Diese Arbeit wurde teilweise durch NIH Forschungsstipendien R01AR062207, R01AR061484, R56AI100901, K01AR053210 und eine Krankheit gezielte Forschungsstipendium Rheumatologie Forschungsgemeinschaft (CJ Liu) unterstützt.

Alle chirurgischen Verfahren in Bezug auf die Tiere sollten durch lokale Institution Tierpflege-und Ethik-Kommission, mit einer Anstrengung, um Schmerzen und Beschwerden durch die Operation zu minimieren genehmigt werden. 1. Vorbereitung <ol><li> Wählen 8-12 Wochen alten männlichen C57BL / 6 Mäusen mit einem Körpergewicht von ca. 25 g für eine Operation. </li><li> Betäuben die Tiere durch intraperitoneale Injektion eines Cocktails, die sowohl Xylazin (5 mg / kg) und Ketamin (40 mg / kg). </li><li> Rasieren Sie die Knie mit Rasierer, dann chirurgisch drapieren das Tier sterilisieren und die Operationsstelle mit Betadine und Alkohol (3x) und decken die Augen mit der Maus Salbe. </li></ol> 2. Chirurgische Verfahren <ol><li> Machen Sie eine 1 cm medial Längs para-Patella-Inzision Kniegelenk aus. </li><li> Öffnen Sie vorsichtig das Kniegelenk durch seitliche Luxation der Kniescheibe und Kniescheibenbandes. <ol><li> Schneiden Sie das Kniegelenk Kapsel längsdurch den medialen Kniescheiben para-Schnitt im Schritt 2.1. </li><li> Dissect Knie Gelenkkapsel mit einer Pinzette. </li><li> Nimm den distalen Teil des Hinterpfote mit der linken Hand und führen seitliche Verschiebung der Kniescheibe und Kniescheibenbandes mit einer Pinzette. Halten Sie die Hinterpfote vorsichtig und stellen Sie sicher, Trauma in der Pfote zu vermeiden. Je besser die Gelenkkapsel wird seziert, desto weniger Kraft erforderlich, um die Verschiebung zu machen. </li></ol></li><li> Tropfen steriler Salz auf der Oberfläche des Gelenkknorpels im Betrieb Austrocknen Knorpeloberfläche zu vermeiden. </li><li> Messer durch die mediale meniskotibialen Band, das mediale Meniskus zum Tibiaplateau verankert. Nicht zu verletzen, den Knorpel unter der medialen Meniskus. <ol><li> Identifizieren Sie die Innenmeniskus, der zwischen medialen Kondylus des Oberschenkelknochens und des medialen Plateau der Tibia lokalisiert. </li><li> Identifizieren Sie die meniskotibialen Band, das mit Eminentia intercondylaris der Tibia lateralen Seite des medialen Meniskus verbindet. </li><li> Halten Sie die hallond Pfote sanft mit der Hand, und schneiden durch die mediale meniskotibialen Bänder vorsichtig mit einem Skalpell Nr. 10. Achten Sie darauf, nicht zu verletzen bewirken, Knorpel und andere Bänder des Gelenkknorpels. In vielen Fällen muss die Abdeckung Fettpolster zur Seite gezogen werden, jedoch nicht, um das Band zu identifizieren entfernt werden. </li></ol></li><li> Schließen Sie das Kniegelenk Kapsel mit einem 6:0-resorbierbares Nahtmaterial. </li><li> Schließen Sie die Haut mit 6-0 Seidenfaden. </li></ol> 3. Postoperative Pflege <ol><li> Einen Tropfen 0,25% Bupivacain in der Operationsstelle jeder Maus, um postoperative Schmerzen zu minimieren. </li><li> Lassen Sie die betrieben Mäusen frei, Wasser und Nahrung zu erhalten. </li></ol> 4. Die histologische Bewertung der chirurgischen DMM Modell <ol><li> Opfere die Mäuse zu den angegebenen Zeitpunkten (zB 4 Wochen, 8 Wochen und 12 Wochen. Hier zeigten wir Ergebnisse von 8 Wochen Vertreter) nach der Operation. </li><li> Die gesamte Kniegelenk fixiert, entkalkt, embevon Paraffin dded und dann seriell auf 5 um Intervall geschnitten. <ol><li> Schneiden Sie die ganze Hinterbeine mit der Nr. 10 Klingen. </li><li> Präparieren Sie die Haut und die Muskeln der Hinterbeine vorsichtig, und fixieren Sie die Proben mit 4% PFA für 3 Tage in der RT. </li><li> Entfernen Sie die PFA, und reinigen Sie die Proben mit Wasser. Danach entkalken die Proben in EDTA bei 4 ° C für 2 Wochen. </li><li> Entwässern der Proben in einem Ethanol-Gradienten. Im Einzelnen halten die Proben in 70% Ethanol für 1 Stunde, dann ändern Sie das Ethanol und halten Sie die Proben in einem neuen Satz von 70% Ethanol für O / N. Danach setzen Sie die Proben in 80% und 90% Ethanol daher und halten sie für 1 Stunde, jeweils gefolgt von 100% Ethanol für O / N. </li><li> Entfernen Sie das Ethanol. Bewahren Sie die Proben in o-Xylol 1 h, und wechseln Sie in einer neuen Reihe von o-Xylol. Wiederholen Sie diesen Schritt 3x. </li><li> Betten Sie die Proben in einem Paraffin Form mit der Leica Einbettung Zentrum. Anschließend werden die Proben bei 5 &amp;mgr; m unter Verwendung eines Rotations Mikrotom (Leica RM225 Schnitt5, Deutschland) und anschließend auf Glasobjektträgern gesammelt. </li><li> Serien Sagittalschnitte werden für jede Probe überspannt einen Bereich von der Mitte der lateralen Kondylus an der Mitte des medialen Kondylus geschnitten. </li></ol></li><li> Safranin-O-Färbung durchgeführt wird, gefolgt von Ritzen und statistische Analysen durch OARSI Scoring-System 25, wie vorher beschrieben. <ol><li> Zunächst entparaffinieren Folien in o-Xylol, und Hydrat sie in einem Ethanol-Gradienten zu destilliertem Wasser. Danach werden die Folien durch Weigert Eisenhämatoxylin Lösung, 0,05% Fast Green (FCF) Lösung, 1% Essigsäure-Lösung und 0,1% Safranin O Lösung gefärbt. Montieren Sie die Folien mit harzigen Medium. </li><li> Ergebnis der Safranin O gefärbten Objektträger, basierend auf Verlust von Proteoglykan (rote Farbe) im Knorpel und den Prozentsatz der Zerstörung im Knorpelstruktur, tun statistischen Analyse für die histologische Score. </li></ol></li></ol>

