Name: biomechanical testing of murine tendons
Uploaded: 2019-10-15T14:30:00
Description: Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

El estudio fue apoyado por los NIH / NIAMS (R01 AR055580, R01 AR057836).

Los estudios en animales fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales de la Universidad de Columbia. Los ratones utilizados en este estudio eran de un fondo C57BL/6J y fueron comprados en the Jackson Laboratory (Bar Harbor, ME, USA). Estaban alojados en condiciones de barrera libre de patógenos y se les proporcionaba alimentos y agua ad libitum.
1. Desarrollo de accesorios impresos en 3D para agarrar hueso
<ol><li>Adquisición de imagen ósea y construcción de...

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Los modelos animales murinos se utilizan comúnmente para estudiar trastornos del tendón, pero la caracterización de sus propiedades mecánicas es desafiante y poco común en la literatura. El propósito de este protocolo es describir un método eficiente en el tiempo y reproducible para las pruebas de tracción de los tendones murinos. Los nuevos métodos redujeron el tiempo necesario para probar una muestra de horas a minutos y eliminaron un artefacto de agarre importante que era un problema común en métodos anteri...

Los trastornos del tendón son comunes y muy frecuentes entre las poblaciones de envejecimiento, atletismo y activa1,2,3. En los Estados Unidos, 16,4 millones de lesiones por tejido conectivo se reportan cada año4 y representan el 30% de todas las visitas al consultorio médico relacionadas con lesiones3,5,6,7, 8. Los sitios más comúnmente afectados incluyen el manguito rotador, tendón de Aquiles, y tendón rotuliano9. Aunque se han explorado una variedad de tratamientos no quirúrgicos y operativos, incluyendo medicamentos antiinflamatorios, rehabilitación y reparación quirúrgica, los resultados siguen siendo pobres, con un retorno limitado a la función y altas tasas de falla5, 6. Estos malos resultados clínicos han motivado los estudios básicos y traslacionales que buscan comprender la tendinopatía y desarrollar nuevos enfoques de tratamiento.
Las propiedades biomecánicas de tracción son los principales resultados cuantitativos que definen la función del tendón. Por lo tanto, la caracterización de laboratorio de la tendinopatía y la eficacia del tratamiento deben incluir una prueba rigurosa de las propiedades de la tensión del tendón. Numerosos estudios han descrito métodos para determinar las propiedades biomecánicas de los tendones a partir de modelos animales como ratas, ovejas, perros y conejos10,11,12. Sin embargo, pocos estudios han probado las propiedades biomecánicas de los tendones murinos, principalmente debido a las dificultades para agarrar los tejidos pequeños para las pruebas de tracción. Como los modelos murinos tienen numerosas ventajas para el estudio mecanicistamente tendinopatía, incluyendo la manipulación genética, amplias opciones de reactivos y bajo costo, se necesita el desarrollo de métodos precisos y eficientes para probar biomecánicamente los tejidos murinos.
Con el fin de probar adecuadamente las propiedades mecánicas de los tendones, el tejido debe ser agarrado eficazmente, sin resbalones o desgarros artifes reales en la interfaz de agarre o fractura de la placa de crecimiento. En muchos casos, particularmente para tendones cortos, el hueso se agarra en un extremo y el tendón se agarra en el otro extremo. Los huesos se fijan típicamente incrustándolos en materiales como resina epoxi13 y polimetilmetacrilato14,15. Los tendones a menudo se colocan entre dos capas de papel de lija, pegados con cianoacrilato, y asegurados mediante abrazaderas de compresión (si la sección transversal es plana) o en un medio congelado (si la sección transversal es grande)15,16,17 . Estos métodos se han aplicado a tendones murinas de ensayo biomecánicos, pero surgen desafíos debido al pequeño tamaño de los especímenes y al cumplimiento de la placa de crecimiento, que nunca osifie18. Por ejemplo, el diámetro de la cabeza hueral murina es de sólo unos pocos milímetros, lo que dificulta el agarre del hueso. Específicamente, las pruebas de tracción de muestras de tendón-hueso de supraespinato murino a menudo resultan en falla en la placa de crecimiento en lugar de en el tendón o en la entesis del tendón. Del mismo modo, las pruebas biomecánicas del tendón de Aquiles son desafiantes. Aunque el tendón de Aquiles es más grande que otros tendones murinos, el calcáneo es pequeño, lo que dificulta el agarre de este hueso. El hueso se puede extraer, seguido de agarrar los dos extremos del tendón; sin embargo, esto impide la prueba de la unión de tendón a hueso. Otros grupos informan de agarrar el hueso de calcáneo utilizando accesorios hechos a medida19,20, anclaje por abrazaderas21, fijación en cemento plástico autocurado22 o utilizando una ranura de forma cónica22, sin embargo, estos métodos anteriores siguen estando limitados por la baja reproducibilidad, las altas tasas de fallas de agarre y los requisitos tediosos de preparación.
El objetivo del estudio actual fue desarrollar un método preciso y eficiente para las pruebas biomecánicas de tracción de los tendones murinos, centrándose en los tendones supraespinato y Aquiles como ejemplos. Utilizando una combinación de reconstrucciones 3D de la anatomía ósea nativa, modelado sólido y fabricación aditiva, se desarrolló un método novedoso para agarrar los huesos. Estos accesorios aseguraron eficazmente los huesos, evitaron fallas en la placa de crecimiento, disminuyeron el tiempo de preparación de la muestra y aumentaron la reproducibilidad de las pruebas. El nuevo método es fácilmente adaptable para probar otros tendones murinos, así como tendones en ratas y otros animales.

