Die Autoren danken der Texas A &amp; M University Engineering and Experiment Station (TEES) für die finanzielle Unterstützung dieser Forschung. Lindsay Nail dankt Unterstützung von der Texas A &amp; M Universität Louis Stokes Allianz für Minderheitsbeteiligung (LSAMP) und der National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP). Dawei Zhang dank der Texas A &amp; M University Dissertation Fellowship.

1. Synthesizing PCL-DA Makromer <ol><li> Führen Sie das Acrylierung Reaktion. <ol><li> Wiegen 20 g PCL-Diol (Mn = ~ 10.000 g / mol) in einem 250 ml Rundkolben, der mit einem mit Teflon überzogen Magnetrührstab ausgestattet. </li><li> Lösen Sie die PCL-diol in DCM. <ol><li> Zugabe von 120 mLlof DCM in den Kolben (Konzentration = 0,17 g / ml). </li><li> Platzieren Gummiseptum lose in den Hals des Kolbens, um Druckaufbau zu vermeiden, während auch Verdampfung von DCM zu verhindern. </li><li> Stir-Lösung für ca. 30 min bei ~ 250 Upm, um das Polymer vollständig aufzulösen. </li></ol></li><li> Hinzufügen ~ 6,6 mg 4-Dimethylaminopyridin (DMAP) zu der Lösung und Rühren lösen. </li><li> Platzieren einem Gummiseptum fest in den Hals des Kolbens. Lassen Sie die Lösung unter Rühren fortgesetzt. </li><li> Durch das Gummiseptum, vorsichtig den Kolben zu spülen mit N 2 ~ 3 min unter Verwendung einer positiven N 2 -Druck Nadel Einlass und einen offenen Nadelals Ventil. </li><li> Nehmen Sie die N 2 Einlass und Auslass. </li><li> Hinzufügen 0,56 ml (4,0 mmol) Triethylamin (Et 3 N) tropfenweise mittels einer Glasspritze mit einer Nadel durch das Gummiseptum eingeführt ausgestattet. </li><li> Hinzufügen 0,65 ml (8,0 mmol) Acrylsäurechlorid tropfenweise mittels einer Glasspritze mit einer Nadel durch das Gummiseptum eingeführt ausgestattet. </li><li> Geben die N 2 Einlass in den Kolben und damit der Inhalt unter einem positiven N 2 Druck für 30 min rühren ~. </li><li> Vorheizen ein Ölbad auf 55 ° C. </li><li> Nach der vorgegebenen ~ 30 Minuten, entfernen Sie die N 2 Einlass und ersetzen Sie das Septum mit einem Kondensator. </li><li> Tauchen Sie den Kolben in den vorgeheizten Ölbad. </li><li> Damit der Inhalt des Kolbens auf 20 h rühren. </li><li> Nach der vorgesehenen 20 Stunden, entfernen Sie den Kolben aus dem Ölbad und ermöglicht Inhalte auf RT abkühlen. </li><li> Mit einem Rotationsverdampfer entfernen DCM Lösungsmittel aus dem Kolben. </li></ol></li><li> Purify das rohe PCL-DA Produkt. <ol><li> In den Kolben, fügen ~ 135 ml Ethylacetat und Auflösen des rohen PCL-DA. </li><li> Gravity Die Lösung wird durch Filterpapier in einen sauberen 250 ml-Rundkolben. (Anmerkung: Die Lösung kann auf das Filterpapier zu verdicken, nicht leicht durchlauf Wenn ja, sorgfältig milder Wärme mit einer Wärmepistole anwendbar.). </li><li> Mit einem Rotationsverdampfer entfernen Ethylacetat-Lösungsmittel aus dem Kolben. </li><li> In den Kolben, fügen ~ 140 ml DCM und lösen das rohe PCL-DA. </li><li> Übertragen Inhalt in einen 500 ml Scheidetrichter gegeben. </li><li> In den Trichter gegeben hinzuzufügen 13,5 ml 2 M Kaliumcarbonat (K 2 CO 3). </li><li> Verschließen Sie den Trichter. Vorsichtig mischen die beiden Schichten durch Invertierung des Trichters und sanft ein- oder zweimal wirbelnden, kümmert sich um den Druck über den Absperrhahn zu lösen. 