Diese Arbeit wurde von der Singapore National Research Foundation von einem seiner Research Centers of Excellence, die Mechanobiologie Institute, Singapur verabreicht unterstützt. MFAC wird von der Agentur für Wissenschaft, Technologie und Forschung (Singapur) und Nationalen Forschungsagentur (Frankreich) gemeinsame Programm unter Projektnummer 1122703037. Bkkt wird durch die Mechanobiologie Institute unterstützt unterstützt. Wir danken Herrn Daniel Wong HC für das Korrekturlesen des Manuskripts und Frau Morgendämmerung JH Neo zur Unterstützung bei der Videoproduktion.

1. Herstellung von Polyelektrolytlösungen <ol><li> Reinige Chitosan, wie in Liao et al beschrieben. Kurz gesagt, erstellen Sie eine 1% (w / v) Lösung von Chitosan in 2% (v / v) Essigsäure und Vakuumfilter mit Grad 93-Filterpapier. Neutralisiert das Filtrat mit 5 M NaOH, bis der pH-Wert stabilisiert bis 7. Zentrifugieren Sie das ausgefallene Chitosan bei 1.200 × g für 10 min. Überstand dekantieren und fügen VE-Wasser, um das Chitosan zu waschen. Wiederholen Sie die Zentrifugation und Waschschritt noch zweimal. Einfrieren der ausgefallene Chitosan bei -80 ° C und lyophilisiere O / N, um die gereinigte Form zu erhalten. Bewahren Sie gereinigte Chitosan in einem entfeuchteten Schrank. </li><li> Abwiegen 1 g gereinigtes Chitosan in eine sterile Gewebekulturschale. Platzieren des Chitosans in der Gewebekulturschale so nahe wie möglich an der UV-Lampe in der Sicherheitswerkbank und freizulegen, um UV-Licht für 15 min. Mit einer sterilen Pinzette, legen Sie die sterilisiert Chitosan in einen Glasbehälter. Chitosan aufzulösen Verwendung gefiltert 0.15M Essigsäure bis zu einer Endkonzentration zwischen 0,5% und 1% (w / v). </li><li> Abwiegen 0,1 g Alginsäure-Natriumsalz und lösen sich in 10 ml destilliertem entionisiertem (DDI) Wasser, um eine 1% (w / v) Lösung zu erhalten. Mischen Sie die Alginsäurenatriumsalz für mindestens 2 Stunden auf dem Vortex-Mischer, um eine vollständige Auflösung zu gewährleisten. Filtern der Alginatlösung durch 0,2 &amp; mgr; m-Spritzenfilter. Bewahren Sie die Alginat-Lösung bei 4 ° C. </li><li> Rekonstituieren humanen rekombinanten Wachstumsfaktoren, wie den vaskulären endothelialen Wachstumsfaktor (VEGF) oder &amp; beta; - Nervenwachstumsfaktor (NGF), wie vom Hersteller empfohlen. </li></ol> 2. Zeichnung der IPC Fibers <ol><li> Mischen Sie Proteine, Wachstumsfaktoren oder andere Biomoleküle in 10-20 ul-Aliquot der Polyelektrolyt-Lösung, die eine ähnliche Nettoladung hat. Biologische Moleküle mit negativen Nettoladung (zB Rinderserumalbumin [BSA]) sollte mit Alginat-Lösung gemischt werden. Biologische Moleküle mit positiven Nettoladung (zB VEGF) sollte mit gemischt werdenChitosan-Lösung. </li><li> Legen Sie kleine Teilmengen (10-20 ul) von Chitosan und Alginat auf eine stabile ebene Fläche, die mit Parafilm abgedeckt ist. Die Tröpfchen aus Chitosan und Alginat sollte in enger Nachbarschaft angeordnet werden, jedoch nicht in Kontakt miteinander. </li><li> Leicht tauchen jede Spitze auf einer Pinzette in die Chitosan und Alginat-Tröpfchen. Bringen Sie die Tropfen von Polyelektrolyten zusammen durch Kneifen der Zange. Wenn die Tröpfchen in Kontakt miteinander kommen, die Zange langsam senkrecht nach oben zu ziehen, um die IPC-Faser von der Schnittstelle der beiden Tröpfchen (1A) ziehen. </li><li> Legen Sie das Ende des gezogenen IPC Faser an der Zange auf einem Kollektor, wie eine flache Polymergerüst auf einem rotierenden Dorn befestigt (siehe Kapitel 3 und 4). Drehen des Dorns mit einer festen Geschwindigkeit von 10 mm / s, um die Bildung einer einheitlichen und wulstfreie IPC Fasern ermöglichen. Die Erhöhung der Geschwindigkeit des Ziehens der Fasern IPC Kügelchen bilden, die eine schlagartige Freisetzung des eingearbeiteten verursachenbiochemischen und vorzeitige Beendigung Faser. 