Die Autoren bedanken sich für die Unterstützung aus New Innovator NIH Director Awards (1DP2 OD006499-01 bis AA und 1DP2 OD006460-01 bis AJE) und King Abdulaziz City for Science and Technology (UC San Diego Center of Excellence in Nanomedizin). Wir möchten Ms. Jessica Moore für ihre kritischen Anmerkungen zum Manuskript danken.

Ein. Synthese von fluoreszenzmarkierten Acryloyl-PEG-RGDS <ol><li> Reagieren des RGDS-Peptids mit Acryloyl-PEG-Succinimidyl carboxymethylester (APEG-SCM, PEG MG: 3400 g / mol) und NN Diisopropylethylamin (DIPEA) in 1.2:1:2 Molverhältnisse in Dimethylsulfoxid (DMSO) unter Argon bei Raumtemperatur Übernachtung. </li><li> Bestätigen Konjugation durch Matrix-Assisted Laser Desorption / Ionisation Time of Flight (MALDI-TOF) Massenspektrometrie. Hinzufügen 1 ul APEG-RGDS Reaktionslösung zu einer Probe Fleck auf das MALDI-Target und trocken. Bereiten Sie eine gesättigte Lösung von Universal MALDI Matrix in Tetrahydrofuran (THF) und Vortex für 1 min. Fügen Sie ~ 1 ul dieser Lösung auf die gleiche Probe vor Ort. Wiederholen Sie den Vorgang für APEG-SCM für den Vergleich. Laden und zu analysieren. Das Molekulargewicht APEG-RGDS sollte größer sein als APEG-SCM (Abbildung 2). </li><li> Um das Fluorophor konjugiert, fügen eine äquimolare Menge von Alexa Fluor 350 carbonsäure (succinimydyl Ester), gelöstin einem minimalen Volumen von DMSO, die RGDS-APEG Reaktionslösung von 1,1 und reagieren unter Argon bei Raumtemperatur über Nacht. </li><li> Läutern APEG-RGDS-350 durch Dialysieren (MW 3500 Da) gegen DI-H 2 O bei 4 ° C für 48 h bei 1.000:1 Volumenverhältnis, Austauschen Dialysat wenigstens zweimal pro Tag. </li><li> Freeze Dry das gereinigte APEG-RGDS-350 in einer Labconco Freezone Plus oder gleichwertige Freeze Dry-System und bei -20 ° C. </li></ol> 2. Herstellung eines 2D-Mold und Anfertigung einer 2D PEG Gel mit alternierenden Schichten RGDS-350 <ol><li> Bereiten hydrophoben Glasträger. Zeigen sauberes Glas gleitet in einer Glasschale in einem Vakuumofen. Wärme auf 80 ° C für 30 min vollständig trocknen die Flächen. Platz Teller mit Folien in einer Abzugshaube und 250 ul Sigmacote zu jeder Folie, sanft schaukelnden für 30 sec zu beschichten gesamte Fläche. Gründlich die beschichteten Objektträger mit 100% Methanol, gefolgt von Waschen in destilliertem Wasser, Einweichen zweimal für 5 min in bei Leasingt 10 ml. </li><li> Cut Silikon-Abstandshalter (0,8 mm dick) mit 10 mm Biopsieausstanzungen. </li><li> Autoklaven werden die Silikon-Abstandshalter und Sigmacote behandelten Objektträger. </li><li> Formulierung von Lösungen für jedes jeweilige Schicht in einzelnen Mikrozentrifugenröhrchen durch Mischen PEG-diacrylat (PEGDA) Vorstufe (Endkonzentration 15% w / v) mit unterschiedlichen Mengen von Iodixanol (60% ige Stammlösung in Wasser) in unterschiedlichem Endkonzentrationen (zB 40% zu ergeben, 30%, 20% und 10%) zur Ergänzung der restliche Volumen mit Phosphat-gepufferter Salzlösung (PBS), um Lösungen von abgestuften Dichten zu erhalten. In einer ähnlichen Weise, zum alternierenden Schichten, um Mischung APEG-RGDS-350 (8 mM Endkonzentration) mit Iodixanol und PBS zu ergeben verschiedenen Konzentrationen (z. B. 35%, 25% und 15%). </li><li> Hinzufügen Fotoinitiator (2-Hydroxy-4&#39;-(2-hydroxyethoxy)-2-methylpropiophenon, 333mg/ml stock in N-vinylpyrrolidon) zu jeder Lösung für verschiedene Schichten (10 ul Stammlösung pro ml jeder Schicht