3 Mal wiederholen. </li><li> Setzen Sie die Kappe mit einer Schicht aus Parafilm und lassen Sie die Mischung zu trennen O / N (~ 12 h). </li><li> Sammeln Sie die unten oderganische Schicht in eine 250 ml-Erlenmeyerkolben. </li><li> Hinzufügen ~ 5 g wasserfreies Magnesiumsulfat (MgSO 4), um den Kolben gegeben und durch vorsichtiges Schwenken. </li><li> Gravity Das Gemisch wird durch qualitative Filterpapier und in einen sauberen 250 ml-Rundkolben. </li><li> Mit einem Rotationsverdampfer entfernen DCM Lösungsmittel aus dem Kolben. </li><li> Trocken unter hohem Vakuum, um restliches DCM zu entfernen. (Hinweis: Die PCL-DA sollte weg von Licht gelagert werden.) </li><li> Bestätigen Acrylierung mit 1 H-NMR 22,23. </li></ol></li></ol> 2. Vorbereitung des SMP Gerüst (Abbildung 1) <ol><li> Bereiten Sie die Salzschmelze-Vorlage. <ol><li> Verwenden ein 425 um Sieb zu Natriumchlorid (NaCl) Partikel mit einem Durchmesser erhalten ~ 460 ± 70 um. (Hinweis: Die mittlere Teilchengröße kann von Rasterelektronenmikroskopie [SEM] Bilder mit ImageJ Software bestätigt werden.) 14 </li><li> In einen 3 ml-Glasfläschchen (ID = 12,9 mm), fügen 1,8 g des zuvor gesiebt NaCl. </li><li> Langsame Zugabe in vier Portionen, 7,5 Gew% (bezogen auf Salzgewicht) DI Wasser (0.146 g) in das Fläschchen. Mix mit einem Metallspatel nach der Zugabe jeder Portion Wasser. </li><li> Verschließe das Röhrchen, wickeln Sie sie in Gewebe und legen vertikal in ein Zentrifugenröhrchen. Zentrifuge für 15 min bei 3.220 x g. </li><li> Entfernen Sie den Deckel und lassen Sie an der Luft trocknen O / N (~ 12 h). </li></ol></li><li> In einer neuen Glasfläschchen, bereiten eine &quot;Makromerlösung&quot; durch Kombination von 0,15 g PCL-DA pro ml DCM. (Hinweis:. Zum einen Gerüst, ~ 1 ml Lösung vorbereitet werden) Cap und mischen Sie die Lösung mit hoher Geschwindigkeit auf einem Vortex-Mischer für ca. 1 min. </li><li> In einer neuen 3-ml-Glasgefäß, bereiten eine &quot;Photoinitiatorlösung&quot; auf Basis von 10 Gew% 2,2-Dimethoxy-2-phenylacetophenon (DMP) in 1-Vinyl-2-pyrrolidinon (NVP). Kombinieren 0,115 g DMP in 1 ml NVP. (Anmerkung: Bei einem Gerüst, ~ 180 &amp; mgr; l ist erforderlich.) Cap, in Alufolie wickeln (um Licht zu blockieren) und mischen Sie die Lösung mit hoher Geschwindigkeit auf einem Vortex-Mischerfür ca. 1 min. (Hinweis: Wenn in fortgeschrittenen vorbereitet, sollte die Lösung im Kühlschrank aufbewahren und vor Licht geschützt werden.) </li><li> Wickeln Sie das Fläschchen mit dem Makromerlösung (ohne Cap) mit Aluminiumfolie (um Licht zu blockieren) und fügen über eine Pipette 15 Vol% (bezogen auf das Gesamtvolumen des Makromerlösung) des Photoinitiatorlösung. Cap und mischen Sie die Lösung mit hoher Geschwindigkeit auf einem Vortex-Mischer für ca. 1 min. </li><li> Wickeln Sie das Fläschchen mit dem geschmolzenen Salz-Vorlage (ohne Cap) mit Aluminiumfolie (um Licht zu blockieren) und fügen über eine Pipette die zuvor hergestellte Makromer / Photoinitiatorlösung (~ 0,6 ml oder bis die Vorlage vollständig bedeckt ist). </li><li> Verschließe das Röhrchen, wickeln Sie sie in Gewebe und legen vertikal in ein Zentrifugenröhrchen. Zentrifugieren für 10 min bei 1.260 · g, um die Makromerlösung ganzen Schablone zu verteilen. </li><li> Entfernen Sie die Aluminiumfolie, uncap das Fläschchen und setzen mit UV-Licht (365 nm, 25 W) für 3 min. Luft trocknen O / N. </li><li> Entfernen Sie die &quot;salzhaltigen scaffold &quot;aus dem Fläschchen mit einer Pinzette nach Scoring und Brechen der Oberseite der Glasfläschchen. </li><li> In einem 400 ml Becherglas, herzustellen ~ 200 ml eines Wasser / Ethanol-Lösungsmittel (1: 1 vol: vol). </li><li> Pflegen Sie