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Wir erklären, dass wir hier keinen Interessenkonflikt.

Es wird berichtet, dass der Stamm von Mäusen ist sehr wichtig für DMM Modell Induktion, als verschiedene Stämme von Mäusen haben unterschiedliche Schwere der OA nach DMM, mit höchstem Niveau in der 129/SvEv Stamm, gefolgt von C57BL6, 129/SvInJ und dann FVB / n 26. Mäuse, die wir in der vorliegenden Studie eingesetzt, die relativ anfällig für DMM sind - Ein großer Teil der Transgene in C57BL/6-Mäuse, wie PGRN-/ etabliert. Wenn das Transgen auf Stamm unempfindlich wie FVB / N-Mäusen basiert, ist es j...

Osteoarthritis (OA), auch als degenerative Arthritis bekannt ist, betrifft 15% der Weltbevölkerung und mehr als 46 Millionen Menschen in den Vereinigten Staaten und wird von Synovitis, Knorpeldegeneration und Osteophytenbildung 1 gekennzeichnet. Es kann ein Ergebnis eines komplexen Zusammenspiels von genetischen, metabolischen, biomechanischen und biochemischen Faktoren. Die zugrunde liegenden Mechanismen der OA auch weiterhin die wissenschaftliche Gemeinschaft zu entziehen. Derzeit gibt es zahlreiche Tiermodelle, die die Pathogenese der OA 2,3 nachahmen kann. Es ist wichtig zu Tiermodellen bei Mäusen, weil sowohl die Verfügbarkeit von verschiedenen genetisch veränderten Mäusen und die Wirtschaftlichkeit Experimentieren herzustellen. Unter den verschiedenen Arten von Versuchs OA Modellen ist die chirurgisch induzierten Destabilisierung des medialen Meniskus (DMM)-Modell ein gut angenommen OA-Modell aufgrund seiner guten Reproduzierbarkeit und einem relativ langsamer Progression bei OA Entwicklung. Beide dieser Attributeder Schlüssel für die Bewertung der Progression OA in verschiedenen Behandlungen oder Transgene 3-8. Jedoch wird die Konsistenz der chirurgischen OA Modell durch verschiedene Faktoren während der Operation und als Ergebnis beeinflusst, ist die Anwendung von chirurgischen Mausmodell beschränkt. Progranulin (PGRN) ist ein multifunktionales Wachstumsfaktor in verschiedenen Zellen exprimiert. Es ist bekannt, dass PGRN spielt eine entscheidende Rolle bei unterschiedlichen physiologischen und Krankheitsprozessen wie der Wundheilung 9, Tumorgenese 10, 11-15 und Entzündungen. Studien auch gezeigt, dass unzureichende PGRN können degenerative Erkrankungen des Nervensystems bei Menschen und Mäusen 16-18 führen. Es ist bekannt, dass PGRN in menschlichen Gelenkknorpel ausgedrückt, und der Wert ist signifikant im Knorpel des Patienten mit Arthrose und rheumatoide Arthritis 19 erhöht. Darüber hinaus spielt PGRN auch eine entscheidende Rolle in Chondrozytenproliferation 20, Differenzierung und endochondralenVerknöcherung der Wachstumsfuge während der Entwicklung 21,22. Vor kurzem berichteten wir, dass PGRN TNF-α antagonisiert durch Bindung an TNF-Rezeptoren und zeigten eine entzündungshemmende Funktion bei entzündlichen Arthritis-Modellen 13,14,23,24. Jedoch ist die Rolle der PGRN in OA, insbesondere in vivo, bleibt ein Rätsel sein. Hier stellen wir das Verfahren zur chirurgischen DMM Modell zu induzieren, und untersuchen die Rolle der PGRN in OA Entwicklung durch Gründung DMM Modell in WT-und PGRN-/ - Mäusen.

