Die Autoren danken für die Unterstützung von NSF Zuschuss CBET-0909678 und Finanzierung von der GW-Zentrum für Bionik und Bioinspirierte Engineering (COBRE). Wir danken den Studenten, Herr Christopher Popma, Frau Leanne Penna, Frau Shannon Callahan, Herr Shadman Hussain, Herr Mohammed R. NAJJARI und Frau Jessica Hinke um Hilfe im Labor und Herr Mathieu Barraja für die Unterstützung in CAD-Zeichnungen.

<ol>
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Das Protokoll in diesem Papier beschreibt den Erwerb von High-Fidelity-experimentellen Daten Particle Image Velocimetry Technik (PIV) und kohärente Struktur Nachweisverfahren unter Verwendung, dh., Kontinuierliche Wavelet-Transformationen, <img alt="Gleichung 1" src="https://neuron2.jove.com/files/ftp_upload/51288/51288eq1.jpg" /> , Geeignet für die Identifizierung von Wirbel und Strömungen schub dominiert. Analyse der experimentellen Daten von physiologischen Zuflüsse in Gegenwart eines idealisierten &quot;Typ IV&q...

Sekundäre Strömungsstrukturen sind verwirbelte Strömungsmuster, die in der internen Strömungsgeometrien mit Krümmungen wie gebogene Rohre und Kanäle auftreten. Diese Wirbelstrukturen entstehen aufgrund der kombinierten Wirkung von Zentrifugalkräften, ungünstige Druckgradienten und Zufluss Eigenschaften. Im Allgemeinen scheinen die sekundäre Strömungsstrukturen in planaren Querschnitte von gebogenen Rohren als symmetrische Dean-Typ Wirbel unter stetigen Zufluss und symmetrische Dean - und Lyne artigen Wirbel unter oszillierenden Einströmverhältnisse . 1 - 3 Sekundärfluss Morphologien von Pulsatilitäts stark betroffen sind und mehrere Harmonische von pulsierender, physiologischen Einströmverhältnisse. Diese Strukturen erfassen deutlich unterschiedlichen Größenfestigkeitsformeigenschaften im Vergleich zu nicht-physiologischen (steady und oszillatorischen) fließt . 1 - 6 atherosklerotischen Läsion Entwicklung in Arterien durch das Vorhandensein von Hochfrequenzscherschwingungen in betroffenen Gebieten erlebt niedrige mittlere Scher 27, 28 </sup&gt;. Sekundärströmungsstrukturen können den Fortschritt von Erkrankungen wie Atherosklerose beeinflussen und möglicherweise die endothelial Reaktion aufgrund pulsatile Blutfluß vermitteln durch Wandschubspannungen verändern und Belichtungszeiten von durch Blut übertragbaren Teilchen. Eine übliche Behandlung der Atherosklerose, eine Komplikation in Verengung der Arterien durch obstruktive Läsionen, ist die Implantation von Stents. Stent Frakturen sind strukturelle Versagen der implantierten Stents , die zur weiteren medizinischen Komplikationen wie In-Stent - Restenose (ISR), Stentthrombose und Aneurysmabildung führen . 9 - 13 Stentfrakturen haben verschiedene Fehler &quot;Typen I-to-IV&quot; eingestuft worden, wobei &quot;Typ IV&quot; den höchsten klinischen Schweregrad charakterisiert und wird als die vollständige Querfraktur der Stentstreben zusammen mit linearen Verschiebungen der Stent Fragmente 14 definiert. das Protokoll in dieser Studie präsentierten beschreibt eine experimental Verfahren zur Visualisierung von sekundären Strömungsstrukturen hinter einer idealisierten &quot;Typ IV&quot; Stentfraktur in einem gekrümmten Arterienmodell. Das vorgeschlagene Protokoll hat die folgenden vier wesentliche Merkmale: Konstruktion und Herstellung von Labormaßstab Stentmodelle: Geometrische Beschreibung von Stents kann mit einer Reihe von Self-Expandable Spiralen (Federn oder Helices) miteinander verflochten mit Nitinol (eine Legierung aus Nickel und Titan) Drähte 29 verbunden sein. Die Länge des Stents und seine Strebe Durchmesser sind abhängig von der Längenskala von arteriellen während der klinischen Implantation 5 aufgetreten Läsionen. Parametric Variation der Strebe Durchmesser und der Aufstand der Wicklung (oder Pech) führt zu Stents aus verschiedenen geometrischen Konfigurationen. Eine Zusammenfassung der Stentkonstruktionsparameter für den 3D - Druck ausgewählt sind in Tabelle 1 dargestellt. Herstellung eines Blut analogen Arbeitsfluid abgestimmtmit kinematischen Viskosität von Blut und der Brechungsindex der Teststrecke: Ein optischer Zugang zu dem gekrümmten Arterie Teststrecke ist erforderlich , um nicht-invasive Geschwindigkeitsmessungen zu machen. Dementsprechend ist ein Newtonsches Blutähnliche Flüssigkeit mit dem Brechungsindex des vaskulären Modells arbeitet und im Idealfall, eine dynamische Viskosität, menschlichem Blut passend verwendet werden , um genaue Blutströmungsmessungen 16 erhalten , -. 