das Gerüst in der Wasser / Ethanol-Lösungsmittel für 4 Tage mit täglichen Änderungen Lösungsmittel getaucht. </li><li> Nehmen Sie das Gerüst von dem Lösungsmittel und der Luft trocknen O / N. </li></ol> 3. Anwenden Polydopamine Coating auf SMP-Gerüst (Abbildung 1) <ol><li> In 400 ml Becherglas mit einem Teflon bedeckten Rührstab, bereiten ~ 200 ml eines Dopamin-Hydrochloridlösung (2 mg / ml in 10 mM Tris-Puffer, pH = 8,5, 25 ºC). Rühre bei ~ 150 Upm. </li><li> Legen Sie eine Einweg-Injektionsnadel (Länge = 40 mm; Gauge = 20) in das Gerüst, ~ die Hälfte der Strecke durch das Schafott. Wickeln Sie ein Draht um den Nadelansatz. </li><li> Unterzutauchen das Gerüst (mit der Nadelnabe über der Oberfläche der Lösung) in die gerührte Lösung Dopamin durch Verankern des Drahtes auf dem Rand des Bechers. </li><li> Entgasen Gerüst indem eine Spritze in das Nadelverbindungsstück und dessen Verwendung, um Luft aus dem Gerüst zu ziehen. (Anmerkung: Die Entgasung ist abgeschlossen, wenn keine Luft mehr entfernt werden kann, und die Lösung wurde komplett das Gerüst infiltriert.) </li><li> Pflegen Sie das Gerüst in der Dopamin-Lösung unter Rühren für 16 Stunden unter Wasser. </li><li> Nehmen Sie das Gerüst aus der Lösung und entfernen Sie die Nadel. Spülen mit DI-Wasser und trocken in einem Vakuumofen bei Raumtemperatur für 24 Stunden. </li><li> Legen Sie das Gerüst in einem 85 ºC Ofen für 1 Stunde. </li><li> Ermöglichen das Gerüst auf RT abkühlen. Die endgültige zylindrische Gerüst wird ~ 6 mm Durchmesser x ~ 5 mm hoch sein. </li></ol> 4. Auswerten &quot;Self-Montage&quot; Verhalten <ol><li> Bereiten Sie eine &quot;irreguläre CMF Defektmodell&quot; mit einem Blatt aus Hartplastik, deren Dicke ca. 5 mm. Mit einem Bohrer, um einen Hohlraum in dem Kunststoffblatt mit einem mittleren Durchmesser von etwas weniger als ca. 6 mm zu erzeugen, wie in 2A gezeigt. </li><li> In abEaker Wärme DI-Wasser (die den klinischen Einsatz von Kochsalzlösung) auf eine Temperatur von ca. 60 ºC. </li><li> Legen Sie das Gerüst in das Becherglas von ~ 60 ° C Wasser. Verwenden einer Pinzette, um das Gerüst unter der Wasseroberfläche zu drücken, Belichten alle Bereiche zum Wasser. Fahren Sie für ca. 2 Min oder bis das Gerüst spürbar gemildert (2B). </li><li> Nehmen Sie das Gerüst aus dem Becherglas und drücken Sie sofort (von Hand) in das Modell Defekt. </li><li> Es wird auf RT (~ 5-10 min) (2C) zu kühlen. </li><li> Von dem Defekt zu entfernen, um den neuen, temporären Form fixiert und die Rückkehr der relativ starren Zustand (2D) zu beobachten. </li></ol> 5. Testen Formgedächtnisverhalten <ol><li> Verwendung eines dynamisch-mechanischen Analysators (DMA, zB einem TA Instruments Q800 wie hier verwendet), und führen eine Spannung-gesteuerte zyklische thermischer mechanischer Kompressionstest auf einem Gerüst über zwei Zyklen (N), die Form fixity (R f) zu bestimmen, undFormwiederherstellung (R r) (Abbildung 3). <ol><li> Äquilibrieren bis 60 ° C (T high) für 5 min. </li><li> Zu komprimieren, um eine maximale Dehnung (ε m = 50%) bei 50% / min. </li><li> Halten Sie bei ε m (5 min). </li><li> Kühlen auf 25 ° C (T low) und für 10 Minuten beibehalten, um die temporäre Form zu fixieren. </li><li> Entfernen Sie die Last. </li><li> Messen Sie die Bruchdehnung im spannungsfreien Zustand (ε u). </li><li> Aufwärmen auf 60 ° C (T hoch) und für 10 Minuten beibehalten, um die permanente Form zurückzugewinnen. </li><li> Messen Sie die wiederhergestellte Dehnung (ε