Ce travail a été soutenu en partie par les NIH subventions de recherche R01AR062207, R01AR061484, R56AI100901, K01AR053210, et une maladie ciblée de subventions de recherche de la Fondation de recherche en rhumatologie (le juge en chef Liu).

Toutes les interventions chirurgicales concernant les animaux devraient être approuvés par le soin des animaux et du Comité d&#39;éthique de l&#39;institution locale, avec un effort pour minimiser la douleur et l&#39;inconfort causé par la chirurgie. Une. Préparation <ol><li> Sélectionner 8-12 semaines mâle souris C57BL / 6 avec un poids corporel d&#39;environ 25 g pour une intervention chirurgicale. </li><li> Anesthésier les animaux par injection intrapéritonéale d&#39;un cocktail contenant à la fois de la xylazine (5 mg / kg) et de la kétamine (40 mg / kg). </li><li> Raser le genou avec des rasoirs, puis draper chirurgicalement l&#39;animal et stériliser le site chirurgical avec de la bétadine et alcool (3x) et couvrir les yeux de souris avec une pommade. </li></ol> 2. Procédures chirurgicales <ol><li> Faire une médiane longitudinale para-patellaire incision de 1 cm pour exposer l&#39;articulation du genou. </li><li> Ouvrez l&#39;articulation du genou doucement à travers la dislocation latérale de la rotule et ligament rotulien. <ol><li> Couper la capsule articulaire du genou longitudinalementà travers la médiane incision para-patellaire à l&#39;étape 2.1. </li><li> Disséquer la capsule articulaire du genou avec une pince. </li><li> Prenez la partie distale de la patte arrière avec la main gauche, et effectuer dislocation latérale de la rotule et ligament rotulien avec une pince. Maintenez la patte arrière doucement et assurez-vous d&#39;éviter les traumatismes dans la patte. Le meilleur de la capsule articulaire est disséqué, moins la force sera nécessaire pour faire la dislocation. </li></ol></li><li> Goutte à goutte une solution saline stérile à la surface du cartilage articulaire lors du fonctionnement afin d&#39;éviter le dessèchement de la surface du cartilage. </li><li> Couper à travers le ligament médial meniscotibial qui ancre ménisque interne au plateau tibial. Éviter de blesser le cartilage sous le ménisque médial. <ol><li> Identifier le ménisque médial qui localise entre condyle médial du fémur et du tibia plateau intermédiaire. </li><li> Identifier le ligament qui relie meniscotibial côté latéral du ménisque médial avec éminence intercondylienne du tibia. </li><li> Tenez le salute patte doucement avec la main, et couper à travers le ligament médial meniscotibial soigneusement à l&#39;aide d&#39;une lame chirurgicale n ° 10. Assurez-vous de ne pas causer de blesser cartilage articulaire et d&#39;autres ligaments. Dans de nombreux cas, le tampon recouvrant la graisse doit être tiré vers le côté mais non d&#39;être retiré dans le but d&#39;identifier le ligament. </li></ol></li><li> Fermez la capsule articulaire du genou avec un fil résorbable 6-0. </li><li> Fermer la peau avec 6-0 suture de soie. </li></ol> 3. Soins post-opératoires <ol><li> Appliquer une goutte de 0,25% de bupivacaïne dans le site chirurgical de chaque souris afin de minimiser la douleur post-opératoire. </li><li> Laissez les souris exploités sans chercher de l&#39;eau et de la nourriture. </li></ol> 4. Marquer histologique de DMM chirurgical Modèle <ol><li> Sacrifier la souris aux points indiqués de temps (par exemple 4 semaines, 8 semaines, et 12 semaines. Ici, nous avons montré les résultats de huit semaines en tant que représentant) après la chirurgie. </li><li> Les articulations des genoux entières sont fixés, décalcifiés, embedded par la paraffine, puis coupe en série à intervalle de 5 um. <ol><li> Couper l&#39;ensemble des membres postérieurs avec n ° 10 lames. </li><li> Disséquer la peau et les muscles des membres postérieurs avec soin, et fixer les échantillons à 4% PFA pendant 3 jours à température ambiante. </li><li> Retirez la PFA, et nettoyer les échantillons avec de l&#39;eau. Ensuite, détartrer les échantillons dans de l&#39;EDTA à 4 ° C pendant 2 semaines. </li><li> Déshydrater les échantillons dans un gradient d&#39;éthanol. Dans le détail, conserver les échantillons dans de l&#39;éthanol à 70% pendant 1 heure, puis changer de l&#39;éthanol et de conserver les échantillons dans un nouvel ensemble de 70% d&#39;éthanol pour O / N. Ensuite, mettre les échantillons dans 80% et 90% d&#39;éthanol, par conséquent, et de les conserver pendant 1 heure, respectivement, suivie par de l&#39;éthanol à 100% pour O / N. </li><li> Éliminer l&#39;éthanol. Conserver les échantillons en o-xylène pendant 1 heure, et passer à une nouvelle série de o-xylène. Répétez cette étape 3x. </li><li> Incorporer les échantillons dans un moule de la paraffine en utilisant le centre enrobage Leica. Par la suite, les échantillons sont coupés à 5 um en utilisant un microtome rotatif (Leica RM2255, Allemagne) et ensuite recueillies sur des lames de verre. </li><li> Des sections sagittales en série sont coupées pour chaque échantillon couvrant une région du centre du condyle latéral vers le centre du condyle interne. </li></ol></li><li> Safranin O coloration est réalisée, suivie par la notation et l&#39;analyse statistique par OARSI système de notation comme décrit précédemment 25. <ol><li> Tout d&#39;abord, Déparaffiner les lames dans o-xylène, et hydrater les dans un gradient d&#39;éthanol à de l&#39;eau distillée. Par la suite, les lames sont colorées par hématoxyline de fer Solution de Weigert, 0,05% vert rapide (FCF) Solution, solution à 1% d&#39;acide acétique et 0,1% safranine O Solution. Monter les lames en utilisant un milieu résineux. </li><li> Note les lames colorées à la safranine O sur la base de la perte de protéoglycanes (de couleur rouge) dans le cartilage et le pourcentage de destruction de la structure du cartilage, faire l&#39;analyse statistique pour le score histologique. </li></ol></li></ol>