10 </li><li> Um zu bestimmen Einbaueffizienz, sammeln alle verbleibenden Flüssigkeiten links auf dem Parafilm durch Verdünnen mit 500 ul 1X phosphatgepufferter Kochsalzlösung (PBS). Messung der Faktor-Inhalts Protein oder das Wachstum in dem Rückstand durch BCA-Assay (für BSA), ELISA (für VEGF und NGF) oder einem entsprechenden Assay eingebaut Biomolekül detektieren. </li></ol> 3. Herstellung von Verbund Hydrogel Gerüst Pullulan-Dextran (PD) Polysaccharide und IPC Fibers <ol><li> Herstellung Opfer Pullulan Rahmen für IPC Fasersammel <ol><li> Abwiegen Pullulan Polysaccharid und mischen mit destilliertem deionisiertem (DDI) Wasser, um eine 20% erstellen (w / v) wässrigen Lösung. Mischen Sie die Pullulanlösung O / N, um die Homogenität zu gewährleisten. </li><li> Guss 15 g Pullulan-Lösung in einen Durchmesser von 10 cm Gewebekultur Polystyrol (TCPS) Gericht. Trocknen Sie die Pullulanlösung O / N bei 37 ° C. Schneiden Sie die Pullulan Filme in 7mm x 7 mm quadratische Rahmen. </li></ol></li><li> Vorbereiten Pullulan-Dextran Polysaccharidlösung <ol><li> Erstellen Sie eine 30% (w / v) Lösung der Polysaccharide Pullulan und Dextran (3: 1-Verhältnis) in DDI Wasser. Mischungs O / N um die Homogenität der Polysaccharidlösung gewährleisten. Langsame Zugabe von Natriumhydrogencarbonat zu der Polysaccharid-Lösung bis zu einer Endkonzentration von 20% zu erreichen (w / v). Mischungs O / N, um die Homogenität der Lösung sicherzustellen. Lagern Sie das Polysaccharid-Lösung bei 4 ° C. </li></ol></li><li> Sammeln IPC Fasern auf Pullulan Rahmen <ol><li> Befestigen Sie das Opfer Pullulan Rahmen (Abschnitt 3.1) mit einem Krokodilklemme. Haften die Krokodilklemme und Pullulan Rahmen auf dem Ende des sich drehenden Dorn mit Kunststoff beschichtetes Klebeband. Drehen des Dorns mit dem befestigten Rahmen mit einer konstanten Geschwindigkeit von 10 mm / sec. Pullulan Rahmen kann auf den sich drehenden Dorn in gewünschten Orientierungen befestigt werden. </li></ol></li><li> Zeichnen Sie die IPC-Fasern mit einer Pinzette (Abschnitt 1) ​​und attach das gezogene Ende der IPC-Fasern auf die rotierende Pullulan Rahmen. Ziehen die IPC Fasern mit einer konstanten Geschwindigkeit. Bei Erreichen der terminalen Ende der IPC Faser, trockne die Fasern gegenüber dem Trägerrahmen die O / N bei RT. </li><li> Einbetten IPC Fasern in PD Hydrogel Gerüst </li><li> Um jedes Gramm Pullulan-Dextran-Lösung zu vernetzen, werden 100 &amp; mgr; l von 11% (w / v) Natrium-Trimetaphosphat wässrige Lösung und 100 ul 10 M Natriumhydroxid. 7 Mischen der Lösung bei 60 UpM unter Verwendung einer Rührplatte für 1 bis 2 min. Nach der Zugabe von Natriumtrimetaphosphat und Natriumhydroxid wird die Polysaccharid-Lösung fast sofort zu vernetzen. Gießen Sie die viskose Polysaccharid-Lösung auf die Fasern-on-Frame-Konstrukt, um die IPC-Fasern vollständig einzubetten. Inkubieren der kombinierten Pullulan-Dextran-IPC Fasern (PD-IPC) bei 60 ° C für 30 min, um eine chemisch vernetzte Verbundgerüste (1B) zu bilden. </li><li> ACHTUNG: Führen Sie Schritt 3.3.2 in der Abzugshaube und verwenden Sie geeignete Schutz equipment wie Essigsäure ist eine aggressive und brennbare. </li><li> Zur Porenbildung in der PD-IPC Gerüst induzieren, tauchen die ganze Gerüst in 20% (w / v) Essigsäure für 20 min. </li><li> Entfernen nicht umgesetzten Reagenzien durch Waschen PD-IPC Gerüste in 1X PBS für 5 min unter Schütteln bei 100 Umdrehungen pro Minute. Wiederholen Sie diesen Schritt 2 mal. </li><li> Entfernen Sie das überschüssige PBS und unverzüglich sperren PD-IPC Gerüste bei -80 ° CO / N. Lyophilisieren die Gerüste mindestens 24 Stunden vor der Verwendung in allen kontrollierten Freisetzung oder Bioaktivitätstests. </li></ol> 4. Herstellung