18, 30 das Arbeitsfluid in dieser Studie verwendet wurde berichtet von Deutsch et al. (2006), das sich aus 79% gesättigtem wässrigem Natriumiodid (NaI), 20% iges Glycerin und 1% Wasser (bezogen auf das Volumen) 16. Versuchsanordnung zum Nachweis von kohärenter sekundären Strömungsstrukturen unter Verwendung eines zweikomponentigen, zweidimensionalen Particle Image Velocimetry (2C-2D - PIV): Die Experimente wurden entworfen , um phasen gemittelten sekundären Strömungsgeschwindigkeitsdaten in verschiedenen planaren Querschnittsstellen erwerben nachgeordnet eine Kombination aus straight und gekrümmte Stentabschnitte verkörpert eine idealisierte &quot;Typ IV&quot; Stentfraktur 5, 6, 9, 14. Die Protokollschritte auf den Erwerb von Sekundärströmungsgeschwindigkeit Felder mit Particle Image Velocimetry (PIV) Technik im Zusammenhang beinhaltet ein PIV - System , das besteht aus ein Laser (Lichtbogen) Quelle, Optik, die Regionen Strömungs eine spezielle Kreuzkorrelationsladungsgekoppelte Vorrichtung (CCD-Sensor oder Kamera) und Markierungspartikel innerhalb eines kurzen Zeitintervalls durch den Lichtbogen ausgeleuchtet werden (&amp; Dgr; t zu fokussieren und zu belichten siehe Tabelle 4) 31, 32;. Die Schritte in dem Protokoll gelten folgende Voraussetzungen: Erstens, eine kalibrierte, experimentelle Aufbau eines Zweikomponenten, zweidimensionale (2C-2D) PIV-System, das Bilder von Doppelrahmen, Einzelbelichtung Aufnahmen auswertet. Zweitens berechnet der 2C-2D-PIV System die mittlere Verschiebungen Markierungspartikel durch Ausführen einer Kreuzkorrelation zwischen zwei Bildrahmen während jeder Aufnahme erworben. Ein brief Zusammenfassung der PIV Spezifikationen und Bildaufnahme-Software ist in der Materialien und Geräte Tabelle dargestellt. Drittens benötigt alle Sicherheitsvorkehrungen um den Laser zu arbeiten, werden von geschultem Laborpersonal gefolgt nach den Richtlinien von der Gastinstitution zur Verfügung gestellt. Die Autoren schlagen vor Refs. 31 und 32 für ein ganzheitliches Verständnis der Umsetzung, Funktionalität und Anwendung von PIV-Technik in der Luft-, Wasser- und mikrofluidischen Dynamik, Korrelationsspitzenerkennung und Verschiebung Schätzung, Material und Dichte der Tracerpartikel und Messrauschen und Genauigkeit. Beachten Sie auch , dass der Laser und die Kamera kann durch die PIV Datenerfassungscomputer (3A) und die Datenverarbeitungssoftware gesteuert werden. Die Datenerfassung und Nachbearbeitung für eine kohärente Strukturerkennung: Phase gemittelten Messungen sekundären Strömungsgeschwindigkeit ein 2C-2D - PIV wurden unter Verwendung erzeugt die Protokollbeschreibung verwendet , die folgt. Nachbearbeitung<html> ing der Daten beteiligt kohärenten sekundären Strömungsstrukturerkennung die folgenden drei Verfahren: kontinuierliche Wavelet-Transformationen, <img alt="Gleichung 1" src="/files/ftp_upload/51288/51288eq1.jpg" /> 5, 6, 19-24, 26. Die Autoren weisen, dass die Geschwindigkeit Gradiententensors ist im Wesentlichen, eine 3 x 3 Matrix, <img alt="Gleichung 2" src="/files/ftp_upload/51288/51288eq2.jpg" /> . Das Protokoll stellt ein Verfahren zum Erfassen zweidimensionaler experimentellen Messungen (von 2C-2D-PIV-Technik). Daher wird die volle experimentellen Zugang zum Geschwindigkeitsgradienten tensor nicht erreichbar sein, mit dieser Methode. Der Geschwindigkeitsgradient Tensor für jedes Pixel <img alt="Gleichung 3" src="/files/ftp_upload/51288/51288eq3.jpg" /> des PIV Bild <img alt="Gleichung 4" src="/files/ftp_upload/51288/51288eq4.jpg" /> sollte eine 2 x 2-Matrix ist, <img alt="Gleichung 5" src="/files/ftp_upload/51288/51288eq5.jpg" /> . Die z-Komponente vorticityquation 6 &quot;src =&quot; / files / ftp_upload / 51288 / 51288eq6.jpg &quot;/&gt; für jedes Pixel <img alt="Gleichung 7" src="/files/ftp_upload/51288/51288eq7.jpg" /> berechnet wird, um die antisymmetrische Teil des Geschwindigkeits Gradiententensors mit <img alt="Gleichung 8" src="/files/ftp_upload/51288/51288eq8.jpg" /> . Das Ergebnis wird ein 2D-Array von vorticity sein <img alt="Gleichung 9" src="/files/ftp_upload/51288/51288eq9.jpg" /> dass in einem Konturdiagramm visualisiert werden. Die Autoren schlagen vor stark Ref. 25 für eine beredte Diskussion experimentellen Zugang zum Geschwindigkeitsgradienten Tensor das Wissen der Verwirbelung Ableitung, Dehnungsraten und kohärente Struktur Erkennung zu verbessern. Weiterhin versuchen die Autoren nicht die Zusammenhänge zwischen den genannten kohärenten Struktur Detektionsverfahren und schlägt Ref zu erkunden. 