Lösung). Photoinitiator is zuletzt zugegeben, um die Polymerisation vor Schichtung der Gele in der Form zu verhindern, da sie lichtempfindlich ist. </li><li> Nachfolgende Schritte dieses Protokoll wird in einer biologischen Sicherheitswerkbank durchgeführt werden, um die Sterilität zu gewährleisten. </li><li> Filtern sterilisieren jede Lösung mit einer sterilen 1-ml-Spritze und 0,2 um Filter. Montieren Sie den Werkzeug-Setups durch Anordnen der Abstandhalter zwischen zwei Sigmacore behandelten Glasplatten und mit Zwingen befestigen Platzierung wie in Abbildung 1 dargestellt. </li><li> Warf die geschichteten Gele, indem das dichteste Lösung (zB PEGDA mit 40% Iodixanol) zunächst durch eine weniger dichte Lösung (zB APEG-RGDS-350 mit 35% Iodixanol) gefolgt. Wiederholen des alternativen Schichtung mehrere Schichten von gewünschten Zusammensetzung und Dichten zu erzielen, wie in Abbildung 1 gezeigt. </li><li> Bestrahlen der Form mit 365 nm Licht für 3 min unter Verwendung eines tragbaren UVR-9000 Lampe. Lassen Sie die polymerisierte Gele für 5 min zu heilen. Entfernen Sie die Klammern, dann heben Sie die obere GlasRutsche und die Form, die geschichtete DGMP Gele werden auf den Folien bleiben. Mit einem sterilen Spatel, vorsichtig die Gele in einem 50 ml Röhrchen mit sterilem PBS oder Kulturmedium für Wäsche. </li><li> Waschen der polymerisierten Gele in PBS bei 1.000:1 Volumenverhältnis, Austauschen Puffer mindestens zweimal pro Tag das Dichtemodifikationsmittel, Photoinitiator und nicht umgesetztem Polymer zu entfernen. Alternativ kann PBS mit Zellwachstumsmedium ausgetauscht werden. Speichern der DGMP Gele in PBS oder Wachstumsmedium für die Zellkultur Experiment in Schritt 3 beschrieben. </li><li> Um abwechselnden Schichten visualisieren, zu veranlassen DGMP Gel (diese Gele nicht verwendbar für die Zellkultur) entlang einer Lineal auf der Probenschale eines VersaDoc Geldokumentation Einheit. Setzen der Gele in 350 nm; die Belichtungszeit variiert in Abhängigkeit von der Konzentration des Fluorophors. Abwechselnd dunklen und blauen Streifen im DGMP Hydrogel demonstrieren Bildung von diskreten Schichten aus verschiedenen chemischen Zusammensetzung (Abbildung 3). </li></ol><ol><li> Für Gele Einbeziehung RGDS-Peptid, verwenden Adhäsion abhängigen Zellen, wie C2C12 Myoblasten. </li><li> Schieben Sie DGMP Gele (gespeichert in PBS) in die Vertiefungen der 48-Well-Zellkultur-Platten mit einer sterilen Zellschaber in einer biologischen Sicherheitswerkbank. </li><li> Vorwärmen Wachstumsmedium (Dulbeccos modifiziertes Eagle-Medium oder DMEM, ergänzt mit 10% v / v fötalem Rinderserum und 1% v / v 100x Penicillin-Streptomycin-Lösung) und PBS in ein Wasserbad gesetzt bei 37 ° C </li><li> Waschen mit 60% konfluenten Platte (10 mm) von C2C12 Zellen dreimal mit PBS. Absaugen PBS und Ernte Zellen durch Zugabe von 1 ml 0,25% Trypsin-EDTA und Inkubation bei 37 ° C für 2 min. Die Zellen in Wachstumsmedium und Anzahl Zellen. Samen der DGMP Gel enthaltenden Zellkultur gut mit C2C12 Myoblasten (20.000 Zellen / cm 2). Inkubieren der Zellen bei 37 ° C in 5% CO 2/95% relative Luftfeuchtigkeit. Gently Tauschmittel nach 4 h, aufpassen, nicht wiederBewegen leicht anhaftenden Zellen. </li><li> Nach 24 Stunden kann die Anbringung von C2C12 Myoblasten auf den RGDS-haltigen Schichten DGMP Gele durch Epifluoreszenz und Phasenkontrast-Mikroskopie (Zeiss Axiovert 200) bestätigt werden. </li></ol>