von Verbundgerüst von PCL und IPC Fibers ACHTUNG: Dichlormethan ist ein Gefahrstoff. Verwenden Sie den Abzug und persönliche Schutzausrüstung beim Umgang mit Dichlormethan. <ol><li> Erstellen unberührten und gemusterten PDMS Substrate <ol><li> Erstellen Sie eine neue, unberührte Polydimethylsiloxan (PDMS) Elastomer-Substrat mit einem Stück TCPS der gewünschten Abmessung mit weichen Lithographieprozess. Erstellen patteingeschaltet PDMS Substrate durch Verwendung von Standard-Weichlithographieverfahren von Poly (methylmethacrylat) Vorlagen mit der gewünschten Topographie. 12 </li></ol></li><li> Herstellung Opfer PCL Rahmen für IPC Fasersammel <ol><li> Abwiegen PCL und lösen in Dichlormethan, um eine 0,9% (w / v) Lösung zu schaffen. Für jede 1 cm 2 Fläche des PDMS-Substrat, fallen 500 ul 0,9% PCL-Lösung. Lassen Sie das gesamte Lösungsmittel Dichlormethan vollständig in der Abzugshaube verdampfen. Wiederholen den Vorgang des Gießens 0,9% PCL, um die Folie auf die gewünschte Dicke zu verdicken. Entfernen Sie die getrockneten PCL-Film aus der PDMS-Substrat. Erstellen Sie ein Loch in der PCL-Rahmen mit einem angemessen dimensionierten Puncher. 2 </li></ol></li><li> Sammeln IPC Fasern auf der PCL-Rahmen <ol><li> Befestigen Sie das Opfer PCL Rahmen mit Loch (von 4.2.1) auf einer Krokodilklemme. Halten Sie die Krokodilklemme auf den sich drehenden Dorn unter Verwendung kunststoffbeschichteten Klebeband. Befestigen Sie den gezogenen Ende des IPC Faser auf die PCL frame vor dem Start der Drehung mit einer konstanten Geschwindigkeit von 10 mm / s (Abschnitt 2). Nach dem Ende der IPC Faserziehen, Trocknen der Faser-on-Frame-Konstrukt O / N bei 4 ° C. </li></ol></li><li> Einbetten von Faser-on-Frame-Konstrukts in gemusterten PCL Substrat <ol><li> Drop 500 ul 0,9% PCL-Lösung auf das PDMS-Substrat, um eine unberührte oder gemusterten PCL Basis zu schaffen, je nach Bedarf. Guss mehrere Schichten von 0,9% PCL-Lösung ein Gerüst mit der gewünschten Dicke zu erhalten. Lassen Sie das gesamte Lösungsmittel Dichlormethan vollständig in der Abzugshaube verdampfen. </li><li> Legen Sie die Faser-on-Frame-Konstrukt (Abschnitt 4.3.1) auf der Oberseite des Basis PCL. In 0,9% PCL-Lösung auf die Faser-auf-Rahmen zu konstruieren mehrmals, um die gewünschte Dicke zu erhalten und vollständig einzubetten die IPC-Fasern, Herstellen einer PCL-IPC Verbundgerüst (Abbildung 1c). </li></ol></li></ol> 5. Messung der Freisetzung biologischer Stoffe aus Verbund IPC Gerüste <ol><li> Zeigen Verbund PD-IPC oderPCL-IPC Gerüsten und Stand-alone-IPC Fasern getrennt in einer 24-Well-Platte. </li><li> Tauchen Sie das Gerüst und Standalone-IPC-Fasern mit 500 ul 1X PBS. Die Proben bei 37 ° C inkubiert. Sammeln PBS zu verschiedenen Zeitpunkten (Release-Medien) und ersetzen mit 500 ul 1X PBS. </li><li> Messen der Menge an Protein oder Wachstumsfaktor in den Freisetzungsmedien mit einem BCA-Test (BSA), ELISA (VEGF und NGF) oder anderen geeigneten Assays für das kumulative Freisetzungsprofil für den einge Biomolekül berechnen. </li></ol> 6. Seeding von Zellen auf Composite-IPC Gerüste auf der freigegeben Biologische Arbeitsstoffe Testen Bioaktivität <ol><li> Sterilisieren Sie die lyophilisierten PD-IPC oder PCL-IPC Verbund Gerüste mit UV-Licht in der biologischen Sicherheitswerkbank für mindestens 20 min. </li><li> Verwenden Standardzellkulturtechniken, um eine Zellsuspension von 2 x 10 5 Zellen in 200 &amp; mgr; l Wachstumsmedium zu erhalten. Samen der konzentrierten Zellsuspension auf die Verbund Gerüste. After 20 min, top-up das Volumen des Wachstumsmediums, um die Gerüste vollständig untertauchen. </li><li> Messen der Zellaktivität, durch Standardtechniken, wie Alamar Blau metabolischen Aktivitätstest PC12 Neuritenwachstum Assay oder Immunfluoreszenz. </li></ol>