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Nous ici déclarer que nous n&#39;avons pas de conflit d&#39;intérêts.

Il est rapporté que la souche de souris est très important pour le modèle de DMM induction, en tant que différentes souches de souris ont différentes gravité de l&#39;arthrose après DMM, avec le plus haut niveau dans la souche 129/SvEv, suivie par C57BL6, 129/SvInJ puis FVB / n 26. Une grande partie des transgènes sont établis dans des souris C57BL6, comme PGRN-/ - souris, nous avons utilisé dans la présente étude, qui sont relativement sensibles à la DMM. Cependant, si le transgène est basé su...

L&#39;arthrose (OA), aussi connu comme l&#39;arthrite dégénérative, affecte 15% de la population mondiale et plus de 46 millions de personnes aux États-Unis, et est caractérisée par une synovite, la dégénérescence du cartilage, et la formation d&#39;ostéophytes 1. Il peut être le résultat d&#39;une interaction complexe de facteurs génétiques, métaboliques, biomécaniques et biochimiques. Les mécanismes sous-jacents de l&#39;arthrose continuent d&#39;échapper à la communauté scientifique. Il existe actuellement de nombreux modèles animaux qui peuvent imiter la pathogenèse de l&#39;arthrose 2,3. Il est important d&#39;établir des modèles animaux chez la souris à la fois en raison de la disponibilité des différentes souris génétiquement modifiées et la rentabilité de l&#39;expérimentation. Parmi les différents types de modèles d&#39;arthrose expérimentale, la déstabilisation induite chirurgicalement du ménisque interne (DMM) de modèle est un modèle OA bien acceptée en raison de sa bonne reproductibilité et une progression relativement lente au cours du développement de l&#39;arthrose. Ces deux attributs ontjoué un rôle clé pour l&#39;évaluation de la progression de l&#39;arthrose dans les différents traitements ou des transgènes 8.3. Toutefois, la cohérence du modèle d&#39;OA chirurgical est affecté par plusieurs facteurs au cours de l&#39;intervention chirurgicale et, par conséquent, l&#39;application du modèle de souris chirurgical est limité. Progranuline (PGRN) est un facteur de croissance multi-fonctionnel exprimé dans diverses cellules. Il est connu que PGRN joue un rôle essentiel dans différents processus physiologiques et pathologiques, tels que la cicatrisation des plaies 9, 10 tumorigenèse, et l&#39;inflammation de 11 à 15. Des études ont également montré que l&#39;insuffisance de PGRN peut provoquer des maladies dégénératives du système nerveux chez les humains et les souris 16-18. Il est connu que PGRN est exprimé dans le cartilage articulaire humain, et son niveau est significativement élevée dans le cartilage de patients atteints d&#39;arthrose et de la polyarthrite rhumatoïde 19. En outre, PGRN joue également un rôle crucial dans la prolifération des chondrocytes 20, la différenciation et endochondraleossification du cartilage de croissance au cours du développement 21,22. Récemment, nous avons signalé que PGRN contrarié TNF-α en se liant à des récepteurs TNF et présentait une fonction anti-inflammatoire dans des modèles inflammatoires d&#39;arthrite 13,14,23,24. Cependant, le rôle de PGRN dans l&#39;arthrose, en particulier in vivo, reste à être une énigme. Ici, nous présentons la procédure pour induire un modèle DMM chirurgicale, et d&#39;enquêter sur le rôle de PGRN en développement de l&#39;OA en établissant modèle de DMM en WT et PGRN-/ - souris.

