Los autores reconocen el apoyo financiero del NIH / NIDCR R00DE018212 (SPH), NIH/NIDCR-R01DE022032 (SPH), NIH / NIDCR T32 DE07306 (AJ, JDL), NIH / CNRR S10RR026645, (SPH) y los Departamentos de Ciencias dentales preventivos y de restauración y Ciencias orofaciales, UCSF. Además, los autores reconocen Xradia Graduate Fellowship (AJ), Xradia Inc., Pleasanton, CA. Los autores agradecen a la Dra. Kathryn Grandfield, UCSF por su ayuda en el procesamiento posterior de los datos, los Dres. Stephen Weiner y Gili Naveh, Weizmann Institute of Science, Rehovot, Israel, el Dr. Ron Shahar, de la Universidad Hebrea de Jerusalén, Israel por sus profundas discusiones específicas para el dispositivo de carga en situ. Los autores también desean agradecer Biomateriales e Instalaciones Bioingeniería MicroCT Imaging en la UCSF para el uso de Micro XCT y el dispositivo de carga en situ.

Alojamiento de animales y la eutanasia: Todos los animales utilizados en esta demostración fueron alojados en condiciones libres de patógenos, de acuerdo a los lineamientos del Cuidado de Animales y el empleo Comisión Institucional (IACUC) y el Instituto Nacional de Salud (NIH). Proporcionar los animales con comida para ratas hard-pellet estándar y agua ad lib. La eutanasia a los animales a través de un método de dos pasos de asfixia con dióxido de carbono, la toracotomía bilateral de acuerdo con el protocolo estándar de la UCSF, aprobado por el IACUC. Realizar pruebas biomecánicas dentro de las 24 horas de sacrificio de animales para evitar la degradación del tejido. 1. Preparación y Disección de una rata o Mandíbula Maxilar <ol><li> Retire las mandíbulas de rata mediante el corte de tejido y suavemente el tejido muscular adjuntos membranosas preservando al mismo tiempo toda la mandíbula, incluyendo el proceso coronoides y el proceso condilar (Figura 1) 15. </li><li> Hemimandíbulas separados en cocheefully cortar el tejido fibroso de la sínfisis mandibular con una hoja de bisturí. Nota: Los procesos coronarios y del cóndilo y rama de la mandíbula (Figura 1) deben ser removidos si obstaculizan físicamente pruebas biomecánicas del 2 º molar. </li><li> Cortar los incisivos sin exponer la cámara de la pulpa de no obstaculizar la carga del molar. </li></ol> 2. Preparación de las muestras in situ para la compresión de carga (Figura 2) <ol><li> Inmovilizar la muestra en un trozo de acero mediante el uso de un material que es significativamente más rígido que el espécimen experimentales antes de cargarlo en un dispositivo de carga en situ (Figura 2A). Nota: polimetilmetacrilato (PMMA) se utiliza para inmovilizar la muestra en este estudio y el exceso, si lo hay, se eliminó usando un explorador dental. </li><li> Alinear la superficie oclusal del molar (s) de interés en paralelo con el disco de muestra de AFM de metal usando un borde recto en tantoaviones (es decir, mesial-distal y bucal-lingual). </li><li> Crear un canal con un objeto contundente que rodea a los molares. Nota: Este espacio debe servir como un &quot;foso&quot; para contener el exceso de líquido y mantener la hidratación de los tejidos durante la carga en el lugar. </li><li> Prepare la superficie del diente a construir para concéntricos (Figura 2B) o excéntrica (Figura 2C) la carga con un compuesto dental. Atacar la superficie del diente de interés con gel de ácido fosfórico al 35% en la superficie oclusal de 15 seg. </li><li> Enjuague el reactivo de ataque a fondo con agua desionizada y secar la superficie utilizando una jeringa de aire / agua o un recipiente de aire comprimido. Con un explorador, se extendió una gota del agente de unión en las cúspides abiertas en una capa delgada. Curar el compuesto con una luz de curado dental. Nota: Todas las etapas que implican compuestos deberán realizarse sin la luz directa de una lámpara. Tales condiciones no deseable de acelerar el proceso de polimerización, y could impedir la colocación apropiada del compuesto. La iluminación de la habitación es aceptable. </li><li> Eliminar el exceso de agente de unión de los dientes adyacentes con un fino bisturí o cuchilla de afeitar. </li><li> Coloque compuesto dental capaz de fluir sobre la superficie después de la preparación de la superficie y se extendió en surcos de los molares (s) de interés utilizando un explorador dental. </li><li> Exponga el material compuesto a la luz de curado dental durante 30 segundos. </li><li> Moldear