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Part A. 13 (2), 423-433 (2007).</li><li>Chaouat, M., Le Visage, C., Autissier, A., Chaubet, F., Letourneur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+a+small-diameter+polysaccharide-based+arterial+graft+in+rats.">The evaluation of a small-diameter polysaccharide-based arterial graft in rats.</a> Biomaterials. 27, 5546-5553 (2006).</li><li>Eshraghi, S., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+microstructural+properties+of+polycaprolactone+scaffolds+with+one-dimensional,+two+dimensional,+and+three-dimensional+orthogonally+oriented+porous+architectures+produced+by+selective+laser+sintering.">Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two dimensional, and three-dimensional orthogonally oriented porous architectures produced by selective laser sintering.</a> Acta Biomater. 6, 2467-2476 (2010).</li></ol>

The authors have nothing to disclose.

IPC Fasern durch die Interaktion von zwei entgegengesetzt geladenen Polyelektrolyten gebildet sind. Das Verfahren nutzt die Gewinnung des Komplexes aus der Schnittstelle der Polyelektrolyte, einen Selbstorganisationsprozess für stabile Faserbildung erleichtert wird. Der Mechanismus der IPC Faserbildung sorgt dafür, dass jedes Biomolekül ergänzt in einen ähnlich geladenen Polyelektrolyten während der Komplexbildung eingearbeitet werden. 10,11 umge Zugabe eines Biomoleküls in die entgegengesetzt geladene...

