skeletonization" está adaptado para utilizar condiciones más suaves, tales como tiempos de incubación más largos a temperaturas más bajas, para eliminar los residuos celulares y para evitar distorsionar significativamente la estructura de tejidos blandos. Para los experimentos detallados en este documento, se utilizaron tres tipos de plantas: Ficus hispida, Pachira aquatica y una especies de Garcinia. Resultados de la cuantificación de ADN, pruebas mecánicas e impacto en la actividad metabólica celular de ambos métodos se describen.

<table border="0" cellpadding="0" cellspacing="0" style="width:771px;" width="771">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Sodium dodecyl sulfate</td>
			<td style="width:111px;">Sigma Life Science</td>
			<td style="width:136px;">75746-1KG</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">Triton X-100</td>
			<td style="width:111px;">MP Biomedicals, LLC</td>
			<td style="width:136px;">807426</td>
			<td style="width:277px;">Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Concentrated bleach (8.25% sodium hypochlorite)</td>
			<td style="width:111px;">Clorox</td>
			<td style="width:136px;">Item #: 31009</td>
			<td style="width:277px;">Standard concentrated bleach.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Sodium bicarbonate</td>
			<td style="width:111px;">Acros Organics</td>
			<td style="width:136px;">217120010</td>
			<td style="width:277px;">Can be substituted with sodium hydroxide or sodium carbonate.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">8 mm Biopunch</td>
			<td style="width:111px;">HealthLink</td>
			<td style="width:136px;">15111-80</td>
			<td style="width:277px;">Cuts samples that fit well in 24 well plate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Belly Dancer-Shake table</td>
			<td style="width:111px;">Stovall Life Sciences</td>
			<td style="width:136px;">BDRAA115S</td>
			<td style="width:277px;">Use low speeds to not damage tissues. Can use any model/brand of shake table.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Isotemp hot/stir plate</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;"></td>
			<td style="width:277px;">Can use any style/brand of hot/stir plate.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Beaker</td>
			<td style="width:111px;">Any</td>
			<td style="width:136px;"></td>
			<td style="width:277px;">Can use any size beaker as long as it will fit your samples and not overcrowd them.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Tris Hydrochloride</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">BP153-500</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DMEM</td>
			<td style="width:111px;">Corning</td>
			<td style="width:136px;">MT50003PC</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Quant-iT Picogreen dsDNA assay</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:136px;">P11496</td>
			<td style="width:277px;">Can use any dsDNA quantification mehtod on hand.</td>
		</tr>
	</tbody>
</table>

two methods for decellularization of plant tissues for tissue engineering applications

Los injertos autólogos, sintéticos y derivados de animales, actualmente utilizados como andamios para reemplazo de tejidos tienen limitaciones debido a la baja disponibilidad, pobre biocompatibilidad y costo. Los tejidos de la planta tienen características favorables que las hacen especialmente adecuado para utilizar como andamios, tales como superficie, excelente transporte y retención, porosidad interconectada, preexistente de redes vasculares y una amplia gama de mecánicas propiedades. Aquí se describen dos métodos exitosos de descelularización de planta para aplicaciones de ingeniería de tejidos. El primer método se basa en baños detergentes para quitar la materia celular, que es similar al previamente establecidos métodos utilizados para eliminar los tejidos mamíferos. El segundo es un método libre de detergente adaptado de un protocolo que aísla la vasculatura de la hoja y consiste en el uso de una lejía caliente y el baño de sal para limpiar las hojas y tallos. Ambos métodos producen andamios con propiedades mecánicas comparables y bajo impacto metabólico celular, lo que permite al usuario seleccionar el protocolo que mejor se adapte a su uso.

Ambos métodos produjeron andamios que eran convenientes para el cultivo de células y tejidos, aplicaciones de ingeniería. La figura 1 muestra el flujo general del proceso de descelularización utilizando una hoja intacta para el método base de detergente y corte muestras (8 mm de diámetro) para el método libre de detergente. Éxito descelularización de Ficus hispida tejidos siguiendo ambos métodos rindió muestras claras e intactas (<strong c...