una acumulación oclusal de unos 3-4 mm usando un compuesto de resina dental, desde el plano oclusal del molar (s) de interés y la cura la luz durante 30 segundos. </li><li> Reducir la parte superior de la acumulación de material compuesto a una superficie paralela plana para permitir un esquema de carga consistente a través de todas las muestras usando un borde recto y una pieza de mano de alta velocidad. Nota: Durante la prueba biomecánica, otras muestras deben almacenarse en Tris-fosfato de solución tamponada (TBS) con 50 mg / ml de penicilina, y estreptomicina 15. </li></ol> 3. Cargando Drift dispositivos yRigidez, Material de Propiedad Diferenciar Capability, in situ La carga del fibroso Conjunta <ol><li> Asegure la muestra con la acumulación de material compuesto en el yunque de la etapa de carga, y la prueba para la carga uniforme como se muestra en la Figura 2B. </li><li> Coloque un papel de articular en la superficie del material compuesto seguido por la carga de la muestra a una carga finita para verificar la carga concéntrica o excéntrica (Figuras 2B y 2C). </li><li> Coloque Kimwipe empapado-TBS alrededor de las muestras para asegurar la hidratación de la muestra. Hacer una depresión alrededor de la muestra y llenarlo con TBS para mantener el órgano hidratado durante la exploración. </li><li> Pico de carga de entrada y la velocidad de desplazamiento en el software Deben comprimir el molar a un pico de carga deseada a una velocidad de desplazamiento después de la inmovilización del hemimandíbula. Nota: las lecturas típicas deben incluir una carga reaccionaria como el material se comprime en el tiempo (sensibilidad del transductor de carga = 0,1N). Desde los tiempos de carga y el tiempo de desplazamiento, una curva de carga-desplazamiento para el material comprimido se debe obtener 16-18. Utilizando los datos recogidos de los ciclos de carga, diversas propiedades de la articulación también pueden ser determinados. La rigidez de la articulación se debe calcular tomando la pendiente de la porción lineal (aproximadamente el último 30% de los datos) de la fase de carga de la carga frente a la curva de desplazamiento 19. </li></ol> 4. La tinción de los tejidos blandos, el PDL, con ácido fosfotúngstico (PTA) Nota: Para mejorar el contraste de atenuación de los rayos X, la PDL debe ser teñido con solución de PTA 5% 20. <ol><li> Solución de tinción de relleno de PTA en una ampolla portadora de vidrio 1,8 ml limpio y colocar ampolla portadora cargado en la jeringa. </li><li> Inyectar la solución lentamente (5 min / ampolla portadora) en el PDL-espacio de los dientes adyacentes para evitar daños estructurales a los tejidos periodontales que rodean molar de interés. Nota: Los pasos anteriores deben abe repite hasta aproximadamente 5 carpules completos (9 ml) de solución se inyecta y se deja fluir dentro de los tejidos circundantes. Los especímenes prepped también pueden ser remojados durante la noche en la solución de PTA restantes (8 h). </li></ol> 5. Configuración de exploración μ-XCT recomendados Realice m-XCT con los siguientes ajustes de escaneo: <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> Aumento Objetivo </td><td> 4X, 10X </td></tr><tr><td></td><td> 1800 imágenes </td></tr><tr><td> Tensión del tubo de rayos X </td><td> 75 kVp (50 kVp para muestras de PTA manchado) </td></tr><tr><td></td><td> 8 W </td></tr><tr><td> Tiempo de Exposición </td><td> ~ 8-25 seg * </td></tr><tr><td></td><td> ~ 4 m (objetivo 4X), ~ 2 micras (objetivo 10X) ** </td></tr></tbody></table> * Tiempo de exposición puede variar en función de la geometría y la densidad óptica de la muestra y VO tubo de rayos Xltaje. ** Píxeles de resolución real será exactamente el mismo en base a la configuración de la fuente, la muestra, y el detector.

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Los autores no tienen nada que revelar.

El primer paso en el establecimiento de este protocolo implicó la evaluación de la rigidez del bastidor de carga mediante el uso de un cuerpo rígido. Basándose en los resultados, la rigidez fue significativamente mayor que permite el uso del dispositivo de carga para la prueba adicional de muestras con valores de rigidez significativamente más bajos. El segundo paso de relieve la capacidad del instrumento para distinguir diferentes valores de rigidez mediante el uso de dos fases de la curva de carga-descarga genera...