<ol>
	<li>Place, E. S., Evans, N. D., Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+in+biomaterials+for+tissue+engineering.">Complexity in biomaterials for tissue engineering.</a> Nat. Mater. 8, 457-470 (2009).</li><li>Liu, V. A., Jastromb, W. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+protein+and+cell+adhesivity+using+PEO-terminated+triblock+polymers.">Engineering protein and cell adhesivity using PEO-terminated triblock polymers.</a> J. Biomed. Mater. Res. 60, 126-134 (2002).</li><li>Sharma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+zonal+organization+into+tissue-engineered+cartilage.">Designing zonal organization into tissue-engineered cartilage.</a> Tissue Eng. 13, 405-414 (2007).</li><li>Hahn, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographic+patterning+of+polyethylene+glycol+hydrogels.">Photolithographic patterning of polyethylene glycol hydrogels.</a> Biomaterials. 27, 2519-2524 (2006).</li><li>Kizilel, S., Sawardecker, E., Teymour, F., Perez-Luna, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+formation+of+covalently+bonded+hydrogel+multilayers+through+surface+initiated+photopolymerization.">Sequential formation of covalently bonded hydrogel multilayers through surface initiated photopolymerization.</a> Biomaterials. 27, 1209-1215 (2006).</li><li>Harley, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+multiphase+osteochondral+scaffold.+II.+Fabrication+of+a+mineralized+collagen-glycosaminoglycan+scaffold.">Design of a multiphase osteochondral scaffold. II. Fabrication of a mineralized collagen-glycosaminoglycan scaffold.</a> J. Biomed. Mater. Res. A. 92, 1066-1077 (2010).</li><li>Cuchiara, M. P., Allen, A. C., Chen, T. M., Miller, J. S., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+microfluidic+PEGDA+hydrogels.">Multilayer microfluidic PEGDA hydrogels.</a> Biomaterials. 31, 5491-5497 (2010).</li><li>Roam, J. L., Xu, H., Nguyen, P. K., Elbert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+protein+concentration+gradients+mediated+by+density+differences+of+poly(ethylene+glycol)+microspheres.">The formation of protein concentration gradients mediated by density differences of poly(ethylene glycol) microspheres.</a> Biomaterials. 31, 8642-8650 (2010).</li><li>Gleghorn, J. P., Lee, C. S., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+properties+of+laminated+alginate+gels+for+tissue+engineering+of+layered+structures.">Adhesive properties of laminated alginate gels for tissue engineering of layered structures.</a> J. Biomed. Mater. Res. A. 85, 611-618 (2008).</li><li>Karpiak, J. V., Ner, Y., Almutairi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+gradient+multilayer+polymerization+for+creating+complex+tissue.">Density gradient multilayer polymerization for creating complex tissue.</a> Adv. Mater. 24, 1466-1470 (2012).</li><li>Pierschbacher, M. D., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+attachment+activity+of+fibronectin+can+be+duplicated+by+small+synthetic+fragments+of+the+molecule.">Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule.</a> Nature. 309, 30-33 (1984).</li><li>Kaushik, G., Fuhrmann, A., Cammarato, A., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Situ+Mechanical+Analysis+of+Myofibrillar+Perturbation+and+Aging+on+Soft,+Bilayered+Drosophila+Myocardium."> In Situ Mechanical Analysis of Myofibrillar Perturbation and Aging on Soft, Bilayered Drosophila Myocardium.</a> Biophysical Journal. 101, 2629-2637 (2011).</li></ol>

Die Autoren haben keine widerstreitenden Interessen offen zu legen.

