Les auteurs reconnaissent l&#39;aide financière NIH / NIDCR R00DE018212 (SPH), NIH/NIDCR-R01DE022032 (SPH), NIH / NIDCR T32 DE07306 (AJ, JDL), NIH / NCRR S10RR026645, (SPH) et les départements des sciences dentaires de prévention et de restauration et Sciences oro-faciales, UCSF. En outre, les auteurs reconnaissent Xradia bourse d&#39;études supérieures (AJ), Xradia Inc., Pleasanton, CA. Les auteurs remercient le Dr Kathryn Grandfield, UCSF pour son aide à la post-traitement de données; Drs. Stephen Weiner et Gili Naveh, Weizmann Institute of Science, Rehovot, Israël, le Dr Ron Shahar, l&#39;Université hébraïque de Jérusalem, Israël pour leurs discussions pertinentes spécifiques au dispositif de chargement in situ. Les auteurs tiennent aussi à remercier les biomatériaux et bioingénierie installation microCT Imaging à l&#39;UCSF pour l&#39;utilisation de micro-XCT et le dispositif de chargement in situ.

Logement des animaux et de l&#39;euthanasie: Tous les animaux utilisés à cette manifestation ont été logés dans des conditions exemptes d&#39;agents pathogènes, conformément aux lignes directrices de l&#39;entretien et l&#39;utilisation des animaux Commission institutionnelle (IACUC) et l&#39;Institut national de la santé (NIH). Offrir aux animaux pour rats dur culot standard et de l&#39;eau ad lib. Euthanasier les animaux par une méthode en deux étapes de dioxyde de carbone asphyxie, thoracotomie bilatérale en conformité avec le protocole standard de l&#39;UCSF approuvé par IACUC. Effectuer des tests biomécaniques dans les 24 heures de sacrifice d&#39;animaux pour éviter la dégradation des tissus. Une. Préparation et Dissection d&#39;une mandibule ou maxillaire Rat <ol><li> Retirer mandibules de rat en coupant délicatement les pièces jointes de tissus et de tissus musculaire membraneuses tout en préservant l&#39;ensemble mandibule, y compris le processus coronoïde et le condyle (figure 1) 15. </li><li> Hemimandibles séparés en voitureefully couper le tissu fibreux de la symphyse mandibulaire avec une lame de scalpel. Remarque: Les processus coronaires et condyliennes et branche de la mandibule (figure 1) devraient être supprimés s&#39;ils gênent physiquement tests biomécaniques de la 2 ème molaire. </li><li> Couper les incisives sans exposer la chambre pulpaire à ne pas gêner le chargement de la molaire. </li></ol> 2. Préparation des échantillons in situ pour la compression chargement (Figure 2) <ol><li> Immobiliser l&#39;échantillon sur un talon de l&#39;acier en utilisant un matériau qui est sensiblement plus rigide que les échantillons expérimentaux avant de le charger dans un dispositif de chargement in situ (Figure 2A) dans. Remarque: polyméthacrylate de méthyle (PMMA) a été utilisé pour immobiliser l&#39;échantillon de cette étude et l&#39;excès, le cas échéant, a été éliminé en utilisant un explorateur dentaire. </li><li> Aligner la surface occlusale de la molaire (s) d&#39;intérêt parallèlement à la platine métallique AFM aide d&#39;une règle à la foisplans (c.-à-mésiodistale et bucco-lingual). </li><li> Créer un creux avec un objet contondant entourant les molaires. Note: Cet espace doit servir de &quot;fossé&quot; pour contenir le liquide en excès et maintenir l&#39;hydratation des tissus pendant le chargement in situ. </li><li> Préparer la surface de la dent à s&#39;accumuler pour concentrique (figure 2B) ou excentrique (figure 2C) en utilisant un chargement composite dentaire. Graver la surface de la dent d&#39;intérêt avec un gel d&#39;acide phosphorique à 35% sur la surface de mastication pendant 15 s. </li><li> Rincer l&#39;agent de gravure à l&#39;eau désionisée et sécher la surface à l&#39;aide d&#39;une seringue air / eau ou une cartouche à air comprimé. Avec un explorateur, répandre une goutte de l&#39;agent de liaison en pointes ouvertes dans une couche mince. Traiter le composite avec une lampe à polymériser dentaire. Remarque: Toutes les étapes impliquant composites devraient être exécutées sans lumière directe d&#39;une lampe. Ces conditions non souhaitable d&#39;accélérer le processus de polymérisation, et could empêcher le positionnement correct du composite. éclairage de la pièce est acceptable. </li><li> Éliminer l&#39;agent de liaison en excès de dents adjacentes avec un scalpel