Este trabajo fue apoyado en parte por el NIH subvenciones de investigación R01AR062207, R01AR061484, R56AI100901, K01AR053210, y una enfermedad objeto de Becas de Investigación de la Fundación de Investigación de Reumatología (a CJ Liu).

Todos los procedimientos quirúrgicos relacionados con los animales debe ser aprobado por el Cuidado de Animales y el Comité de Ética de la institución local con un esfuerzo realizado para minimizar el dolor y la incomodidad causada por la cirugía. 1. Preparación <ol><li> Seleccionar 8-12 semanas de edad macho C57BL / 6 ratones con un peso corporal de aproximadamente 25 g para la cirugía. </li><li> Se anestesia a los animales mediante la inyección intraperitoneal de un cóctel que contiene tanto xilazina (5 mg / kg) y ketamina (40 mg / kg). </li><li> Afeitado de la rodilla con las maquinillas de afeitar, a continuación, cuelgue quirúrgicamente el animal y esterilizar el sitio quirúrgico con betadine y alcohol (3x) y cubrir los ojos del ratón con el ungüento. </li></ol> 2. Proceso quirúrgico <ol><li> Hacer un 1 cm de la incisión longitudinal medial para-patelar para exponer articulación de la rodilla. </li><li> Abra la articulación de la rodilla suavemente a través de la luxación lateral de la rótula y el tendón rotuliano. <ol><li> Cortar la cápsula de la articulación de la rodilla longitudinalmentea través de la incisión medial para-patelar en el paso 2.1. </li><li> Diseccionar cápsula articular de la rodilla con una pinza. </li><li> Coge la parte distal de la pata trasera con la mano izquierda, y le realizará luxación lateral de la rótula y el tendón rotuliano con fórceps. Sostenga la pata trasera con cuidado y asegúrese de evitar el trauma en la pata. Cuanto mejor sea la cápsula de la articulación se disecciona, se requiere menos fuerza para hacer que la dislocación. </li></ol></li><li> Por goteo de solución salina estéril en la superficie del cartílago articular durante la operación para evitar la desecación de la superficie del cartílago. </li><li> Cortar el ligamento medial meniscotibial que ancla menisco medial de la meseta tibial. Evitar lesiones en el cartílago debajo del menisco medial. <ol><li> Identificar el menisco medial que se localiza entre el cóndilo medial del fémur y la meseta medial de la tibia. </li><li> Identificar el ligamento que conecta meniscotibial lado lateral del menisco medial con eminencia intercondílea de la tibia. </li><li> Sostenga la hiND pata suavemente con la mano, y cortar a través del ligamento meniscotibial medial cuidadosamente mediante el uso de una cuchilla quirúrgica N º 10. Asegúrese de no causar herir al cartílago articular y otros ligamentos. En muchos casos, la almohadilla de grasa que cubre necesita ser tirada hacia el lado, pero no ser retirado con el fin de identificar el ligamento. </li></ol></li><li> Cierre la cápsula de la articulación de la rodilla con una sutura absorbible 6-0. </li><li> Cierre la piel con sutura de seda 6-0. </li></ol> 3. Cuidados después de la operación <ol><li> Aplicar una gota de 0,25% de bupivacaína en el sitio quirúrgico de cada ratón para minimizar el dolor post-operatorio. </li><li> Deje los ratones operados gratis para conseguir agua y comida. </li></ol> 4. Scoring histológico de Surgical Modelo DMM <ol><li> Sacrificar los ratones en los puntos indicados de tiempo (por ejemplo, 4 semanas, 8 semanas y 12 semanas. Aquí mostramos los resultados de 8 semanas como representante) después de la cirugía. </li><li> Las articulaciones de la rodilla enteras son fijos, descalcificada, embedded por parafina y luego realizaron cortes seriados 5 intervalo de micras. <ol><li> Cortar todo los miembros posteriores con N º 10 cuchillas. </li><li> Diseccionar la piel y los músculos de las extremidades traseras cuidadosamente, y fijar las muestras con 4% PFA durante 3 días en RT. </li><li> Retire la PFA, y limpiar las muestras con agua. Después, las muestras descalcificar en EDTA a 4 ° C durante 2 semanas. </li><li> Deshidratar las muestras en un gradiente de etanol. En detalle, mantener las muestras en etanol al 70% durante 1 hora, a continuación, cambiar el etanol y mantener las muestras en un nuevo conjunto de etanol al 70% para el S / N. Después, poner las muestras en el 80% y etanol al 90%, en consecuencia, y mantenerlos durante 1 hora, respectivamente, seguido de etanol al 100% para el S / N. </li><li> Retire el etanol. Mantener las muestras en oxylene durante 1 hora, y el cambio a un nuevo conjunto de oxylene. Repita este paso 3x. </li><li> Incorporar las muestras en un molde de parafina utilizando el centro de la incrustación de Leica. Después, las muestras se seccionaron a 5 micras usando un micrótomo rotatorio (Leica RM2255, Alemania) y después se recogieron en portaobjetos de vidrio. </li><li> Secciones sagitales en serie se cortan para cada muestra que abarca una región desde el centro del cóndilo lateral al centro del cóndilo medial. </li></ol></li><li> Tinción de safranina O se realiza, seguido de puntuación y análisis estadístico a través del sistema de puntuación OARSI lo descrito previamente 25. <ol><li> En primer lugar, Desparafinar las diapositivas en oxylene, e hidratar en un gradiente de etanol a agua destilada. Posteriormente, los portaobjetos se tiñen mediante hematoxilina de Weigert solución, 0,05% verde rápido (FCF) de soluciones, solución al 1% de ácido acético y 0,1% Solución de safranina O. Montar las diapositivas utilizando medio resinoso. </li><li> Puntuación de los portaobjetos con tinción de safranina O basado en la pérdida de proteoglicanos (color rojo) en el cartílago y el porcentaje de destrucción de la estructura del cartílago, hacer el análisis estadístico para la puntuación histológica. </li></ol></li></ol>