23, 24 für eine umfassende Diskussion zu diesem Thema. Der Fokus der Schritte in dem Protokoll ist die quantitative Ermittlung der Sekundärströmung (verwirbelten) structures (auch als kohärente Strukturen bekannt). Drei Methoden der kohärenten Strukturerkennung nämlich., <img alt="Gleichung 10" src="/files/ftp_upload/51288/51288eq10.jpg" /> und Wavelet-transformierten vorticity <img alt="Gleichung 11" src="/files/ftp_upload/51288/51288eq11.jpg" /> sind auf Geschwindigkeit Felddaten zu Detektion von stromabwärts der idealisierten &quot;Typ IV&quot; Stentfraktur Multi-Skala, Multi-Stärke Vorkommen von sekundären Strömungsstrukturen angewendet. Das <img alt="Gleichung 12" src="/files/ftp_upload/51288/51288eq12.jpg" /> Definiert einen Wirbel als räumliche Bereich , in dem die euklidische Norm des vorticity tensor dass 19 der Rate der Dehnungs dominiert, 23, 24 .Die Geschwindigkeitsgradienten Matrix in symmetrische (Dehnungsrate) zerlegt wird und antisymmetrische (Rotation) Teilen. Eigenwerte der Dehnungsrate Matrix berechnet werden; <img alt="Gleichung 13" src="/files/ftp_upload/51288/51288eq13.jpg" /> . Norm der Verformungsgeschwindigkeit wird dann berechnet; <img alt = &quot;Gleichung 14&quot; src = &quot;/ files / ftp_upload / 51288 / 51288eq14.jpg&quot; /&gt;. Vorticity aus dem anti- symmetrischen Teil berechnet. Enstrophie oder Quadrat von z-Komponente Verwirbelung, <img alt="Gleichung 15" src="/files/ftp_upload/51288/51288eq15.jpg" /> ) Wird dann berechnet. Das <img alt="Gleichung 16" src="/files/ftp_upload/51288/51288eq16.jpg" /> schließlich ist berechnet; <img alt="Gleichung 17" src="/files/ftp_upload/51288/51288eq17.jpg" /> . Ein Konturdiagramm des gesamten Satzes von <img alt="Gleichung 18" src="/files/ftp_upload/51288/51288eq18.jpg" /> mit iso-Regionen <img alt="Gleichung 19" src="/files/ftp_upload/51288/51288eq19.jpg" /> Zeigt an , 19 sekundäre Strömungsstrukturen. Das <img alt="Gleichung 20" src="/files/ftp_upload/51288/51288eq20.jpg" /> , Die auch als &quot;wirbelnde Kraft&quot; genannt , ist eine Methode Wirbel Identifizierung von kritischen Stellenanalyse des lokalen Geschwindigkeits Gradiententensors und seine entsprechenden Eigenwerte durchgeführt von 20 bis 24 <strong&gt;. Eigenwerte des Geschwindigkeits Gradiententensors bei jedem Pixel <img alt="Gleichung 21" src="/files/ftp_upload/51288/51288eq21.jpg" /> berechnet. Die Eigenwerte sollten von der Form sein, <img alt="Gleichung 22" src="/files/ftp_upload/51288/51288eq22.jpg" /> . Ein Konturplot <img alt="Gleichung 23" src="/files/ftp_upload/51288/51288eq23.jpg" /> mit iso-Regionen <img alt="Gleichung 24" src="/files/ftp_upload/51288/51288eq24.jpg" /> zeigt an Strukturen Nebenstrom 20-22. Wavelet-Transformationsverfahren verwendet eine Analysefunktion (oder wavelet), die Glätte in physikalischen und spektralen Räume hat, zulässig ist (oder nur Null-Mittelwert) und hat eine finite <img alt="Gleichung 25" src="/files/ftp_upload/51288/51288eq25.jpg" /> 5, 6, 26. Durch Falten einer dilatiert oder kontrahiert mit einer 2D vorticity Feld Wavelet - transformierten vorticity Wavelet <img alt="Gleichung 26" src="/files/ftp_upload/51288/51288eq26.jpg" /> Feld erzeugt comprising kohärenter Strukturen mit einer Vielzahl von Skalen und Stärken 5, 6, 26. Shannon Entropie des 2D - Wavelet-transformierten vorticity Feld wird berechnet , um die optimale Wavelet - Skala abzuschätzen bei dem alle kohärenten Strukturen angemessen gelöst werden. Diese Entropieschätzung beinhaltet eine Reihe von Wahrscheinlichkeiten <img alt="Gleichung 27" src="/files/ftp_upload/51288/51288eq27.jpg" /> für jedes Pixel, <img alt="Gleichung 21" src="/files/ftp_upload/51288/51288eq21.jpg" /> so dass <img alt="Gleichung 28" src="/files/ftp_upload/51288/51288eq28.jpg" /> Der normierte Quadratmodul der Verwirbelung mit dem Pixel an der Stelle m zugeordnet ist , n 5, 6. Die Verfahrensschritte sind in Figur 6 graphisch dargestellt. Die Einschränkungen bei der Wahl der Wavelet platziert werden ausführlich in Ref dargestellt. 26. Dieses Protokoll Schritt beschreibt das Verfahren für eine kohärente Struktur Erkennung eines 2D-Ricker-Wavelet verwendet wird. Die Begründung für die Verwendung dieses wavelet für vortical Musterabgleich wird in Ref dargestellt. 5, 6 und die zugehörigen darin zitierten Referenzen.