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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

Destabilisierung des medialen Meniskus (DMM) Modell ist ein wichtiges Werkzeug für die Untersuchung der pathophysiologischen Rollen von zahlreichen Arthritis assoziierten Molekülen in der Pathogenese von Osteoarthritis (OA) in vivo. Allerdings ist die detaillierte, vor allem die visualisiert Protokoll für die Einrichtung dieses kompliziertes Modell bei Mäusen, ist nicht verfügbar. Dabei nutzten wir die Wildtyp-und progranulin (PGRN) - / - Mäuse als Beispiele ein Protokoll zur Induktion DMM Modell bei Mäusen, und verglichen das Auftreten von OA folgende Einrichtung dieser chirurgisch induzierten Modell einzuführen. Die an Mäusen durchgeführten Operationen waren entweder Schein-Operation, die gerade geöffnet Gelenkkapsel oder DMM-Operation, die die menisco-Schienbeinbandes geschnitten und verursachte Destabilisierung des medialen Meniskus. Schwere Arthrose wurde mit histologischen Test (z. B. Safranin-O-Färbung), Ausdrücke der OA-assoziierten Gene, Abbau von Knorpel extrazellulären Matrix-Moleküle und Osteophyten Form ausgewertetation. Mäuse, und der Verlust der PGNR Wachstumsfaktor führte zu einer schweren OA Phänotyp in dieser chirurgisch induzierten Modell - DMM Vorgang erfolgreich OA Initiierung und Progression sowohl in Wildtyp-und PGRN-/ induziert.

DMM Modell wurde erfolgreich an Mäusen etabliert und Mangel an PGRN übertrieben chirurgisch-induzierten OA Entwicklung. Mäuse - Sham und DMM-Operationen (Abbildung 1) wurden in WT-und PGRN / durchgeführt. 8 Wochen nach der Operation wurden die Mäuse getötet, und Safranin-O-Färbung wurde in den Kniegelenken von Abschnitten durchgeführt wird, gefolgt von einer statistischen Analyse der Arthritis-Score auf Basis der Histologie. Wie in 2A</strong...

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

Wir beschreiben ein Protokoll für die Destabilisierung des medialen Meniskus (DMM)-Modell bei Mäusen, ein wirksames Instrument für Osteoarthritis (OA) Forschung. Darüber hinaus haben wir diesen Mangel an progranulincan übertreiben OA Entwicklung und Progression von mit diesem Modell gezeigt, das anzeigt, dass progranulin spielt eine schützende Rolle in der Pathogenese der OA.

Einrichtung einer chirurgisch-induzierten Modell bei Mäusen, die Rolle der Schutz Progranulin in Osteoarthritis Untersuchen

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

27.3K Aufrufe.  NYU Hospital for Joint Diseases.  Wir beschreiben ein Protokoll für die Destabilisierung des medialen Meniskus (DMM)-Modell bei Mäusen, ein wirksames Instrument für Osteoarthritis (OA) Forschung. Darüber hinaus haben wir diesen Mangel an progranulincan übertreiben OA Entwicklung und Progression von mit diesem Modell gezeigt, das anzeigt, dass progranulin spielt eine schützende Rolle in der Pathogenese der OA. 

Serie: 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.

Multiparametrische Optical Mapping der Langendorff-perfundierten Rabbit Heart

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

Forschung

Biotechnik

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.

Erstellen Zweidimensionale Patterned Substrate für Protein-und Zell-Confinement

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.

Entwurf eines zyklischen Druck Bioreaktor für die Ex Vivo Studium der Aorten-Herzklappen

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.

Quantifizierung der globalen diastolischen Funktion durch kinematische Modellierung basierende Analyse der transmitralen Fluss über die diastolische Füllung Parametrisierte Formalismus

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

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 Diffusionssystem, um Chemotaxis in einem 3D-Kollagen-Matrix Untersuchen

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.

Ein<em&gt; In Vitro</em&gt; Organ Culture Modell der Murine Bandscheiben

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.

Erstellen eines strukturell realistische Finite-Elemente geometrischen Modells von einer Cardiomyocyte zur Untersuchung der Rolle von zellulären Architektur in Cardiomyocyte Systembiologie

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: Eine starke, nicht-invasive Ansatz zur zellulären Energiestoffwechsel untersuchen

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.

Ein Tripeptid-stabilisierten Nanoemulsion Ölsäure

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 ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Ein menschliches 3D extrazelluläres Matrix-Adipozyten-Kulturmodell zum Studium der Matrix-Zell Metabolischen Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...