p). </li><li> Während noch bei 60 ° C (T high), starten Sie den 2. Zyklus (N = 2) durch Zusammendrücken des Gerüsts zu 50% der Höhe nach dem 1. Zyklus gewonnen (N = 1). </li><li> Wiederholen 5.1.3-5.1.8 Für N = 2. </li><li> Berechnen R f und R r für N = 1 und 2 unter Verwendung der folgenden Gleichungen: R f (N) = [ε u (n) / ε m] r R (N) = [ε m - ε p (N)] / [ε m - ε p (N -1)] </li></ol></li></ol> 6. Visualisierung Porengröße und Interkonnektivität <ol><li> Mit Hilfe der Rasterelektronenmikroskopie (SEM; zB eine FEI Quanta SEM, wie hier verwendet), beobachten die Porengröße und Interkonnektivität. <ol><li> Mit einer Pinzette die SMP-Gerüst zu halten, tauchen in flüssigem N 2 für 1 min. </li><li> Von flüssigem N 2 und Bruch entlang der Mitte des Gerüsts mit einer Rasierklinge zu entfernen. </li><li> Verwendung von Kohlenstoffband, befestigen eine der SMP Gerüsthälften auf die ProbeStufe mit dem Bruchfläche nach oben zeigt. </li><li> Sputter-Mantel mit Au-Pt (~ 4 nm). </li><li> Erfassen die REM-Aufnahme bei einer empfohlenen Beschleunigungsspannung von 10-15 kV (4A). </li></ol></li></ol> 7. Prüfung von in-vitro-Bioaktivität <ol><li> In ein 50 ml Zentrifugenröhrchen, fügen ~ 30 ml 1X SBF 24. </li><li> Besorgen Sie sich ein Gerüst in seiner ursprünglichen, zylindrisch geformten permanenten Form. Schneiden Sie das Gerüst in die Hälfte (über den kreisförmigen Rand) mit einem sauberen Klinge. </li><li> Legen Sie eine einzelne Gerüsthälfte in die vorbereitete Zentrifugenröhrchen und Kappe. </li><li> Aufrechterhaltung des Rohrs bei 37 ° C in einem Wasserbad unter statischen Bedingungen ohne SBF ändert. </li><li> Nach 14 Tagen, entfernen Sie das Gerüst aus dem SBF und der Luft trocknen für 24 Stunden. </li><li> Verwendung von Kohlenstoffband, bringen Sie das Gerüst auf den Probentisch mit der Bruchfläche nach oben zeigt. </li><li> Sputter-Mantel mit Au-Pt (~ 4 nm). </li><li> Nehmen Sie das SEM-Bild bei einer Wiedergelobt Beschleunigungsspannung von 10-15 kV (4B). </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+fusion:+an+approach+to+improve+pore+interconnectivity+within+tissue+engineering+scaffolds.">Salt fusion: an approach to improve pore interconnectivity within tissue engineering scaffolds.</a> Tissue Eng. 8, 43-52 (2002).</li><li>Yang, Q., Chen, L., Shen, X., Tan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+polycaprolactone+tissue+engineering+scaffolds+by+improved+solvent+casting/particulate+leaching+method.">Preparation of polycaprolactone tissue engineering scaffolds by improved solvent casting/particulate leaching method.</a> J Macromol Sci Phys. 45, 1171-1181 (2006).</li><li>Madbouly, S. A., Kratz, K., Klein, F., L&#252;zow, K., Lendlein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermomechanical+behaviour+of+biodegradable+shape-memory+polymer+foams.">Thermomechanical behaviour of biodegradable shape-memory polymer foams.</a> 1190, (2009).</li><li>Luo, X., Mather, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+shape+memory+elastomeric+composites.">Preparation and characterization of shape memory elastomeric composites.</a> Macromolecules. 42, 7251-7253 (2009).</li><li>Lendlein, A., Schmidt, A. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AB-polymer+networks+based+on+oligo(&#603;-caprolactone)+segments+showing+shape-memory+properties.">AB-polymer networks based on oligo(&#603;-caprolactone) segments showing shape-memory properties.</a> Proc Natl Acad Sci. 98, 842-847 (2001).</li></ol>