DGMP ist eine einfache Strategie zur Vorbereitung vielschichtigen Gele, die nicht auf teure Instrumente angewiesen ist. Dieses Protokoll kann zum Erstellen von Gerüsten mit anderen biokompatiblen Materialien, wie beispielsweise Collagen und Hyaluronsäure angepasst werden. Bioaktiven kleinen Molekülen, zum Beispiel Zell haftvermittelnde RGDS-Peptid, können an die Polymermatrix angebunden werden zur Verhinderung der Vermischung von Stimmungen zwischen den Schichten. Proteine ​​können in getrennten Schichten ohne chemische Konjugation eingekapselt werden, da sie je nach Matrix Maschengröße, weniger anfällig für durch Hydrogele 10 diffundieren können. Hier verwendet Iodixanol (Nycoprep), eine inerte Dichtemodifikationsmittel, die zuvor für lebensfähige Zelle Anwendungen verwendet worden. Andere Dichtemodifizierer wie Saccharose und Dextrose können ebenfalls verwendet werden. Durch Variation der Einschwingzeit (t s), kann man die Feinabstimmung der Schnittstellen zwischen zwei Schichten auf glatten oder scharfe Übergänge zu erzeugen, wie erforderlich ist (längere Einschwingzeit gibt weichere Übergänge) &lt;sup&gt; 10. Zum Beispiel könnte weichere Übergänge zwischen DGMP Gelschichten verwendet, um einen kontinuierlichen Gradienten eines biologischen Cue zu Zelle Prozesse wie Chemotaxis studieren zu erzeugen. Die Wirkung des Gels auf Dichtemodifikationsmittel Steifigkeit ist in 5 für einen 15%-Gel aPEGda gezeigt; eine vollständige Charakterisierung der Steifigkeit und Porosität als Funktion der PEGDA und Iodixanol Konzentrationen wird derzeit untersucht. Während die PEGDA Konzentration in diesem Beispiel ist relativ hoch, beobachteten wir eine 60% größer Elastizitätsmodul in Gelen mit 30% im Vergleich zu Iodixanol Gelen ohne. Die Veränderung der Steifigkeit Gel kann durch Modulation der Konzentration oder Makromer Vernetzungsdichte einstellbar. Wir haben auch die DGMP Technik 3D vielschichtigen Gele mit Polyacrylamid und PEG-Vorstufen 10 zu schaffen angewendet. Variieren der Konzentration oder der Grad der Vernetzung des Prepolymers ermöglicht strukturellen Variation in derGerüste, die zur Zell-Verhalten wie polarisierten Wachstum und Migration in 3D erforschen kann. Zusammenfassend ist DGMP eine anpassungsfähige Technik, die angewendet 2D-und 3D-Gerüste aus einer Vielzahl von biokompatiblen Werkstoffen für eine Vielzahl von biomedizinischen Anwendungen und Grundlagenforschung fabrizieren lassen.

<table border="1"><tbody><tr><td> Reagenz oder Instrument </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Polyethylenglykol succinimydyl carboxymethyl (a-PEG-SCM) </td><td> Laysan </td><td> 120-64 </td></tr><tr><td> Polyethelyene Glykoldiacrylat (PEGDA) </td><td> Dajac Labs </td><td> 9359 </td></tr><tr><td> Arginin-Glycin-Asparaginsäure-Serin (RGDS) </td><td> American Peptide </td><td> 49-01-4 </td></tr><tr><td> N, N - Diisopropylethylamin (DIPEA) </td><td> Sigma </td><td> D125806 </td></tr><tr><td> Dimethylsulfoxid (DMSO) </td><td> Sigma </td><td> D2438 </td></tr><tr><td> N, N-Dimethylformamid (DMF) </td><td> Fischer </td><td> D119-4 </td></tr><tr><td> Tetrahydrofuran (THF) </td><td> Fischer </td><td> T397 </td></tr><tr><td> Dialysekassette (3500Da) </td><td> Thermo Scientific </td><td> 66330 </td></tr><tr><td> Alexa Fluor 350 Carbonsäure succinimydyl Ester </td><td> Life Technologies </td><td> A-10168 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> Silikon Abstandshalter </td><td> Grainger </td><td> 1MWA4 </td></tr><tr><td> Biopsieausstanzungen </td><td> Acuderm </td><td> P1025 (10 mm) P850 (8 mm) </td></tr><tr><td> Dulbeccos phosphatgepufferter Salzlösung (DPBS) </td><td> Hyclone </td><td> SH30028 </td></tr><tr><td> Iodixanol (NycoPrep) </td><td> Fischer </td><td> NC9388846 </td></tr><tr><td> 2-Hydroxy-4&#39;-(2-hydroxyethoxy)-2-methylpropiophenon </td><td> Sigma </td><td> 410896 </td></tr><tr><td> Dulbecco modifiziertem Eagle-Medium (DMEM) </td><td> Life Technologies </td><td> 11054 </td></tr><tr><td> Fötales Rinderserum </td><td> Life Technologies </td><td> 10082 </td></tr><tr><td> Penicillin-Streptomycin </td><td> Life Technologies </td><td> 15140 </td></tr><tr><td> C2C12 Myoblasten </td><td> ATCC </td><td> CRL-1772 </td></tr><tr><td> MALDI </td><td> Bruker </td><td> N / A </td></tr><tr><td> UVR-9000 </td><td> Bayco </td><td> UVR-9000 </td></tr><tr><td> VersaDoc </td><td> Bio-Rad </td><td> N / A </td></tr></tbody></table>

