Wir danken Dr. Jeffrey Caplan für seine Ausbildung und Erfahrungsberichte zu konfokale Bildgebung. Wir danken auch dem Keck Electron Microscopy Lab und Dr. Chaoying Ni für die SEM-Unterstützung. Diese Arbeit wird von National Institutes of Health (NIDCD, R01DC008965 und R01DC011377) finanziert. ABZ erkennt NSF Integrative Graduate Education &amp; Research Traineeship (IGERT)-Programm für die Finanzierung.

1. Bioreaktor Versammlung (Video 1) <ol><li> Machen Sie eine Aluminiumform (kreisförmige Düse + Abstandsstift) mit vorgegebenen Innen-und Außenmaße (Abbildung 1). </li><li> Mit der Form aus Schritt 1.1, fabrizieren eine Silikonmembran (Durchmesser: 42 mm, Dicke: 1,5 mm, Abbildung 1) mit einem verschanzten Hülse (Durchmesser: 12 mm, Dicke ~ 0,25 mm, von der Abstandsstift in Abbildung 1 förmig) in die mittlere mit einem handelsüblichen Silikon-Elastomer-Kit. </li><li> Machen Sie ein Paar Acrylblöcke (Abbildung 2 (4, 5)) mit einer kreisförmigen Öffnung (Durchmesser: 24 mm) in der Mitte, gravieren die Spitze (1,8 cm dick) und unten (0,9 cm dick) Blöcke mit passenden Rippen und Rillen . 10. </li><li> Sandwich die Silikonmembran zwischen den gepaarten Acrylblöcke. Sichern Sie die Montage mit vier Schrauben Ecke mit einer Micro-Drehmomentschrauber einer konstanten Kraft (35 cN · m) eingestellt. Als Ergebnis wird eine wasserdichte, 24 mm breit und 18 mm tief Vibrationskammer erzeugt wird (Fig. 2C). </li><li> Montieren Sie einen 3 &quot;erweiterten Bereich Mini-Woofer (2D, 8 Ω/20 W) unter der Vibrationskammer durch einen anderen Satz von Ecke Schrauben auf der Unterseite Acrylblock. An diesem Punkt wird eine einzelne Vibrationsmodul montiert. </li><li> Replizieren sieben zusätzliche Schwingungsmodule. Anbringen vier davon mit einem von zwei feststehenden Aluminiumstangen (40 cm x 10 cm x 2,5 cm), indem die Lautsprecher Basen in gleichmäßig beabstandeten kreisförmigen Öffnungen (Durchmesser: 7 cm, Dicke: 2 cm) in die Stäbe zu schneiden. Stabilisieren Sie jeden Lautsprecher durch Einsetzen einer Schraube durch die Seite der Aluminiumstange in jeder kreisförmigen Loch. </li><li> Individuell steuern die Lautsprecher durch einen Lautsprecher Selector. Verbinden einzelnen Lautsprecher an den Selektor durch Anbringen Drähte an die positiven und negativen Eingänge des Lautsprechers Körper dann an die entsprechenden Ausgänge des Wählers. Das Lautsprecherwähler ermöglicht das Signal von ter Funktionsgenerator, nachdem es durch einen Leistungsverstärker, um alle acht Lautsprecher auf einmal (2E) zu erreichen. </li><li> Platzieren Sie die beiden Kammeranordnungen, die Lautsprecherauswahl und die zugehörige Elektronik in einem Gehäuse zum Schutz gegen Feuchtigkeit. Haus die gesamte Baugruppe in einem kommerziellen Zellkulturbrutschrank. </li><li> Führen Sie die Hauptkabel (durch eine medizinische Qualität PVC-Schlauch), die den Leistungsverstärker und Lautsprecherwähler durch die Filteranordnung auf der Rückseite des Inkubators. </li></ol> 2. Gerüst Herstellung und Charakterisierung <ol><li> Auflösen PCL-Pellets in Chloroform bei einer Konzentration von 15 Gew.%. Laden Sie die Lösung in eine 10 ml Spritze mit einer 21 G Nadel stumpfen Enden gekappt. </li><li> Sperren Sie die Spritze auf einem programmierbaren Spritzenpumpe und stellen Sie die Durchflussmenge bei 1 ml / Std. </li><li> Platzieren der Aluminiumfolie bedeckten Kollektor gegenüber der Nadel horizontal, mit einer Nadelspitze-zu-Kollektor-Strecke von ca. 18 cm. </li><li> Klemmen Sie die positive alliAlligator-Clip in der Mitte der Nadel und dem Boden Krokodilklemme an die Aluminiumkollektor, dann die Spannung auf der Hochspannungsversorgung