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	<tbody>
		<tr >
			<td >Agarose</td>
			<td >Fisher Scientific</td>
			<td >BP160-100</td>
			<td >Dissovle 1g in 100 ml ultrapure water to make 1% agarose&#160;</td>
		</tr>
		<tr >
			<td >Bruker microCT&#160;</td>
			<td >Bruker BioSpin Corp</td>
			<td >Skyscan 1272&#160;</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >ElectroForce&#160;</td>
			<td >TA Instruments</td>
			<td >3200</td>
			<td>Testing platform</td>
		</tr>
		<tr >
			<td >Ethanol 200 Proof</td>
			<td >Fisher Scientific</td>
			<td >A4094</td>
			<td>Dilute to 70% and use as suggested in protocol</td>
		</tr>
		<tr >
			<td >Fixture to attach grips</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Kimwipes</td>
			<td >Kimberly-Clark</td>
			<td >&#160;S-8115</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >MicroCT CT-Analyser (Ctan)</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >MilliQ water (Ultrapure water)</td>
			<td>Millipore Sigma</td>
			<td>QGARD00R1 (or related purifier)</td>
			<td>100 ml&#160;</td>
		</tr>
		<tr >
			<td >Meshmixer</td>
			<td >Autodesk</td>
			<td >http://www.meshmixer.com/</td>
			<td >Free engineering software used by authors to refine mesh</td>
		</tr>
		<tr >
			<td >Objet EDEN 260VS&#160;</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Precision Prototyping</td>
		</tr>
		<tr >
			<td >Objet Studio</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Used by authors with 3D printer</td>
		</tr>
		<tr >
			<td >PBS - Phosphate-Buffered Saline</td>
			<td >ThermoFisher Scientific</td>
			<td >10010031</td>
			<td >2.5 L of 10% PBS&#160;</td>
		</tr>
		<tr >
			<td >S&#38;T Forceps</td>
			<td >Fine Science Tools</td>
			<td >00108-11</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Blade - #11</td>
			<td >Fine Science Tools</td>
			<td >10011-00</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Handle - #3</td>
			<td >Fine Science Tools</td>
			<td >10003-12</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >SkyScan 1272</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >Skyscan CT-Vox</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SkyScan NRecon</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SolidWorks CAD</td>
			<td >Dassault Syst&#232;mes</td>
			<td >SolidWorks Research Subsription</td>
			<td>Solid modeling computer-aided design used by authors</td>
		</tr>
		<tr >
			<td >SuperGlue</td>
			<td >Loctite</td>
			<td >234790</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >Testing bath</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Thin film grips&#160;</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >VeroWhitePlus</td>
			<td >Stratasys LTD</td>
			<td >NA</td>
			<td>3D printing material used by authors</td>
		</tr>
		<tr >
			<td >WinTest&#160;</td>
			<td >WinTest Software</td>
			<td ></td>
			<td>Used by authors to collect data</td>
		</tr>
	</tbody>
</table>