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			<td style="width:155px;">Cole Parmer</td>
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			<td style="width:208px;">Toward measurement of kinematic viscosity of the blood-analog fluid</td>
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			<td height="21" style="height:21px;width:216px;">VELP scientifica - ESP stirrer&#160;</td>
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			<td style="width:155px;">Sigma-Aldrich</td>
			<td>72049-250G</td>
			<td style="width:208px;">Powder</td>
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			<td height="42" style="height:42px;width:216px;">PIV Recording medium</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">Imager Intense 10Hz</td>
			<td style="width:208px;">PIV Image acquisition CCD camera</td>
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			<td style="width:155px;">New Wave Research</td>
			<td style="width:119px;">Solo III-15</td>
			<td style="width:208px;">PIV Laser source, Nd:YAG laser, 532 nm, dual pulse 70 mJ/pulse</td>
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			<td height="42" style="height:42px;width:216px;">PIV Imaging software</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">DaVis 7.2&#160;</td>
			<td style="width:208px;">PIV data acquisition and instrument control&#160;</td>
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			<td height="211" style="height:211px;width:216px;">PIV Seeding material</td>
			<td style="width:155px;">Thermo-scientific&#160;&#160;</td>
			<td style="width:119px;">Flouro-Max</td>
			<td style="width:208px;">Red fluorescent polymer microspheres (&#8776; 7 &#181;m); Dry dyed polystyrene (DVB) fluorescent microspheres emit bright and distinct colors when illuminated by the light of shorter&#160; wavelengths than the emission wavelength.&#160;</td>
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experimental investigation of secondary flow structures downstream of a model type iv stent failure in a 180&#176; curved artery test section