density gradient multilayered polymerization (dgmp): a novel technique for creating multi-compartment, customizable scaffolds for tissue engineering

Komplex Gewebekultur Matrizen, in denen Arten und Konzentrationen der biologische Reize (zB Wachstumsfaktoren, Inhibitoren, oder kleinen Molekülen) oder Matrix-Struktur (zB Zusammensetzung, Konzentration oder Steifigkeit der Matrix) variieren über Raum, würde es ermöglichen, eine Vielzahl von Untersuchungen darüber, wie diese Variablen Zelldifferenzierung, Migration und andere Phänomene beeinflussen. Die größte Herausforderung beim Erstellen geschichteten Matrices ist die Aufrechterhaltung der strukturellen Integrität der Schicht Schnittstellen ohne Diffusion einzelner Komponenten aus jeder Schicht ein. Aktuelle Methoden, dies zu erreichen, zählen Photostrukturierung 2-3, Lithographie 4, sequentielle functionalization5, Gefriertrocknung 6, Mikrofluidik 7 oder Zentrifugation 8, von denen viele erfordern anspruchsvolle Mess-und technischen Fähigkeiten. Andere verlassen sich auf sequentielle Befestigung der einzelnen Schichten, die kann dazu führen, Schichten 9 Delamination </sup&gt;. DGMP überwindet diese Probleme durch die Verwendung eines inerten Dichtemodifikationsmittel wie Iodixanol, um Schichten unterschiedlicher Dichte 10 zu erzeugen. Da das Dichtemodifikationsmittel mit jeder Prepolymer oder bioaktive Molekül vermischt werden können, ermöglicht DGMP jedes Gerüst Schicht angefertigt werden. Einfache Variation der Konzentration des Dichtemodifikationsmittel verhindert eine Vermischung benachbarter Schichten während sie wässrigen bleiben. Anschließende Polymerisation einzigen Schritt führt zu einer strukturell kontinuierlichen mehrschichtigen Gerüst, in der jede Schicht unterschiedlichen chemischen und mechanischen Eigenschaften. Die Dichtemodifizierer können leicht mit ausreichend Spülen ohne Störung der einzelnen Lagen oder deren Bestandteile entfernt werden. Diese Technik ist daher gut für die Erstellung von Hydrogelen in verschiedenen Größen, Formen und Materialien. Ein Protokoll für die Herstellung eines 2D-Polyethylenglykol (PEG)-Gel, in dem alternierende Schichten aufzunehmen RGDS-350, wird im Folgenden erläutert. Wir verwenden PEG beil es ist biokompatibel und inert. RGDS, eine Zelladhäsion Peptid 11 wird verwendet, um räumliche Beschränkung einer biologischen Cue demonstrieren, und die Konjugation von einem Fluorophor (Alexa Fluor 350) ermöglicht es, visuell zu unterscheiden verschiedenen Schichten. Dieses Verfahren kann auch für andere Materialien angepasst werden (z. B. Kollagen, Hyaluronsäure, etc.) und kann um 3D Gele mit einigen Modifikationen 10 herzustellen.