Varios métodos experimentales siguen siendo utilizados para investigar la biomecánica de las articulaciones diartrodiales y fibrosos. Métodos específicos para la biomecánica de órganos de dientes incluyen el uso de medidores de deformación 1-3, métodos fotoelasticidad 4, 5, interferometría moiré 6, 7, patrón de moteado electrónico interferometría 8, y la correlación de la imagen digital (DIC) 9-14. En este estudio, el enfoque innovador incluye imágenes no invasivo que usa rayos X para exponer las estructuras internas de la articulación fibrosa (tejidos mineralizados y sus interfaces consistentes en zonas más suaves, y la interconexión tejidos como ligamentos) con cargas equivalentes a las condiciones in vivo. Se realizará una dispositivo de carga in situ en acoplada a un microscopio de micro-radiografía. El tiempo de carga y las curvas de carga-desplazamiento se recogerán como el molar de interés dentro de una rata hemimandíbula recién cosechado se carga. La mmeta ain del enfoque presentado en este estudio es hacer hincapié en el efecto de la morfología tridimensional de dientes de hueso mediante la comparación de las condiciones en: 1) sin carga y una vez cargado, y cuando 2) cargado concéntrica y excéntrica. La eliminación de la necesidad de que los especímenes de corte, y para llevar a cabo experimentos en los órganos intactos enteras en condiciones de humedad permitirá la máxima preservación del estado de estrés en 3D. Esto abre una nueva área de investigación en la comprensión de los procesos dinámicos del complejo bajo varios escenarios de carga. En este estudio, los métodos para las pruebas de biomecánica PDL dentro de una articulación fibrosa intacta de una rata Sprague Dawley, se detallarán una empresa considerada como un sistema óptimo modelo de la bioingeniería. Los experimentos incluyen la simulación de cargas de masticación en condiciones hidratadas con el fin de destacar tres características importantes de la articulación que se relacionan con la biomecánica a nivel de órganos. Los tres puntos serán los siguientes: 1) fuerza reaccionaria contra el desplazamiento:desplazamiento del diente en el alveolo y su respuesta reaccionaria a la carga, 2) en tres dimensiones (3D) la configuración espacial y morfometría: relación geométrica del diente con el alveolo, y 3) los cambios en las lecturas 1 y 2 debido a un cambio en la eje de carga, es decir, desde concéntrica a cargas excéntricas. Los tres lecturas fundamentales de la técnica propuesta se pueden aplicar para investigar la naturaleza adaptativa de las articulaciones en los vertebrados, ya sea debido a los cambios en las demandas funcionales, y / o enfermedad. Los cambios en las lecturas antes mencionadas, específicamente la correlación entre las cargas reaccionarios con el desplazamiento, y que resulta en tiempo de carga y de carga-desplazamiento curvas reaccionarios a diferentes tasas de carga se pueden aplicar para resaltar los cambios globales en la biomecánica articular. La eficacia del protocolo propuesto será evaluado mediante el acoplamiento de las lecturas de ensayos mecánicos para la morfometría en 3D y la biomecánica globales de la empresa.

<table border="0" cellpadding="0" cellspacing="0" width="652">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Bard Parker Blade</td>
			<td style="width:101px;">BD</td>
			<td style="width:123px;">MEDC-001054</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">AFM metal disk</td>
			<td style="width:101px;">Ted Pella</td>
			<td style="width:123px;">16218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Polymethyl methacrylate&#160;</td>
			<td style="width:101px;">GC America</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Uni-Etch</td>
			<td style="width:101px;">Bisco</td>
			<td style="width:123px;">E5502EBM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Optibond Solo Plus</td>
			<td style="width:101px;">Kerr Corp</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Filtek Flow</td>
			<td style="width:101px;">3M</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Hurculite Ultra</td>
			<td style="width:101px;">Kerr</td>
			<td style="width:123px;">34346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Tris buffer</td>
			<td style="width:101px;">Mediatech Inc.</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Articulating paper</td>
			<td style="width:101px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Phosphotungstic Acid</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">HT152</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ compressive loading and correlative noninvasive imaging of the bone-periodontal ligament-tooth fibrous joint