The extracellular matrix has inherent biochemical and biophysical cues that direct cell behaviors. Mimicking this physiological three-dimensional (3D) microenvironment is a widely explored strategy for regenerative medicine and tissue engineering applications. For example, both naturally-derived and synthetic substrates have been modified with topographical cues as a means to mimic the biophysical cellular environment.1 For example, polycaprolactone (PCL) scaffolds can be easily patterned by casting on patterned PDMS substrates.2 However, most synthetic scaffolds inadequately recapitulate the controlled biochemical environment in vivo. Bulk or surface modification of synthetic materials only present biochemical cues for cell attachment but still lack temporal regulation of biochemical delivery.3 Thus, there is a need for optimal scaffolds that can mimic the temporally regulated biochemical delivery system of the extracellular matrix. 
Biochemical delivery systems such as microspheres are plagued by problems of loss of bioactivity and low incorporation efficiency due to the severity and complexity of multi-step synthesis process.4-6 Alternative methods that use a one-step fabrication and incorporation method were proven to have excellent potential to create a favorable biochemical microenvironment without the accompanying inefficiency in incorporation and loss of bioactivity. One viable solution is the use of interfacial polyelectrolyte complexation (IPC) fibers to deliver and protect biological agents. When two oppositely charged polyelectrolyte aqueous solutions are brought together, IPC fibers can be drawn out from the interface. Virtually any type of hydrophlic biomolecule in aqueous solution can be added into either the negatively- or positively-charged polyelectrolyte solution, thus facilitating the incorporation of useful biomolecules into the IPC fiber during the complexation process. Furthermore, this process only requires aqueous and ambient conditions, thereby decreasing the risk of loss of bioactivity. Using this method, active growth factors2,7 even cells8,9 have been successfully delivered. In addition, the simple method of forming IPC fibers allows molding into any shape or orientation. The stability of such fibers has been advantageous in its incorporation into both hydrophobic2 and hydrophilic polymers7 to create composite scaffolds. These composite scaffolds with IPC fibers are beneficial for creating a physiologically relevant biochemical environment while providing physical anchorage for cells. 
In this study, we show a method to incorporate IPC fibers into a hydrophilic and a hydrophobic scaffold with topography for controlled release of active biomolecules. As a proof-of-concept, we incorporate IPC fibers made from chitosan and alginate into the biocompatible, non-immunogenic and non-antigenic pullulan-dextran hydrophilic hydrogel or the biocompatible polycaprolactone hydrophobic scaffold.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pullulan&#160;</td>
			<td>Hayashibara Inc Okayama Japan</td>
			<td></td>
			<td>Molecular weight (MW) 200 kDa. This material is pharmaceutical grade pullulan used to make pullulan frames and PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma Aldrich</td>
			<td>D1037</td>
			<td>MW 500 kDa. This material is no longer being produced by Sigma Aldrich. Alternative suggested is catalog number 31392 (Sigma Aldrich). This material is used to make PD-IPC scaffolds.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Sodium bicarbonate must be slowly added to the pullulan-dextran polysaccharide solution. Rapid addition of sodium bicarbonate will result in precipitation.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Trimetaphosphate</td>
			<td>Sigma Aldrich</td>
			<td>T5508</td>
			<td>This chemical is hygroscopic and must be stored in the dehumidifying cabinet. Aqueous solution of sodium trimetaphosphate must always be made fresh.</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td>This material is hazardous and must be handled with proper protective equipment such as nitrile gloves.</td>
		</tr>
		<tr>
			<td>Chitosan</td>
			<td>Sigma Aldrich</td>
			<td>448877</td>
			<td>MW 190-310 kDa. Acetylation degree of 75 to 85%. Purification of chitosan is required to create stable IPC fibers.</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Merck</td>
			<td></td>
			<td>This can be replaced by another brand type. This material is corrosive and flammable. Protective equipment such as face shield, nitrile gloves, lab coat and shoe cover must be worn when handling this chemical in the fume hood.&#160;</td>
		</tr>
		<tr>
			<td>Alginic acid sodium salt from brown algae, low viscosity</td>
			<td>Sigma Aldrich</td>
			<td>A2158</td>