mit 15 kV. ACHTUNG: Hochspannung, halten Sie Abstand von der Nadel. </li><li> Nacheinander auf dem Spritzenpumpe und Stromversorgung drehen; schnell reinigen / entfernen Sie die Restpolymerlösung rund um die Spitze der Nadel mit einem trockenen Papiertuch vor stabile Faserdüsen und Taylor-Konus 19 gebildet werden. </li><li> Die Fasern auf der Sammel Al in einer Dicke von ca. 250-300 &amp;mgr; m akkumulieren (~ 7 Stunden unter den derzeitigen Spinnbedingungen). Speichern Sie die resultierende Gerüste im Vakuumtrockenschrank für 1-2 Tage, um restliche Lösungsmittel zu entfernen. </li><li> Bild die Gerüste, mit Gold beschichtet Sputtern mit einem Raster-Elektronen-Mikroskop, um konsistente Fasermorphologie zeigen. 10. </li></ol> 3. Bioreaktor Assemblierung und Charakterisierung <ol><li> Lochen Sie ein zylindrische Scheibe (Durchmesser: 8 mm) mit vier Armen (Länge: 2mm) aus der wie gesponnenen PCL Matte (2A), indem zuerst mit einem Durchmesser von 12 mm Stanzbiopsie, um den äußeren Durchmesser der Platte geschnitten. Dann mit einem zweiten, 8 mm Stanzbiopsie zu vier 2 mm langen Kerben gleichmäßig um den Kreismesser Abstand zu punkten, wo die Arme zu schneiden sind zu machen. Nach der Wertung mit der 8 mm-Stanz, verwenden Sie ein Skalpell, um die Kanten der Arme nach außen abgeschnitten. Einfügen des Gerüsts in die Nut der Silikonmembran über den verlängerten Armen (Video 1). Glätten Sie das Gerüst eingefügt durch sanftes Drücken Sie die Oberfläche mit Flachkopf-Pinzette. </li><li> Bringen Sie ein kleines Stück von dünnen Al-Folie (8 mm x 2 mm, senkrecht Form, 2B) mit dem PCL-Gerüst Laserreflexion unterstützen. </li><li> Befestigen Sie den zusammengebauten Silikonmembran / PCL Gerüst (wie in Schritt 1.4 beschrieben) in der Vibrationskammer. 1,5 ml Wasser in der Kammer, um die PCL-Gerüst vor Vibrationen Hydrat. </li><li> Mit dem Funktionsgenerator, einzuführen Vibrations ZeichenALS (z. B. 200 Hz-Sinuswellen mit einer Spitze-zu-Spitze-Spannung Vpp von 0,1 V) an die sandwichartig Acrylkammer. Mit einem Voltmeter die Spannung exakt messen an jedem Lautsprechereingang. Hinweis: die Vpp Auslesen aus dem Funktionsgenerator von der eventuellen Spannungs an den Lautsprecher geliefert abweichen. </li><li> Montieren Sie den Ein-Punkt-LDV und sichern die Laserlichtsensorkopf auf eine Schwenk-Neige-Kopf Stativ. Angle den Sensorkopf, so dass sie senkrecht auf der Tischplatte zeigt. Schließen Sie das LDV Sensorkopf mit dem Datenerfassungsmodul über Koaxialkabel dann das Modul an den Laptop über USB. </li><li> Fokussierung des Laserstrahls senkrecht an verschiedenen vorbestimmten Stellen auf der Silikon-Membran (Fig. 2B und Fig. 3). </li><li> Mit der Software zur Datenerfassung, notieren Sie die Mitte Membranverschiebung. Klicken Sie auf &quot;Übernahme-Einstellungen&quot; aus dem Menü &quot;Optionen&quot;; ändern Sie dann den Messmodus auf &quot;FFT221;. Als nächstes klicken Sie auf die &quot;Dauermessung&quot; in der Hauptsymbolleiste klicken Sie dann auf die Spitze, die bei der gewählten Frequenz (6D) bildet, um eine Verschiebung zu erfassen. </li><li> Zur Erstellung der normalen Halbmembranverschiebung (w 0) als Funktion der relativen Position gegenüber dem Substrat. Erstellen Sie ein 3D-Farbtabelle der Oberfläche Vibrations Profil mit Origin 8.5 Datenanalyse-Software. </li></ol> 4. Vibrationszellkultur <ol><li> Sub-Kultur menschlichen Knochenmark abgeleiteten MSCs in T150 Zellkulturflaschen mit einer Anfangsaussaatdichte von 4000-5000 Zellen / cm 2 in MSC Wartung Medien. </li><li> Nach 7-8 Tagen Zellkultur (um ~ 85% Konfluenz), trypsinize die Zellen mit einer Zellspalt Reagens wie Accutase zählen mit