Este estudio demuestra un protocolo de pruebas novedosas biomecánica. La ventaja de este protocolo incluye el uso de un dispositivo de carga en situ acoplada a un microscopio de rayos X de alta resolución, lo que permite la visualización de elementos estructurales internos bajo cargas fisiológicas simuladas y condiciones de humedad. Muestras experimentales incluirán ligamento-hueso periodontal intacta (PDL) de diente de articulaciones fibrosas. Los resultados se ilustran tres características importantes del protocolo, ya que se pueden aplicar a la biomecánica a nivel de órganos: 1) la fuerza reaccionaria contra el desplazamiento: desplazamiento del diente en el alveolo y su respuesta reaccionaria a la carga, 2) en tres dimensiones (3D) configuración espacial y morfometría geométrica: la relación del diente con el alveolo, y 3) cambios en las lecturas 1 y 2 debido a un cambio en el eje de carga, es decir, desde concéntrica a cargas excéntricas. La eficacia del protocolo propuesto será evaluado mediante el acoplamiento mecánico del telecturas picadura a la morfometría en 3D y la biomecánica globales de la empresa. Además, esta técnica se hará hincapié en la necesidad de equilibrar las condiciones experimentales, cargas específicamente reaccionarios antes de la adquisición de tomografías de articulaciones fibrosas. Debe tenerse en cuenta que el protocolo propuesto se limita a analizar muestras bajo condiciones ex vivo, y que el uso de agentes de contraste para visualizar la respuesta mecánica de los tejidos blandos puede llevar a conclusiones erróneas acerca del nivel de órganos tejidos y biomecánica.

Estimación de la carga de dispositivo &quot;reacción violenta&quot;, &quot;retroceso&quot;, la rigidez y la deriva del sistema bajo una carga constante Backlash: Entre raciones de carga y descarga del ciclo, no existe una pausa de 3 segundos durante la cual marchas atrás en el motor antes de la verdadera descarga comienza, esto es, como la muestra se separa de la mandíbula superior (Figura 3). Este periodo se conoce como una rea...

Watch this Scientific Journal Video about In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint at JoVE.com

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

En este estudio, se discutirá el uso de un dispositivo de carga en situ junto con la tomografía de rayos micro-X calculado para la biomecánica conjuntos fibrosos. Lecturas experimentales identificables con un cambio general en la biomecánica articular serán los siguientes: 1) fuerza reaccionaria contra el desplazamiento, es decir, el desplazamiento del diente en el alveolo y su respuesta reaccionaria a la carga, 2) en tres dimensiones (3D) la configuración espacial y la morfometría, es decir geométrica relación de los dientes con el alvéolo, y 3) cambios en las lecturas 1 y 2 debido a un cambio en el eje de carga, es decir, las cargas concéntricas o excéntricas.

In situ carga de compresión y de imágenes no invasiva correlativa del hueso-ligamento periodontal de dientes fibroso Conjunta

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

in-situ-compressive-loading-correlative-noninvasive-imaging-bone

Research

JoVE Journal

Bioengineering

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

13.2K vistas.  University of California San Francisco.  En este estudio, se discutirá el uso de un dispositivo de carga en situ junto con la tomografía de rayos micro-X calculado para la biomecánica conjuntos fibrosos. Lecturas experimentales identificables con un cambio general en la biomecánica articular serán los siguientes: 1) fuerza reaccionaria contra el desplazamiento, es decir, el desplazamiento del diente en el alveolo y su respuesta reaccionaria a la carga, 2) en tres dimensiones (3D) la confi...

Video: In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

De alta resolución de fibra óptica para Microendoscopy In situ Celular imágenes

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Investigación

Bioingeniería

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

Demostrando la Utilización del Espectrómetro de Novela de la Fuerza gravitacional para estirar y medir las proteínas fibrosas

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

La síntesis in vitro de Espaciado nativo, y colágeno fibroso Long Long segmentaria Espaciado

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Diseño de un Carga mecánica Biaxial biorreactor para Ingeniería de Tejidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS habilitadas no invasiva perfiles metabólicos

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Factor de Crecimiento de electrospinning Liberar microesferas en Fibrosa andamios

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

El estudio de la estequiometría de factor de crecimiento epidérmico en células intactas utilizando microscopía correlativa

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

Configuración optimizada y protocolo para la proyección de imagen de dominio magnético con In Situ la medición histéresis

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensores para detectar actividad proteasa en Vivo para el diagnóstico no invasivo

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Correlativos luz microscopia electrónica (CLEM) para el seguimiento y proyección de imagen proteína Viral asociada a estructuras en las células inmovilizadas de Cryo

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...