biomechanical testing of murine tendons

Los trastornos del tendón son comunes, afectan a personas de todas las edades y a menudo son debilitantes. Los tratamientos estándar, como los antiinflamatorios, la rehabilitación y la reparación quirúrgica, a menudo fallan. Con el fin de definir la función del tendón y demostrar la eficacia de los nuevos tratamientos, las propiedades mecánicas de los tendones de los modelos animales deben determinarse con precisión. Los modelos animales murinos ahora se utilizan ampliamente para estudiar trastornos tendinosos y evaluar nuevos tratamientos para las tendinopatías; sin embargo, determinar las propiedades mecánicas de los tendones del ratón ha sido un reto. En este estudio, se desarrolló un nuevo sistema para pruebas mecánicas de tendones que incluye accesorios impresos en 3D que coinciden exactamente con las anatomías del húmero y el calcáneo para probar mecánicamente los tendones supraespinatus y los tendones de Aquiles, respectivamente. Estos accesorios fueron desarrollados utilizando reconstrucciones 3D de anatomía ósea nativa, modelado sólido y fabricación aditiva. El nuevo enfoque eliminó las fallas de agarre artifreales (por ejemplo, fallas en la falla de la placa de crecimiento en lugar de en el tendón), disminuyó el tiempo total de prueba y aumentó la reproducibilidad. Además, este nuevo método es fácilmente adaptable para probar otros tendones y tendones murinos de otros animales.

Los accesorios impresos en 3D se utilizaron para probar el supraespinato murino de 8 semanas de edad y los tendones de Aquiles. Todas las muestras analizadas mecánicamente fallaron en la entesis, como se caracteriza por escaneos microCT, inspección visual y análisis de video después de pruebas de tracción. En la Figura 3se muestra una comparación uno a uno de los métodos anteriores y actuales para las pruebas de tendón supraespinato en nuestro laboratorio. En el método anterior<sup ...

Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

Biomechanical Testing of Murine Tendons

El protocolo describe métodos de ensayo biomecánicos de tracción eficientes y reproducibles para tendones murinos mediante el uso de accesorios impresos en 3D de ajuste personalizado.