The authors have nothing to disclose.

Dieses Protokoll beschreibt die Herstellung eines polydopamine beschichtet, PCL basierende Gerüst deren Selbstschlüssige Verhalten sowie Osteoinduktivität und Bioaktivität, macht es von Interesse bei der Behandlung von irregulären CMF Knochendefekten. Aspekte des Protokolls kann verändert werden, um verschiedene Gerüst Merkmale ändern. Das Protokoll beginnt mit Acrylierung eines PCL-diol zur UV-Härtung zu ermöglichen. In der dargestellten Beispiel ist das PCL-diol M n ~ ...

Zur Zeit als Goldstandard der Kiefer- und Gesichts (CMF) Knochendefekt-Behandlungen, ist die Transplantation von autologen Transplantaten geerntet durch komplexe Transplantation Verfahren, Entnahmemorbidität und der begrenzten Verfügbarkeit 1 behindert. Eine besondere Schwierigkeit ist die Gestaltung und Befestigung des starren Autotransplantat fest in den Defekt, um die Osseointegration zu erhalten und Transplantat Resorption verhindern. Tissue Engineering ist als alternative Strategie zur Autotransplantation und synthetischen Knochenersatzmaterialien (zB Knochenzement) 2,3 untersucht. Entscheidend für den Erfolg einer Tissue-Engineering-Ansatz ist ein Gerüst mit einem bestimmten Satz von Eigenschaften. Erstens, um die Osseointegration zu erreichen, muss das Gerüst engem Kontakt mit angrenzenden Knochengewebe 4 zu bilden. Das Gerüst sollte auch osteokonduktive werden, wodurch die Zellmigration, Nährstoffdiffusion und neotissue Abscheidung 4,5. Dieses Verhalten ist in der Regel mit biologisch abbaubaren sca erreichtffolds die eine hochgradig vernetzten Porenmorphologie. Schließlich sollte das Gerüst bioaktiven sein, um die Integration und Bindung mit umgebenden Knochengewebe 5 zu fördern. Hier präsentieren wir ein Protokoll, um eine Gewebetechnik Gerüst mit diesen Eigenschaften herzustellen. Wichtig ist, weist dieses Gerüst die Fähigkeit, &quot;self-fit&quot; in unregelmäßige CMF Defekte aufgrund seiner Formgedächtnisverhalten 6. Thermoresponsive Formgedächtnispolymere (SMP) sind dafür bekannt, Formänderung bei Belichtung unterziehen, um zu heizen 7,8. SMPs sind von &quot;Netzpunkte&quot; (dh chemische oder physikalische Vernetzungen), die die permanente Form und &quot;Schaltsegmente&quot;, die die temporäre Form zu erhalten und wiederherzustellen die permanente Form festzustellen, besteht. Die Schaltsegmente weisen eine Wärmeübergangstemperatur (T trans) entspricht, entweder die Glasübergangs (Tg) oder Schmelzübergang (Tm) des Polymers. AlsFolglich können SMPs nacheinander in eine temporäre Form bei T&gt; T trans, in der temporären Form bei T &lt;T trans fixiert verformt werden und wiedergewonnen, um die permanente Form bei T&gt; T trans. Somit wird ein SMP-Gerüst &quot;Selbst-Montage&quot; in einem CMF Defekt erreichen könnte wie folgt 6. Nach der Belichtung mit warmer Kochsalzlösung (T&gt; T trans), wäre ein SMP-Gerüst werden zu formbaren, wodurch eine allgemein vorbereiteten zylindrischen Gerüst Hand gepressten in einem unregelmäßigen defekt sein, mit Formrück Förderung Expansion des Gerüsts des Mangels Grenze. Beim Abkühlen (T &lt;T trans), würde das Gerüst seiner relativ starrer Zustand zurück, mit Formstarrheit seiner neuen temporären Form innerhalb des Defekts erhalten bleibt. In diesem Protokoll wird ein SMP-Gerüst von Polycaprolacton (PCL) hergestellt, ein biologisch abbaubares Polymer intensiv untersucht für die Geweberegeneration und andere biomedizinische Anwendungen 9-11. Für Formgedächtnis, the T m von PCL dient als die T trans und variiert zwischen 43 und 60 ºC, abhängig vom Molekulargewicht des PCL 12. In diesem Protokoll wird die T trans (dh T m) des Gerüstes beträgt 56,6 ± 0,3 ºC 6. Um Osteokonduktivität zu erreichen, wurde ein Protokoll entwickelt, um PCL-basierten SMP Gerüste mit stark miteinander verbundenen Poren auf der Basis eines Lösungsmittelgießen Partikel-Laugung (SCPL) Methode 6,13,14 zu machen. Polycaprolacton-diacrylat (PCL-DA) (M n = ~ 10.000 g / mol) wurde verwendet, um eine schnelle, photochemischen Vernetzung zu ermöglichen, und wurde in Dichlormethan (DCM) gelöst, um Lösungsmittel-Gießen über der Salzvorlage ermöglichen. Nach der photochemischen Härtung und Lösungsmittelverdampfung wurde das Salz Vorlage durch Auslaugen in Wasser entfernt. Die