amende ou une lame de rasoir. </li><li> Placez composite dentaire fluide sur la surface suite à la préparation de la surface et l&#39;étaler dans les rainures de la molaire (s) d&#39;intérêt en utilisant un explorateur dentaire. </li><li> Exposer le composite à la lumière de durcissement dentaire pendant 30 secondes. </li><li> Mouler une accumulation d&#39;occlusion de l&#39;ordre de 4.3 mm en utilisant une résine composite dentaire, à partir du plan d&#39;occlusion de la molaire (s) d&#39;intérêt et photopolymériser pendant 30 sec. </li><li> Réduire le haut de l&#39;accumulation composite sur une surface plane parallèle pour permettre un système de chargement uniforme dans tous les échantillons à l&#39;aide d&#39;une règle et une pièce à main haute vitesse. Remarque: Lors de tests biomécaniques, d&#39;autres échantillons doivent être stockés dans tris-solution tamponnée au phosphate (SCT) avec 50 mg / ml de pénicilline, la streptomycine et 15. </li></ol> 3. Chargement dérive de l&#39;appareil etRigidité, Matériel propriété de différenciation des capacités, in situ Chargement de la commune Fort <ol><li> Fixer le spécimen avec l&#39;accumulation de synthèse sur l&#39;enclume de l&#39;étage de chargement, et le test de charge uniforme comme le montre la figure 2B. </li><li> Placez un papier à articuler sur la surface du composite suivi d&#39;un chargement de l&#39;échantillon à une charge finie pour vérifier le chargement concentrique ou excentrique (figures 2B et 2C). </li><li> Placez Kimwipe SCT trempé dans l&#39;échantillon pour assurer spécimen hydratation. Faire un creux autour de l&#39;échantillon et de le remplir avec le SCT pour maintenir l&#39;organe hydraté pendant l&#39;imagerie. </li><li> une pointe de charge d&#39;entrée et la vitesse de déplacement dans le logiciel Deben pour comprimer la molaire à une charge de crête souhaité à une vitesse de déplacement suivant l&#39;immobilisation du hemimandible. Remarque: lectures typiques devraient inclure une charge réactionnaire que le matériau est comprimé au cours du temps (sensibilité du capteur de charge = 0,1N). De charge-temps et le déplacement en temps, une courbe charge-déplacement pour le matériau comprimé doit être obtenu 16-18. En utilisant les données recueillies à partir des cycles de chargement, diverses propriétés du joint peuvent également être déterminés. La raideur de l&#39;articulation doit être calculée en prenant la pente de la partie linéaire (environ le dernier 30% de données) de la phase de chargement de la charge en fonction de la courbe de déplacement 19. </li></ol> 4. La coloration des tissus mous, le PDL, avec de l&#39;acide phosphotungstique (PTA) Remarque: Pour améliorer X-ray atténuation revanche, le PDL doit être coloré avec une solution de PTA de 5% 20. <ol><li> Solution de coloration remblai PTA dans un récipient propre de 1,8 ml de cartouche en verre et placer de cartouche chargée dans la seringue. </li><li> Injecter la solution lentement (5 min / de cartouche) dans l&#39;espace PDL des dents adjacentes pour empêcher des dommages structurels aux tissus parodontaux molaire d&#39;intérêt environnants. Remarque: Les étapes ci-dessus devraient be répété jusqu&#39;à environ 5 carpules plein (9 ml) de solution sont injectés et autorisé à circuler dans les tissus environnants. Les spécimens prepped peuvent également être trempées pendant une nuit dans la solution d&#39;ATP restant (8 h). </li></ol> 5. Paramètres de numérisation μ-XCT recommandés Effectuer m-XCT avec les paramètres de numérisation suivants: <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> Grossissement de l&#39;objectif </td><td> 4X, 10X </td></tr><tr><td></td><td> 1800 images </td></tr><tr><td> X-ray tension du tube </td><td> 75 kV (50 kV pour les échantillons de la ZEP souillé) </td></tr><tr><td></td><td> 8 W </td></tr><tr><td> Temps d&#39;exposition </td><td> ~ 8-25 sec * </td></tr><tr><td></td><td> ~ 4 pm (objectif 4X), ~ 2 pm (objectif 10X) ** </td></tr></tbody></table> * Le temps d&#39;exposition peut varier en fonction de la géométrie et de la densité optique de l&#39;échantillon et de tube à rayons X voltage. ** Pixels de résolution réelle sera légèrement différer en fonction de la configuration de la source, l&#39;échantillon, et le détecteur.