Das arterielle Netzwerk im menschlichen Gefäßsystem besteht aus ubiquitär Blutgefäße mit komplexen Geometrien (Filialen, Krümmungen und Verwindung). Sekundäre Strömungsstrukturen sind verwirbelte Strömungsmuster, die in gekrümmten Arterien aufgrund der kombinierten Wirkung von Zentrifugalkräften, ungünstige Druckgradienten und Zufluss Merkmale auftreten. Solche Fließ Morphologien sind durch Pulsatilität und mehreren Harmonischen der physiologischen Zuströmbedingungen und variieren stark in Größe stärkeFormEigenschaften im Vergleich zu nicht-physiologischen (steady und oszillatorischen) stark beeinflusst fließt 1 bis 7. Sekundäre Strömungsstrukturen können beeinflussen letztlich die Wandschubspannung und Belichtungszeit von Blut getragenen Partikeln in Richtung Progression der Atherosklerose, Restenose, Sensibilisierung von Blutplättchen und Thrombose 4 - 6, 8 - 13. Deshalb ist die Fähigkeit zu erkennen und zu charakterisieren , diese Strukturen im Labor. -kontrollierten Bedingungen ist precursoder klinische Untersuchungen zu fördern. Eine gemeinsame chirurgische Behandlung von Atherosklerose ist der Stent-Implantation, verengte Arterien für einen ungehinderten Blutfluss zu öffnen. Aber die gleichzeitige Flussstörungen aufgrund Stent Installationen führen in mehrskaligen Sekundärströmung Morphologien . 4 - 6 Progressiv höhere Ordnung Komplexitäten wie Asymmetrie und der Verlust in der Kohärenz kann 5 durch folgenden Stent Ausfälle gegenüber den unter ungestörte Ströme induziert werden. Diese Stents wurden Fehler als &quot;Typ I-to-IV&quot; eingestuft basierend auf Versagen Erwägungen und klinischen Schweregrad 14. Diese Studie stellt ein Protokoll für die experimentelle Untersuchung der komplexen Sekundärströmungsstrukturen aufgrund von Quer Stent Fraktur und lineare Verschiebung des gebrochenen Teile ( &quot;Typ IV&quot;) in einer gekrümmten Arterienmodell vervollständigen. Das experimentelle Verfahren beinhaltet die Durchführung von Particle Image Velocimetry (2C-2D PIV) Techniken mit einer archetypischen Arteria carotis Einströmen Wellenform, einen Brechungsindex angepaßten Blut analogen Arbeitsfluid für phasen gemittelten Messungen . 15 - 18 Quantitative Identifizierung der Sekundärströmungsstrukturen erreicht Konzepte der Strömungsphysik verwendet, kritische Punkt Theorie und eine neuartige 26 - Wavelet - Algorithmus angewendet , um experimentelle Daten PIV 5, 6, 19 zu transformieren.