durchschnittliche Salzgröße regelt Gerüstporengröße. Wichtig ist, das Salz-Vorlage mit Wasser verschmolzen wurde vor der Lösungsmittel-Gießen zur Poren interconnectivi erreichenty. Bioaktivität wurde auf Porenwänden 6 zu der SMP-Gerüst durch die in situ-Bildung eines polydopamine Beschichtung verliehen wird. Bioaktivität wird häufig durch Einschluß von Glas oder Glaskeramik-Füllstoffe 15 in Gerüste eingebracht. Doch diese können zu unerwünschten spröden mechanischen Eigenschaften zu geben. Dopamin ist gezeigt worden, um eine haftende, dünne polydopamine Schicht auf einer Vielzahl von Substraten 16-19 bilden. In diesem Protokoll wurde die SMP Gerüst einer leicht basischen Lösung (pH = 8,5) von Dopamin zogen, um eine Beschichtung von polydopamine nanothick auf allen Porenwandoberflächen 6 bilden. Zusätzlich zur Verbesserung Oberflächenhydrophilie für verbesserte Zelladhäsion und Ausbreitung hat polydopamine gezeigt worden bioaktiven in Bezug auf die Bildung von Hydroxyapatit (HAp) bei Belichtung mit simulierter Körperflüssigkeit (SBF) 18,20,21 sind. In einem letzten Schritt wird das beschichtete Gerüst ausgesetzt Behandlung bei 85 ºC (T&gt; T trans) wh erhitzenich führt zu Verdichtung Schafott. Wärmebehandlung wurde bereits erwähnt die für Gerüstformgedächtnisverhalten zu sein, möglicherweise aufgrund PCL kristallinen Domänen Reorganisation um eine größere Nähe 14. Wir beschreiben außerdem die Methoden, um die Selbstsitz Verhalten innerhalb einer unregelmäßigen Modell Defekt zu charakterisieren, Formgedächtnisverhalten in Bezug auf Belastung gesteuerte zyklische thermomechanische Druckversuche (dh Wiederherstellung der Form und Gestalt Festigkeit), Porenmorphologie und In-vitro-Bioaktivität. Strategien zur Gerüsteigenschaften maßzuschneidern werden ebenfalls vorgestellt.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polycaprolactone-diol (Mn ~ 10,000 g/mol)</td>
			<td>Sigma-Aldrich</td>
			<td>440752</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>D65100</td>
			<td>Dried over 4A molecular sieves</td>
		</tr>
		<tr>
			<td>4-dimethylaminopyridine (DMAP)</td>
			<td>Sigma-Aldrich</td>
			<td>D5640</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine (Et3N)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryloyl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A24109</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Carbonate (K2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>209619</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous magnesium sulfate (MgSO4)</td>
			<td>Fisher</td>
			<td>M65</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2-dimethoxy-2-phenyl acetophenone (DMP)</td>
			<td>Sigma-Aldrich</td>
			<td>196118</td>
			<td></td>
		</tr>
		<tr>
			<td>1-vinyl-2-pyrrolidinone (NVP)</td>
			<td>Sigma-Aldrich</td>
			<td>V3409</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine Hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H8502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer (2mol/L)</td>
			<td>Fisher</td>
			<td>BP1759</td>
			<td>Used at 10 mM concentration, pH = 8.5</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>VWR</td>
			<td>47729-972</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Transilluminator (365 nm, 25 W)</td>
			<td>UVP</td>
			<td>95-0426-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic Mechanical Analyzer (DMA)</td>
			<td>TA Instruments</td>
			<td>Q800</td>
			<td></td>
		</tr>
		<tr>
			<td>High Resolution Sputter Coater</td>
			<td>Cressington</td>
			<td>208HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope (SEM)</td>
			<td>FEI</td>
			<td>Quanta 600</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a bioactive, pcl-based "self-fitting" shape memory polymer scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Die resultierende PCL basierende MMP-Gerüst ist in der Lage selbst passend in einem Modell CMF Defekt (Abbildung 2). Nach kurzer Einwirkung von Salzlösung (~ 60 ° C) erwärmen, erweicht die zylindrische Gerüst ermöglicht das Gerüst manuell in gedrückt werden und innerhalb des Modells Defekt zu erweitern. Nach dem Abkühlen auf RT wird das Gerüst in die neue temporäre Form, die nach der Entnahme aus dem Defekt zurückgehalten wird fixiert. Die Formgedächtnisverhalte...