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Estamos aquí declaramos que no tenemos ningún conflicto de intereses.

Se ha informado de que la cepa de ratones es muy importante para el modelo de DMM de inducción, como diferentes cepas de ratones han diversa gravedad de la OA después de DMM, con más alto nivel en la cepa 129/SvEv, seguido de C57BL6, 129/SvInJ y luego FVB / n 26. Una gran parte de los transgenes se establecen en ratones C57BL6, como PGRN-/ - ratones que usamos en el presente estudio, que son relativamente susceptibles a DMM. Sin embargo, si el transgén se basa en la cepa insensible tales como FVB / N rato...

La osteoartritis (OA), también conocida como artritis degenerativa, afecta al 15% de la población mundial y más de 46 millones de personas dentro de los Estados Unidos, y se caracteriza por una sinovitis, la degeneración del cartílago y formación de osteofitos 1. Puede ser el resultado de una interacción compleja de factores genéticos, metabólicos, biomecánicas y bioquímicas. Los mecanismos subyacentes de la OA continúan para evadir la comunidad científica. Actualmente existen numerosos modelos animales que pueden imitar la patogénesis de la OA 2,3. Es importante establecer modelos animales en ratones tanto por la disponibilidad de varios ratones modificados genéticamente y la rentabilidad de la experimentación. Entre los diferentes tipos de modelos experimentales de OA, la desestabilización inducida quirúrgicamente de menisco medial modelo (DMM) es un modelo bien aceptado OA-debido a su buena reproducibilidad y una progresión relativamente más lenta durante el desarrollo de la OA. Ambos atributos tienensido clave para la evaluación de la progresión de la OA en diferentes tratamientos o transgenes 3-8. Sin embargo, la consistencia de OA modelo quirúrgico se ve afectada por varios factores durante la cirugía y como resultado, la aplicación de modelo de ratón quirúrgica está limitado. Progranulina (PGRN) es un factor de crecimiento multifuncional expresado en varias células. Se sabe que PGRN desempeña un papel fundamental en diversos procesos fisiológicos y patológicos tales como la cicatrización de heridas 9, la tumorigénesis 10, y la inflamación 11-15. Los estudios también muestran que la insuficiencia de PGRN puede causar enfermedades degenerativas del sistema nervioso en los seres humanos y los ratones 16-18. Se sabe que PGRN se expresa en el cartílago articular humano, y su nivel es significativamente elevado en el cartílago de pacientes con OA y la artritis reumatoide 19. Además, PGRN también juega un papel crucial en la proliferación de condrocitos 20, diferenciación y endocondralosificación del cartílago de crecimiento durante el desarrollo 21,22. Recientemente, se informó de que PGRN antagoniza el TNF-α a través de la unión a los receptores de TNF y exhibió una función anti-inflamatoria en modelos de artritis inflamatorias 13,14,23,24. Sin embargo, el papel de PGRN en la OA, especialmente in vivo, sigue siendo un enigma. Aquí, se presenta el procedimiento para inducir un modelo DMM quirúrgica, e investigar el papel de PGRN en el desarrollo de OA a través del establecimiento del modelo DMM en WT y PGRN-/ - ratones.