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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>
			<td style="width:167px;"></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

Déstabilisation du ménisque interne (DMM) modèle est un outil important pour étudier les rôles physiopathologiques de nombreuses molécules associées à l&#39;arthrite dans la pathogenèse de l&#39;arthrose (OA) in vivo. Cependant, le détail, en particulier le protocole visualisée pour établir ce modèle compliqué chez la souris, ne sont pas disponibles. Ici, nous avons profité de type sauvage et progranuline (PGRN) - / - souris comme exemples d&#39;introduire un protocole pour induire modèle DMM chez la souris, et contre l&#39;apparition de l&#39;arthrose suite à la création de ce modèle induit chirurgicalement. Les opérations effectuées sur des souris ont un fonctionnement en trompe-l&#39;œil, qui vient d&#39;ouvrir la capsule articulaire, ou opération de DMM, qui a coupé le ligament menisco-tibiale et provoqué la déstabilisation du ménisque interne. Arthrose gravité a été évaluée à l&#39;aide dosage histologique (par exemple, la safranine O de coloration), des expressions de gènes OA-associés, la dégradation de cartilage molécules de la matrice extracellulaire, et ostéophytes formeation. DMM opération induite avec succès OA initiation et la progression à la fois de type sauvage et PGRN-/ - souris, et la perte de facteur de croissance PGNR conduit à un phénotype plus sévère OA dans ce modèle induit chirurgicalement.

Modèle DMM a été créé avec succès chez la souris, et la carence de PGRN exagéré développement de l&#39;OA chirurgicalement induite. Opérations fictives et DMM (figure 1) ont été effectuées dans le document WT et PGRN-/ - souris. 8 semaines après l&#39;opération, les souris ont été sacrifiées, et la safranine O coloration a été réalisée sur des sections de l&#39;articulation du genou, suivies d&#39;une analyse statistique de score sur la...

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

Nous décrivons un protocole pour la déstabilisation du ménisque médial (DMM) de modèle chez la souris, un outil efficace pour l&#39;arthrose (OA) de recherche. De plus, nous avons démontré que la carence de progranulincan exaggerate développement de l&#39;OA et de la progression à l&#39;aide de ce modèle, ce qui indique que progranulin joue un rôle protecteur dans la pathogenèse de l&#39;arthrose.

Mise en place d&#39;un modèle chirurgicalement induite chez la souris pour étudier le rôle de protection de progranuline dans l&#39;arthrose

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

establishment-surgically-induced-model-mice-to-investigate-protective

Research

JoVE Journal

Bioengineering

27.1K vues.  NYU Hospital for Joint Diseases.  Nous décrivons un protocole pour la déstabilisation du ménisque médial (DMM) de modèle chez la souris, un outil efficace pour l'arthrose (OA) de recherche. De plus, nous avons démontré que la carence de progranulincan exaggerate développement de l'OA et de la progression à l'aide de ce modèle, ce qui indique que progranulin joue un rôle protecteur dans la pathogenèse de l'arthrose. 

Vidéo: 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.

Multiparamétrique cartographie optique du Cœur lapin perfusé de Langendorff

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

Recherche

Bio-ingénierie

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.

Créer deux dimensions Substrats motifs pour le confinement des protéines et de cellules

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.

Conception d&#39;un bioréacteur pour la pression cyclique Ex Vivo Étude des valves cardiaques aortiques

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.

Quantification de Global diastolique fonction de l&#39;analyse basée sur la modélisation cinématique de transmitral débit via le formalisme de remplissage diastolique paramétrée

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.

Production, la caractérisation et utilisations potentielles d&#39;un humain de l&#39;oesophage muqueux modèle 3D de l&#39;ingénierie tissulaire

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 Gradient système de diffusion d&#39;enquêter Chimiotactisme dans une matrice de collagène 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; Orgue Culture Modèle de la murine disque intervertébral

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.

Création d’un modèle géométrique structurellement réaliste par éléments finis d’un Cardiomyocyte pour étudier le rôle de l’Architecture cellulaire en biologie des systèmes Cardiomyocyte

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 : Une approche puissante et non invasive pour étudier le métabolisme énergétique cellulaire

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.

Une Nanoémulsion Tripeptide-stabilisé de l’acide oléique

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 ...