Watch this Scientific Journal Video about Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold at JoVE.com

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Herstellung eines bioaktiven, PCL-basierte &quot;Self-Montage&quot; Formgedächtnispolymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

fabrication-bioactive-pcl-based-self-fitting-shape-memory-polymer

Research

JoVE Journal

Bioengineering

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

12.5K Aufrufe.  Texas A&M University. Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Serie: Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Formgedächtnis-Polymere für Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Forschung

Biotechnik

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive &quot;SurgiLux &#39;: Vorbereitung und Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Umweltfreundliche kontrollierten Microtensile Testing von mechanisch-adaptive Polymer-Nanokomposite für<em&gt; Ex vivo</em&gt; Charakterisierung

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Mikrofluidische Herstellung von Polymer- und Biohybridfasern mit vorgefertigter Größe und Form

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

Herstellung von robusten Naturfaser-Preforms Verwendung Bakteriencellulose als Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Herstellung von mechanisch Tunable und Bioactive Metallgerüste für biomedizinische Anwendungen

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Die Herstellung von Inverted Kolloid-Kristallen Poly (Ethylenglycol) Scaffold: Eine dreidimensionale Zellkultur-Plattform für Leber-Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Herstellung und Charakterisierung von optischen Gewebe Phantome, die Makrostruktur enthalten

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Mikrodissektion primäre renale Gewebe Segmente und Einbindung mit neuartigen Gerüst-freie Konstrukt-Technologie

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Skalierbare Fertigung von dehnbar, Dual Channel, mikrofluidischen Orgel Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...