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Les auteurs n&#39;ont rien à révéler.

La première étape dans l&#39;établissement de ce protocole a consisté à évaluer la rigidité du cadre de chargement à l&#39;aide d&#39;un corps rigide. D&#39;après les résultats, la rigidité était significativement plus élevée permettant l&#39;utilisation du dispositif de chargement pour un test supplémentaire d&#39;échantillons avec des valeurs de rigidité nettement plus faibles. La deuxième étape a mis en évidence la capacité de l&#39;instrument de distinguer différentes valeurs de rigidité à l&...

Plusieurs méthodes expérimentales continuent d&#39;être utilisés pour étudier la biomécanique de diarthroses et fibreux. Les méthodes spécifiques pour la biomécanique d&#39;organes de dents comprennent l&#39;utilisation de jauges de contrainte de 1 à 3, les méthodes de photoélasticité 4, 5, l&#39;interférométrie de moiré 6, 7, électronique mouchetis interférométrie 8, et de la corrélation d&#39;images numériques (DIC) 9-14. Dans cette étude, l&#39;approche novatrice comprend imagerie non invasive utilisant des rayons X d&#39;exposer les structures internes d&#39;un joint fibreux (tissus minéralisés et de leurs interfaces, comprenant des zones plus doux, et l&#39;interfaçage des tissus tels que les ligaments) à des charges équivalentes à des conditions in vivo. Un dispositif de chargement in situ dans un microscope couplé à micro-rayons X est utilisé. Le temps de charge et courbes charge-déplacement seront recueillies que la molaire d&#39;intérêt au sein d&#39;un hémi-mandibule rat fraîchement récolté est chargé. Le main objectif de l&#39;approche présentée dans cette étude est de mettre l&#39;accent sur l&#39;effet de la morphologie tridimensionnelle de la dent à l&#39;os en comparant les conditions à: 1) pas de charge et lorsqu&#39;il est chargé, et quand 2) concentrique et de façon excentrée. Éliminant ainsi la nécessité pour les spécimens coupés, et de réaliser des expériences sur les organes entières intactes dans des conditions humides permettra de préserver au maximum de l&#39;état de contrainte 3D. Cela ouvre un nouveau champ d&#39;investigation dans la compréhension des processus dynamiques du complexe sous différents scénarios de chargement. Dans cette étude, les méthodes de test biomécanique de PDL au sein d&#39;une joint fibreux intact d&#39;un rat Sprague Dawley, une collaboration considérée comme un système modèle de la bio-ingénierie optimale seront détaillées. Expériences comprennent la simulation de charges de mastication dans des conditions hydratées afin de mettre en évidence trois caractéristiques importantes de la commune en ce qui concerne à la biomécanique au niveau des organes. Les trois points seront les suivants: 1) les forces réactionnaires en fonction du déplacement:dent déplacement dans l&#39;alvéole et sa réponse réactionnaire à charge, 2) en trois dimensions (3D) configuration spatiale et morphométriques: relation géométrique de la dent avec l&#39;alvéole, et 3) les changements dans les affichages 1 et 2 en raison d&#39;un changement de axe de chargement, c&#39;est à dire à partir de concentrique aux charges excentrées. Les trois lectures fondamentaux de la technique proposée peut être appliquée pour étudier la nature adaptative des articulations chez les vertébrés, soit en raison de changements dans les demandes fonctionnelles et / ou de la maladie. Changements dans les affichages mentionnés ci-dessus, en particulier la corrélation entre les charges réactionnaires de déplacement, et résultant courbes réactionnaires charge à temps et charge-déplacement à différents taux de charge peuvent être appliquées pour mettre en évidence les changements globaux dans la biomécanique articulaire. L&#39;efficacité du protocole proposé sera évaluée en couplant lectures d&#39;essais mécaniques de morphométrie 3D et de la biomécanique ensemble de la commune.