MALDI-TOF-Analyse bestätigt die Konjugation von RGDS-Peptid um Acryloyl-PEG (Abbildung 2). Gel Imaging offenbart alternierenden RGDS-350 (blau) Schichten nach Photopolymerisation (Abbildung 3A). Wie in 3A gezeigt, können 2D DGMP Gelgröße basierend auf dem Durchmesser der Silikonformen (10 mm, links; 8 mm, rechts) variiert werden, und sind daher leicht anpassbar für die Verwendung in mehreren Assays - in diesem Fall um einen passenden 48 well Zellkulturplatte (3B). Epifluoreszenz und Phasenkontrast-Mikroskopie von C2C12 Myoblasten auf einem DGMP Gel kultiviert zeigt selektiver Anbringung auf RGDS-350-enthaltenden PEG Schichten (Abbildung 4), was zeigt, Kompartimentierung der Zelladhäsion Peptid (RGDS). <img src="/files/ftp_upload/50018/50018fig1.jpg" alt="Abbildung 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50018/50018fig1highres.jpg"/> Abbildung 1. Molecular Weight Analyse durch MALDI-TOF Vergleich APEG-SCM APEG-RGDS nach Konjugation RGDS Peptid erhalten. <img src="/files/ftp_upload/50018/50018fig2.jpg" alt="Abbildung 2" fo:content-width="6in" fo:src="/files/ftp_upload/50018/50018fig2highres.jpg"/> Abbildung 2. Schematische Darstellung DGMP Gelherstellung. Nachdem die Gradienten geschichtet sind, können sie gestattet, um die verschiedenen Zeiträume (t s) zur abgestuften Schnittstellen, durch Photopolymerisation gefolgt erstellen begleichen. Geschichtete DGMP Gele können leicht aus der Form zur weiteren Verwendung entnommen werden. <a href="https://www.jove.com/files/ftp_upload/50018/50018fig2large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen</a> . <img src="/files/ftp_upload/50018/50018fig3.jpg" alt="Abbildung 3" /> Abbildung 3. A) 2D vielschichtigen Gelen nach Photopolymerisation erhalten abgebildet mit 350 nm und weißes Licht Kanäle VersaDoc Gel-Dokumentation Einheit. Das Graustufenbild zeigt abwechselnden Schichten mit RGDS in weiß. B) Insertion von DGMP Gel in 48-Well-Zellkulturschalen. <img src="/files/ftp_upload/50018/50018fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Merged Phasenkontrast und Epifluoreszenz Bild von C2C12 Myoblasten auf DGMP Gele (Maßstab 50 um) gewachsen. <img src="/files/ftp_upload/50018/50018fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Wirkung von Iodixanol auf Geloberfläche Elastizität. Atomic force microscopy Messungen von statischen Proben von vernetzten PEG Substraten unter Verwendung von bereits etablierten Methoden mit einem 2 nN Kraft Trigger 12. * P &lt;0,05 und ** p &lt;0,01.

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Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Hier beschreiben wir ein einzigartiges Strategie zur Erzeugung biokompatiblen, schichtförmige Matrices mit kontinuierlicher Schnittstellen zwischen unterschiedlichen Schichten für das Tissue Engineering. Ein solches Gerüst könnte eine ideale anpassbare Umgebung zu modulieren Zellverhalten durch verschiedene biologische, chemische oder mechanische Signale

Density Gradient Multilayered Polymerisation (DGMP): A Novel Technique für das Erstellen von Multi-Fach, Anpassbare Scaffolds für das Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

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Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

12.4K Aufrufe.  University of California, San Diego. Hier beschreiben wir ein einzigartiges Strategie zur Erzeugung biokompatiblen, schichtförmige Matrices mit kontinuierlicher Schnittstellen zwischen unterschiedlichen Schichten für das Tissue Engineering. Ein solches Gerüst könnte eine ideale anpassbare Umgebung zu modulieren Zellverhalten durch verschiedene biologische, chemische oder mechanische Signale

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Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Elastomer-PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Forschung

Biotechnik

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproduction Verarbeitung von Elektrogesponnene Fasern für Tissue Engineering

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Kapillar-Force-Lithographie für Herz-Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Selbstberichts Gerüste für 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A Novel Bioreaktor für High Density Anbau von Diverse mikrobiellen Gemeinschaften

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

Rod-basierte Herstellung von Anpassbare Weiche Robotic Pneumatic Gripper Geräte für empfindliche Gewebe Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Neonatal Cardiac Scaffolds: Neuartige Matrices für Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Elektrospinnen Schreiben von dreidimensionalen Poly(ε-caprolactone) Gerüste mit steuerbaren Morphologien für Tissue Engineering Anwendungen zu schmelzen

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting von Knorpel und Haut-Gewebe-Analoga nutzen eine neuartige Passive Einheit Mischtechnik für Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

Ein Protokoll für einem Maus Cochleae für das Innenohr Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...