Pruebas biomecánicas de tendones murinos

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

El protocolo describe un método eficiente y reproducible para las pruebas biomecánicas de tendones de ratón a través de accesorios impresos 3D reutilizables personalizados. Los nuevos métodos reducen el tiempo necesario para probar la muestra de horas a minutos y eliminan un artefacto de agarre importante que era un problema común en los métodos anteriores. Este método no se limita a los supraespinatus y tendones de Aquiles.Se puede adaptar fácilmente a probar otros tendones de ratón y tendones de animales más grandes. Para garantizar la reproducibilidad y la eficiencia del enfoque, pueden ser necesarios varios ciclos de creación de prototipos de diseño y validación para accesorios que no se describen aquí. Para comenzar, realice una tomografía microcomputada de todo el hueso en el gel de agarosa.Después de la reconstrucción en imágenes de sección transversal, seleccione el comando Abrir archivo para abrir el conjunto de datos de archivos. Abra el archivo de diálogo Preferencias y seleccione la pestaña Avanzadas. Utilice el algoritmo de renderizado adaptable para construir los modelos 3D que proporcionan un detalle de superficie más suave.Utilice 10 como parámetro de localidad que define la distancia en píxeles al punto vecino utilizado para encontrar el borde del objeto. Minimice la tolerancia a 0.1 para reducir el tamaño del archivo. Para especificar el volumen de interés, seleccione manualmente dos imágenes para establecer como la parte superior e inferior del rango de VOI seleccionado y, a continuación, vaya a la segunda página, Región de interés.Seleccione manualmente la región de interés en una sola imagen de sección transversal. Repita la selección del ROI para cada 10 a 15 imágenes de sección transversal. A continuación, vaya a la tercera página, Selección binaria.En el menú Histograma, haga clic en Desde conjunto de datos para mostrar la distribución del histograma del brillo de todas las imágenes del conjunto de datos. También en el menú Histograma, haga clic en el menú Crear un archivo de modelo 3D. Guarde un modelo 3D del hueso en formato de archivo STL.En el software Meshmixer, importe la malla y seleccione Todo para editar. Reduzca el tricount y acepte los cambios.Vuelva a guardar el archivo recién reducido en formato STL eligiendo Exportar As.To diseñar un hueso humeral del tendón supraespinatus, utilice un programa de diseño asistido por computadora de modelado sólido para crear un modelo personalizado de accesorio de agarre del húmero. Abra el archivo de formato STL del hueso del húmero en un programa de modelado sólido y guárdelo como archivo de pieza en formato SLDPRT. A continuación, abra el archivo Pieza para crear manualmente tres planos anatómicamente relevantes, por ejemplo, sagital, coronal y transversal.Haga clic en Nuevo archivo para crear la pieza de componente de bloque sólido. Haga clic en Archivo nuevo para crear un modelo de ensamblaje con dos componentes, el bloque sólido y el hueso del húmero derecho o izquierdo. Defina la orientación del hueso dentro del bloque para asegurar que el ángulo entre el tendón y el hueso sea de 180 grados.Asegúrese de que todo el volumen óseo se ajuste dentro del bloque. En la ventana Ensamblaje, seleccione el bloque y haga clic en Editar componente en la barra de herramientas Ensamblaje. Haga clic en Insertar, Operaciones, Cavidad.Seleccione Escala uniforme e introduzca 0% como valor para escalar en todas las direcciones. Suprima la pieza ósea y guarde el ensamblaje como pieza. Abra el cilindro con la parte de la cavidad.Cree un croquis, asegúrese de dejar sólo 0,5 milímetros por encima de la cabeza del húmero. En Operaciones, seleccione Corte extruido. Corte el ensamblaje a lo largo del plano sagital para crear dos componentes simétricos que se ajusten al hueso antes y después.Corte la pieza del ensamblaje a lo largo del plano sagital para crear dos componentes simétricos que se ajusten al hueso antes y después. Es fundamental asegurar la cabeza humeral para evitar el fallo de la placa de crecimiento y definir un espacio libre ajustado que evite el desenganchamiento del húmero del modelo durante las pruebas. Proceda como se describe en el manuscrito para finalizar el componente posterior y el componente anterior.Guarde ambos componentes como archivos de pieza independientes. Ahora presione Insertar patrón, Espejo y seleccione Espejo para crear modelos de espejo 3D para cada componente de la luminaria para la extremidad opuesta. Seleccione la cara frontal como una cara de simetría.Seleccione la pieza como el sólido que desea reflejar. Seleccione el plano de simetría y cree un croquis que incluya todo el material. En Operaciones, seleccione Corte extruido para eliminar la pieza original y mantener solo la pieza simétrica.Haga clic en la parte inferior de la pieza y utilice como plano para crear un croquis. Haga clic en Texto de boceto y agregue L o R.Esto añade un grabado en la parte inferior de los accesorios para distinguir entre los lados izquierdo y derecho. Guarde todas las piezas de accesorios en formato de archivo estándar STL como preparación para la impresión 3D.Para diseccionar el supraespinatus músculo-tendón, muestra de hueso del húmero, primero coloque el ratón eutanizado en una posición propensa y haga una incisión en la piel desde arriba del codo de la parte delantera hacia el hombro. Utilice fórceps para eliminar cuidadosamente la piel con disección contundente de modo que la musculatura del hombro sea visible. Retire el tejido que rodea el húmero hasta que el hueso quede expuesto.Sostenga el húmero con fórceps y retire cuidadosamente los músculos deltoides y trapezius para exponer el arco coracoacromial. Identifique la articulación ac y separe cuidadosamente la clavícula del acromion con una cuchilla de bisturí. Teniendo cuidado de no dañar el tendón supraespinatus y su apego óseo, retire el músculo de su apego escapular usando una cuchilla de bisturí.Separe la cabeza del húmero del glenoide y lacera la cápsula articular y los tendones menores infraespinatus subscapularis y teres. Desarticular la articulación del codo para separar el húmero del cúbito y el radio. Aísle la muestra del músculo tendón humerus-supraspinatus y limpie el exceso de tejidos blandos en el húmero y la cabeza del húmero.Para diseccionar el tendón de Aquiles, el hueso calcáneo, coloque un ratón eutanizado en una posición propensa. Teniendo cuidado de no dañar el tendón de Aquiles y su apego óseo, retire la piel con disección contundente para que la musculatura alrededor de las articulaciones del tobillo y la rodilla quede expuesta. Usando una cuchilla de bisturí, comenzando en el apego tendón-calcáneo de Aquiles, desasocie cuidadosamente el músculo gastrocnemius de sus accesorios proximales.Desarticular cuidadosamente el calcáneo de los diversos huesos adyacentes y aislar la muestra de tendón-calcáneo de Aquiles y limpiar el exceso de tejidos blandos. Para determinar el área transversal del tendón, inserte el hueso boca abajo para suspender el espécimen en el criotubo lleno de gel de agarosa con el hueso en el gel de agarosa y el tendón y el músculo exterior. Después de la exploración con tomografía microcomputada, retire suavemente el músculo del tendón usando una cuchilla de bisturí.Inserte el hueso en el accesorio impreso 3D y adjúntelo a las rejillas de prueba. Inserte y pegue el tendón entre un papel de tejido delgado doblado y fíjelo con finos agarres de película. Inserte la muestra y las empuñaduras en un baño de prueba de PBS a 37 grados Celsius y realice una prueba mecánica.En este estudio, utilizando accesorios impresos en 3D reutilizables para agarrar el hueso, el tiempo de preparación de la muestra se redujo de horas a minutos. Las curvas representativas de deformación de carga para las pruebas de tracción del tendón supraespinato utilizando métodos actuales eliminaron artefactos de agarre principales, como la falla de la placa de crecimiento. Las propiedades mecánicas de los supraespinatus y tendones de Aquiles demuestran un efecto significativo del sexo basado en pruebas T no pares con un valor P inferior a 0,05.Micro CT midió con éxito el área transversal a lo largo de la longitud del tendón supraespinatus a lo largo de la longitud del tendón de Aquiles. Es importante recordar que cada sitio anatómico tiene criterios de diseño específicos necesarios para un agarre efectivo. Por lo tanto, el diseño de cada accesorio debe adaptarse en consecuencia.Además de las pruebas de tracción al fallo, estos accesorios se pueden utilizar para pruebas de carga cíclica, que proporcionan información sobre la fatiga del tendón y las propiedades viscoelásticas. Caracterizar las propiedades mecánicas de los tendones murinos es poco común en la literatura debido a una serie de razones, incluyendo la dificultad para agarrar estos pequeños tejidos, métodos de preparación de muestras tediosos y lentos, y fracturas de placas de crecimiento. Este protocolo presenta un método reproducible y eficiente en el tiempo para probar los tendones del ratón que ha eliminado los errores de agarre artefacto y ha triplicado el número de muestras que se pueden probar en un día.