			<td>Dissolve in water overnight. Filter through sterile 0.2&#181;m syringe filter before use. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sinopharm Chemical Reagent</td>
			<td></td>
			<td>Dissolve in sterile PBS and filter using 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td>This kit was used to measure BSA release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Human Recombinant Vascular Endothelial Growth Factor</td>
			<td>R&#38;D systems</td>
			<td>293-VE</td>
			<td>Dissolve growth factor in 0.2% heparin solution to a final concentration of 5 mg/ml.</td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt From Porcine</td>
			<td>Sigma Aldrich</td>
			<td>H3393</td>
			<td>This can be replaced by another brand type. Dissolve heparin salt in sterile water at a final concentration of 1% and filter through 0.2 &#181;m syringe filter before use.&#160;</td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td>Lonza</td>
			<td>C2517A</td>
			<td>This primary cell type was used in the assay to determine VEGF bioactivity after release from PD-IPC scaffolds.&#160;</td>
		</tr>
		<tr>
			<td>Alamar blue</td>
			<td>Life Technologies</td>
			<td>DAL1025</td>
			<td>This is used to measure cell metabolic activity. Incubate Alamar blue with cells and maintain in standard cell culture conditions for 2 to 4 hours. Measure absorbance at 570 nm to determine Alamar blue percent reduction, which is correlated to the cell activity.&#160;</td>
		</tr>
		<tr>
			<td>ScanVac Coolsafe Lyophilizer</td>
			<td>Labogene</td>
			<td>7.001.200.060</td>
			<td>This is a non-programmable freeze dryer that operates at -105 to -110 &#176;C, This can be replaced by other standard lab lyophilizers.</td>
		</tr>
		<tr>
			<td>Polycaprolactone (PCL)</td>
			<td>Sigma Aldrich</td>
			<td>181609</td>
			<td>MW 65 kDa. This is no longer being manufactured by Sigma Aldrich. This can be replaced by Sigma Aldrich catalog number 704105.</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>V800151</td>
			<td>This can be replaced by another brand type. This material is hazardous and must be handled in the fume hood. Protective equipment must be worn at all times when handling this chemical.</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS; 184 Silicone Elastomer Kit)</td>
			<td>Dow Corning</td>
			<td>(240)4019862</td>
			<td>The elastomer kit comes with polymer base and crosslinker. Mixing the polymer base and crosslinker in different ratios will result in different stiffness of the PDMS.</td>
		</tr>
		<tr>
			<td>Human Recombinant Beta-Nerve Growth Factor (NGF)</td>
			<td>R&#38;D systems</td>
			<td>256-GF</td>
			<td>Reconstituted in sterile DI water to a final concentration of 100 &#181;g/ml. Aliquot and store in -20 &#176;C until use.</td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells (hMSC)</td>
			<td>Cambrex</td>
			<td></td>
			<td>This cell type was used in the assay to determine synergistic effect of NGF and nanotopography.</td>
		</tr>
		<tr>
			<td>Rat PC12 Pheochromocytoma Cells&#160;</td>
			<td>ATCC</td>
			<td></td>
			<td>This cell type was used in the neurite outgrowth assay to determine bioactivity of NGF. After exposure to release media with NGF, measure number of cells with neurite extensions and normalize to total number of cells.</td>
		</tr>
		<tr>
			<td>Grade 93 filter paper</td>
			<td>Whatman</td>
			<td>Z699675</td>
			<td>This is used for the purification of chitosan after its precipitation with sodium hydroxide at pH 7.</td>
		</tr>
		<tr>
			<td>Swing bucket centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>To be used during the purification of chitosan using 1200 x g speed.&#160;</td>
		</tr>
		<tr>
			<td>Motor with mandrel rotating at constant speed</td>
			<td>Rhymebus</td>
			<td>RM5E</td>
			<td>The motor is used for the fabrication of IPC fibers on pullulan or PCL frame.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>FirstBase</td>
			<td></td>
			<td>Sterilize through filtration (0.2 &#181;m filter) and autoclave.&#160;</td>
		</tr>
		<tr>
			<td>10-mm diameter Tissue Culture Polystyrene Dish (TCPS)</td>
			<td>Greiner</td>
			<td></td>
			<td>The TCPS dish is used for casting of pullulan frame.&#160;</td>
		</tr>
		<tr>
			<td>Human VEGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DVE00</td>
			<td>The ELISA kit is used for detection of VEGF in the release medium.</td>
		</tr>
		<tr>
			<td>Human NGF ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY256</td>
			<td>&#160;The ELISA kit is used for detection of NGF in the release medium.</td>
		</tr>
		<tr>
			<td>Plastic Coated Adhesive Tape</td>
			<td>Bel-Art</td>
			<td>9040336</td>
			<td>The adhesive tape is used to securely stick the alligator clip to the rotating mandrel</td>
		</tr>
	</tbody>
</table>