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	<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

Cette étude démontre un protocole de test de nouveaux biomécanique. L&#39;avantage de ce protocole comprend l&#39;utilisation d&#39;un dispositif de chargement in situ couplée à un microscope à haute résolution des rayons X dans, permettant ainsi la visualisation des éléments structurels internes au titre des charges physiologiques simulées et des conditions humides. Échantillons expérimentaux comprendront intact ligament à l&#39;os parodontal (PDL)-dent articulations fibreuses. Résultats illustrent trois aspects importants du protocole car ils peuvent être appliqués à la biomécanique au niveau d&#39;organes: 1) la force réactionnaire en fonction du déplacement: dent déplacement dans l&#39;alvéole et sa réponse réactionnaire à charge, 2) en trois dimensions (3D) configuration spatiale et morphométriques: la relation géométrique de la dent avec l&#39;alvéole, et 3) les changements dans les affichages 1 et 2 en raison d&#39;un changement dans l&#39;axe de chargement, c&#39;est à dire à partir de concentrique aux charges excentrées. L&#39;efficacité du protocole proposé sera évaluée en couplant te mécaniquepiqûre lectures à la morphométrie 3D et de la biomécanique ensemble de la commune. En outre, cette technique mettra l&#39;accent sur la nécessité d&#39;équilibrer les conditions expérimentales, des charges particulièrement réactionnaires avant d&#39;acquérir des tomographies de joints fibreux. Il convient de noter que le protocole proposé est limité à l&#39;analyse des échantillons sous des conditions ex vivo, et que l&#39;utilisation d&#39;agents de contraste pour visualiser les tissus mous réponse mécanique pourrait conduire à des conclusions erronées sur la biomécanique des tissus et au niveau des organes.

Estimation de la charge dispositif &quot;jeu&quot;, &quot;refoulement&quot;, la raideur et la dérive du système sous une charge constante Backlash: Entre chargement et de déchargement des parties du cycle, il existe une pause de 3 sec au cours de laquelle se prépare inverse dans le moteur avant le déchargement commence vrai, à savoir que l&#39;échantillon s&#39;éloigne de la mâchoire supérieure (Figure 3). Cette période e...

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

Dans cette étude, l&#39;utilisation d&#39;un dispositif de chargement in situ couplée à la tomographie micro-ordinateur à rayons X pour la biomécanique articulaire fibreux en sera discuté. Lectures expérimentales identifiables avec un changement global de la biomécanique articulaire seront les suivants: 1) les forces réactionnaires en fonction du déplacement, c&#39;est-dent déplacement dans l&#39;alvéole et sa réponse réactionnaire à charge, 2) en trois dimensions (3D) configuration spatiale et morphométriques, soit géométrique relation de la dent avec l&#39;alvéole, et 3) les changements dans les affichages 1 et 2 en raison d&#39;un changement dans l&#39;axe de chargement, c&#39;est à dire les charges concentriques ou excentriques.

In situ à la compression de chargement et d&#39;imagerie non invasive corrélative de l&#39;os-ligament parodontal-dent Fort commune

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 vues.  University of California San Francisco.  Dans cette étude, l'utilisation d'un dispositif de chargement in situ couplée à la tomographie micro-ordinateur à rayons X pour la biomécanique articulaire fibreux en sera discuté. Lectures expérimentales identifiables avec un changement global de la biomécanique articulaire seront les suivants: 1) les forces réactionnaires en fonction du déplacement, c'est-dent déplacement dans l'alvéole et sa réponse réactionnair...

Vidéo: 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.

Haute résolution à fibre optique pour les Microendoscopy In situ Cellular Imaging

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

Recherche

Bio-ingénierie

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.

Démonstration des utilisations du spectromètre force gravitationnelle roman de s&#39;étirer et de mesurer les protéines fibreuses

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.

En synthèse in vitro de la maternelle, de l&#39;espacement de longue fibreux et segmentaire collagène Espacement long

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.

Conception d&#39;un bioréacteur de chargement mécanique biaxial pour l&#39;ingénierie tissulaire

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-permis non invasive au profil métabolique

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 ...

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.

L&#39;étude de la Stœchiométrie Epidermal Growth Factor Receptor dans des cellules intactes en utilisant la microscopie corrélative

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.

Installation optimisée et protocole d’imagerie de domaines magnétiques sur mesure In Situ de hystérésis

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.

Nano-capteurs pour détecter l’activité protéase In Vivo pour le diagnostic non invasif

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.

Corrélatives microscopie électronique léger (CLEM) de suivi et d’imagerie protéine virale associés Structures des cellules immobilisées Cryo

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

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

Isolement, expansion et différenciation des cellules souches mésenchymateuses du coussinet adipeux infra-patellaire de l’articulation étouffante de chèvre

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...