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			<td height="21" style="height:21px;width:216px;">No. 10 Surgical blades</td>
			<td style="width:135px;">FEATHER</td>
			<td style="width:119px;">25-2976#10</td>
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			<td height="21" style="height:21px;">6-0 suture</td>
			<td>APPLIED DENTAL</td>
			<td>WG-N53133</td>
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establishment of a surgically-induced model in mice to investigate the protective role of progranulin in osteoarthritis

La desestabilización del menisco medial modelo (DMM) es una herramienta importante para el estudio de las funciones fisiopatológicas de numerosas moléculas asociadas con artritis en la patogénesis de la osteoartritis (OA) in vivo. Sin embargo, la detallada, especialmente el protocolo visualizado para establecer este modelo complicado en ratones, no está disponible. Aquí nos aprovechamos de tipo salvaje y progranulina (PGRN) - / - ratones como ejemplos de introducir un protocolo para inducir modelo DMM en ratones, y se comparó la aparición de OA después del establecimiento de este modelo inducida quirúrgicamente. Las operaciones realizadas en los ratones eran o simulacro de operación, que acaba de abrir la cápsula articular, o la operación DMM, que cortó el ligamento menisco tibial y causó la desestabilización del menisco medial. Severidad osteoartritis se evaluó usando el ensayo histológico (por ejemplo, tinción de safranina O), las expresiones de genes asociados a la OA, la forma de degradación del cartílago moléculas de la matriz extracelular, y osteofitosción. Operación DMM indujo con éxito la iniciación y la progresión de la OA en tanto de tipo salvaje y PGRN-/ - ratones, y la pérdida de factor de crecimiento PGNR dado lugar a un fenotipo más grave en la OA este modelo inducida quirúrgicamente.

Modelo DMM se estableció con éxito en ratones, y la deficiencia de PGRN exagerado desarrollo OA inducida quirúrgicamente. Sham y DMM operaciones (Figura 1) se realizaron en WT y PGRN-/ - ratones. 8 semanas después de la operación, los ratones fueron sacrificados, y la tinción de safranina O se realizó en las secciones de las articulaciones de rodilla, seguidos por análisis estadístico de la puntuación de artritis basada en la histología. Como se m...

Watch this Scientific Journal Video about Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis at JoVE.com

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Se describe un protocolo para la desestabilización del menisco medial modelo (DMM) en ratones, una herramienta eficaz para la osteoartritis (OA) de la investigación. Además, hemos demostrado que la deficiencia de desarrollo progranulincan OA exagera y la progresión mediante el uso de este modelo, lo que indica que progranulina juega un papel protector en la patogénesis de la OA.

Establecimiento de un modelo quirúrgicamente inducida en ratones para investigar el papel de protección de progranulina en la osteoartritis

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

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JoVE Journal

Bioengineering

27.1K vistas.  NYU Hospital for Joint Diseases.  Se describe un protocolo para la desestabilización del menisco medial modelo (DMM) en ratones, una herramienta eficaz para la osteoartritis (OA) de la investigación. Además, hemos demostrado que la deficiencia de desarrollo progranulincan OA exagera y la progresión mediante el uso de este modelo, lo que indica que progranulina juega un papel protector en la patogénesis de la OA. 

Video: Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparamétrico óptico Cartografía del corazón de conejo Langendorff perfundidos

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Investigación

Bioingeniería

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creación de dos dimensiones con dibujos sustratos para el confinamiento de proteínas y células

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Diseño de un biorreactor de presión cíclica de la Ex Vivo Estudio de las válvulas aórtica del corazón

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

La cuantificación de la función diastólica por Global Análisis basado en Modelado cinemático de flujo transmitral mediante el formalismo de llenado diastólico parametrizada

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Producción, Caracterización y posibles usos de un Humano esofágica mucosas Modelo ingeniería tisular 3D

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar Sistema de Difusión de degradado para Investigar quimiotaxis en una matriz de colágeno 3D

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In Vitro</em&gt; Órgano Cultura Modelo Murino del disco intervertebral

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

Creando un modelo geométrico realista estructural de elementos finitos de cardiomiocitos para estudiar el papel de la arquitectura celular de cardiomiocitos biología de sistemas

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: Es un enfoque potente y no invasivo para investigar el metabolismo energético celular

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

Una nanoemulsión tripéptido-estabilizado del ácido oleico

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...