composite scaffolds of interfacial polyelectrolyte fibers for temporally controlled release of biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

In diesem Artikel haben wir versucht, Verbund Gerüste mit IPC-Fasern für die verzögerte Freisetzung verschiedener Biomoleküle zu schaffen. Eigenschaften der in dieser Studie verwendeten Biomoleküle sind in Tabelle 1 gefunden. IPC Faser wurde in eine hydrophile PD Hydrogel eingebettet ist, um einen PD-IPC Verbundgerüst (1B) zu schaffen. Modellmolekuls BSA wurde zuerst untersucht, um die Durchführbarkeit der Verwendung eines Verbundgerüst zur kontrollierten Freisetzung Biomolekül...

Watch this Scientific Journal Video about Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules at JoVE.com

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Verbund Scaffolds für Grenzflächen Polyelectrolyte Fasern für zeitlich kontrollierte Freisetzung von Biomolekülen

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

composite-scaffolds-interfacial-polyelectrolyte-fibers-for-temporally

Research

JoVE Journal

Bioengineering

8.3K Aufrufe.  National University of Singapore. Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Serie: Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

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

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Erstellung von 3D-Fibrin-Gerüste für Stem Cell Culture Anwendungen

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Berührungslose, Label-freie Überwachung von Zellen und extrazellulären Matrix mittels Raman-Spektroskopie

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

Herstellung der lichtreaktions Membranen durch eine Kombinierte Oberflächen-Pfropfen und Postmodification Prozess

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Erzeugung eines dreidimensionalen Vollhaut Equivalent und automatisierte Verwundung

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetisches Wechselfeld-Responsive Hybrid Gelatine Mikrogele der gezielten Medikamentenapplikation

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

Polyelektrolytkomplex für Heparin-Bindungsdomäne Osteogenisches Growth Factor Lieferung

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Herstellung von extrazellulären Matrix Proteinfasern für Brillouin-Spektroskopie

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Magnetisches und thermisch empfindliches Poly (<em&gt; N.</em&gt; -isopropylacrylamid) -basierte Mikrogele für die magnetisch getriggerte kontrollierte Freisetzung

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Herstellung von Decellularized Knorpel abgeleitet Matrix Gerüste

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...