Watch this Scientific Journal Video about Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications at JoVE.com

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Dos métodos de descelularización de tejidos vegetales para aplicaciones de ingeniería de tejidos

Aquí presentamos, y protocolos de contraste dos utilizan para decellularize los tejidos de la planta: un enfoque de base de detergente y un libre de detergente. Ambos métodos dejan la matriz extracelular de los tejidos de la planta utilizada, que luego puede ser utilizada como andamios para aplicaciones de ingeniería de tejidos.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

These methods can help address key questions in the biomaterials field such as the development of a scaffold with excellent fluid transfer capabilities with sophisticated ultra-structures and scalable manufacturing. The main advantage of these techniques is that they can be applied to most plants for stem tissues without the need for specialized equipment. First, collect fresh or frozen samples of F.hispida leaves.Store any remaining fresh sample at minus 20 degrees Celsius for future use. Let the leaf remain emersed in deionized water at 20 to 25 degrees Celsius. Use a clean biopsy punch to cut the leaf into eight millimeter diameter discs.Next, to wash the leaf samples, incubate them on a shake plate at low speed for five to 10 minutes, emersed in deionized water. Then prepare 10%weight by volume of SDS in deionized water. After preparing the SDS solution, place the leaf samples on a plastic dish.Then add the SDS solution to completely cover the samples. Place the SDS emersed leaf samples, covered, on a shake plate to prevent evaporation. Then incubate the leaf sample at room temperature for five days.After five days of incubation, replace the SDS solution with deionized water. Incubate the samples soaked in deionized water for 10 to 15 minutes on a shake plate. Next, mix five milliliters of non-ionic surfactant with 50 milliliters of bleach.Add this mixture to 445 milliliters of deionized water. Then emerse the samples in the freshly prepared non-ionic surfactant bleach solution. Continue changing the non-ionic surfactant bleach solution every 24 hours till the samples are cleared.Then leave the cleared samples emersed in deionized water for two minutes on a shake plate. For detergent free decellularization, prepare five percent volume by volume bleach in 3%weight by volume sodium bicarbonate solution. Next, warm the solution to 60 to 70 degrees Celsius on a hot plate within a fume hood.As soon as the solution reaches the desired temperature, soak the leaf samples. Then reduce the speed of the stir plate to prevent damaging the samples and look for the visibly cleared samples and transfer them from the bath carefully. Finally, incubate the samples in deionized water for one to two minutes.As all the samples are intrinsically different, it's recommended to check on the progress of decellularization approximately every 15 minutes to ensure the samples are not being damaged. MTM, SAF, and UTS parameters are first studied to investigate the unique decellularized scaffold properties of both P.aquatica and F.hispida species. In fact, all three parameters display similar mechanical properties using both the detergent based and detergent free decellularization methods.However, the UTS mechanical property of F.hispida shows a significantly higher average value using SDS rather than bleach for decellularization. The genomic DNA content of the cleared plant species studied is also significantly reduced upon decellularization when compared to the non-decellularized samples. Next, the metabolic activity of human dermal thyroblasts is also measured in the presence of decellularized P.aquatica or Garcinia scaffolds.Interestingly, detergent free based scaffolds elicited a reduced impact on cellular metabolic activity, but washing with Trishydrochloricde buffer, followed by serum-free medium after SDS clearing decreases the impact on cellular metabolic activity When compared to washing with deionized water alone. Finally, growth of the mesenchymal stem cells stained with calcine on the surface of the F.hispida leaf structure is shown here. Once mastered, these techniques can be completed from around seven days to as little as 15 minutes when performed properly.When performing these procedures, it's important to remember to monitor the samples as they decellularize because the samples can vary from batch to batch which can result in different clearing times. Following this procedure, other methods such as dequantification or mechanical testing can be applied to answer additional questions such as to what extent are the samples cleared and to what other tissues the samples are mechanically similar to. After its development, this technique has helped pave the way for researchers in the field of tissue engineering to manufacture perfusable scaffolds.After watching this video you should have a good understanding of how to decellularize plant tissues for use as scaffolds. Don't forget that working with detergents and bleach fumes can be hazardous and precautions such as using proper protective equipment and using a fume hood should always be taken while performing these procedures.

Research

JoVE Journal

Bioengineering

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The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Investigación

Bioingeniería

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

PGS elastomérico andamios en ingeniería de tejidos arteriales

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Postproduction Processing of Electrospun Fibres for Tissue Engineering

Postproducción de procesamiento de las Fibras electrospun de Ingeniería de Tejidos

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

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Los tejidos de ingeniería músculo esquelético de células mioblastos murinos progenitoras y aplicación de estimulación eléctrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Capilar Fuerza Litografía Cardiaca de Ingeniería de Tejidos

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Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

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Recuperación Quirúrgica, aislamiento y<em&gt; In vitro</em&gt; Ampliación del cruzado anterior humana derivados de las células del ligamento para aplicaciones de ingeniería de tejidos

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

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Extraction of Plant-based Capsules for Microencapsulation Applications

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Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...