Research

JoVE Journal

Bioengineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Dirigida celular de auto-ensamblaje para fabricar células derivadas de tejido para el análisis de anillos de Biomecánica e Ingeniería de Tejidos

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Investigación

Bioingeniería

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinación de la velocidad de transporte de xenobióticos y Nanomateriales través de la placenta mediante el<em&gt; Ex vivo</em&gt; Modelo perfusión placentaria humana

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Caracterización biomecánica de los tejidos blandos humanos utilizando sangría y Ensayo de tracción

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Un experimental y el Protocolo de elementos finitos para investigar el transporte de solutos neutros y cargadas a través de cartílago articular

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during an impact are important factors in protecting the helmet wearer from impact-induced injury. This manuscript aims to investigate impact-induced injury mechanisms in different helmet fit scenarios through analysis of simulated helmeted impacts with an anthropometric test device (ATD), an array of headform acceleration transducers and neck force/moment transducers, a dual high speed camera system, and helmet-fit force sensors developed in our research group based on Bragg gratings in optical fiber. To simulate impacts, an instrumented headform and flexible neck fall along a linear guide rail onto an anvil. The test bed allows simulation of head impact at speeds up to 8.3 m/s, onto impact surfaces that are both flat and angled. The headform is fit with a crash helmet and several fit scenarios can be simulated by making context specific adjustments to the helmet position index and/or helmet size. To quantify helmet retention, the movement of the helmet on the head is quantified using post-hoc image analysis. To quantify head and neck injury potential, biomechanical measures based on headform acceleration and neck force/moment are measured. These biomechanical measures, through comparison with established human tolerance curves, can estimate the risk of severe life threatening and/or mild diffuse brain injury and osteoligamentous neck injury. To our knowledge, the presented test-bed is the first developed specifically to assess biomechanical effects on head and neck injury relative to helmet fit and retention.

Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact.

Un banco de pruebas para examinar el casco ajuste y retención y medidas biomecánicas de cabeza y cuello, lesión en impacto simulado

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

Métodos para ensayos biomecánicos In Vivo en el plexo braquial en lechones neonatales

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

Tono Basal Biaxial y Pruebas Pasivas del Sistema Reproductivo Murino Usando un Miógrafo de Presión

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

La cuantificación de la inyección mediante pruebas mecánicas

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

Tomografía de coherencia óptica basada en el análisis de interacción fluido-estructura biomecánica de la progresión de la aterosclerosis coronaria

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Construcción de un modelo de órgano en un chip de células musculares lisas de la aorta humana para recapitular la tensión biomecánica en la pared aórtica

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...