Ergebnisse in 7A-D wurden nach der Nachbearbeitung sekundären Strömungsgeschwindigkeitsdaten (siehe Abbildungen 5, 6) erzeugt präsentiert von 2C-2D - PIV - System in 3A gezeigt erworben. Der Zufluss Zustand der gekrümmten Arterie Meßstrecke mit einer idealisierten &quot;Typ IV&quot; Stent Fraktur zugeführt wurde , die Arteria carotis Wellenform in Figur 4B gezeigt. Unsere frühere Studien haben die Empfindlichkeit ...

Watch this Scientific Journal Video about Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180Ã‚Â° Curved Artery Test Section at JoVE.com

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Experimentelle Untersuchung von Sekundärströmungsstrukturen hinter einem Modell Typ IV Stent Failure in einem 180 ° Curved Artery Test Sektion

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

experimental-investigation-secondary-flow-structures-downstream-model

Research

JoVE Journal

Bioengineering

11.5K Aufrufe.  The George Washington University. Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Serie: Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180° Curved Artery Test Section

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 ...

Forschung

Biotechnik

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

Parallel-Platten-Flow-Kammer und Continuous Flow Circuit endothelialen Vorläuferzellen unter Laminar Flow Schubspannung Bewerten

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Die Überwachung der Wand Mechanik Während der Entfaltung des Stents in einem Gefäß

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Tissue Engineering eines Menschen 3D<em&gt; In-vitro-</em&gt; Tumor-Testsystem

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Ein verbessertes Verfahren zur Herstellung von Typ I-Kollagen aus der Haut

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

Charakterisierung von mehrschichtigen Fischschuppen (<em&gt; Atractosteus Spachtel</em&gt;) Verwendung Nanoindentierung, Röntgen-CT-, FTIR-und SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Test auf Augenreizung (EIT) zur Kennzeichnung der Gefahr von Augen Reizt Chemicals mit rekonstruierter menschlicher Hornhaut-ähnlichen Epithelial (RhCE) Gewebemodell

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Starre Einbettung von fixiert und gefärbt, im großen und ganzen Millimeter-Skala Exemplare für Abschnitt-free 3D Histologie durch Mikro-Computertomographie

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Gen Expressionsanalyse von Endothelzellen ausgesetzt um Stress mit mehreren Parallel-Platte Fluss Kammern Scheren

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

Eine automatisierte Methode zur Durchführung des In-Vitro-Mikrokern-Assays mit multispektraler Bildflusszytometrie

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...