einer Zählkammer, Zentrifuge (440 × g für 5 min) und Resuspension der Zellpellets in Frisch MSC Erhaltungsmedium bei einer Konzentration von 4,5 x 10 6 Zellen / ml. </li><li> Tauchen Sie den PCL scaffold in 70% Ethanol O / N. Nachdem das Lösungsmittel verdampft ist, setzen beide Seiten des Gerüsts mit UV-Licht (254 nm) für 5-8 min keimtötende. </li><li> Einweichen des PCL-Gerüst in einem 20 ug / ml Fibronektin-Lösung bei 37 ° C für 1 Stunde. Legen Sie die Fibronektin-beschichtete Gerüst in die Silikonmembran. Montieren Sie den Bioreaktor wie in Schritt 1 beschrieben. </li><li> Verteilen 40 ul der Zellsuspension gleichmäßig auf die gesicherte PCL Gerüst. Lassen Sie die Zellen für 1 bis 1,5 Stunden, bevor Sie weitere 1,5 ml frisches Medium mit der Vibrationskammer zu befestigen. </li><li> Kultur der MSC-beladenen PCL Gerüst statisch für 3 Tage und aktualisieren Sie die Medien nach Abschluss der statischen Kultur. </li><li> Impose ausgewählten Vibrations Regime auf die Zellkonstrukte. Anmerkung: Als Beispiel werden die Zellen in einen 1-Stunden-on-1-h-aus (OF) Schwingung bei 200 Hz mit einem w von 0 ~ 40 &amp;mgr; m für 12 Stunden pro Tag für bis zu 7 Tage unterzogen. Konstrukte, um Schwingungsanregungen unterzogen werden, wie Vib Proben und diesen kul bezeichnetrot statisch in identischen Schwingungs Kammern dienen als statische Kontrollen (Stat). </li></ol> 5. Biologische Bewertung <ol><li> Sammeln Sie 200 ul Zellkulturmedien aus jeder Kammer jeden zweiten Tag (Tag 1, 3, 5 und 7) und bündeln die Portionen aus der gleichen Probe zusammen (jeweils 800 ul). </li><li> Quantifizierung der zellulären Produktion von Matrix-Metalloproteinase-1 (MMP1) und HA mit einem MMP1 ELISA-Entwicklung-Kit und eine Hyaluronsäure kompetitiven ELISA-Kit, jeweils folgende Verfahren der Hersteller. Unterdessen untersuchen die Produktion von löslichem Elastin-Vorläufer nach dem zuvor berichtet ELISA-Verfahren. 8 </li><li> Nach Abschluss der letzten Schwingungszyklus am Tag 7, schnell zu entfernen, die zellulären Konstrukte aus den Schwingungs Kammern mit scharfen Pinzette und spülen Sie sie kurz mit kaltem Phosphat-gepufferte Salzlösung (PBS, 4 ° C). </li><li> Für die Live / Dead-Färbung, brüten die Konstrukte mit Propidiumiodid (1:2000 in PBS) undSyto-13 (1:1000 in PBS) gleichzeitig für 5 min bei RT. Bild die gefärbten Konstrukte mit einem Multiphotonen-konfokalen Mikroskop. </li><li> Getrennt Snap-Einfrieren der PBS-gespült Zellkonstrukte auf Trockeneis und extrahieren Sie die gesamte zelluläre RNA nach einer zuvor berichtet Protokoll für die Gen-Analyse. 9 </li><li> Überprüfen Sie, ob die Quantität und Qualität der extrahierten RNA mit Hilfe eines UV-Vis-Spektrophotometer. RNA-Proben mit A260/A280 und A260/A230 Verhältnisse von 1,8 bis 2,2 sind für die anschließende qPCR-Analyse verwendet. </li><li> Reverse Transkription der RNA (500 ng / Probe) in cDNA mit einem kommerziell erhältlichen Kit der reversen Transkription. </li><li> Führen Sie die PCR-Reaktion auf einer Echtzeit-Sequenz-Detektionssystem mit einem handelsüblichen PCR Master Mix nach der vorher beschrieben Verfahren. 8 </li><li> Analysieren Sie die qPCR Ergebnisse mit kommerziellen qPCR Datenanalyse-Software. Um die Zuverlässigkeit der Datenanalyse zu gewährleisten, werden mehrere Referenzgene (YWHAZ, TBP, PPIA) als int verwendetern Kontrollen und die Varianz der spezifischen Primer Effizienz berücksichtigt wird. 8 </li></ol>

<ol>
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Erfolgreiche Engineering von funktionellen Stimmlippengewebe in vitro erfordert die Erholung von einem Stimmlippenartigen Mikroumgebung, um die Verhaltensweisen der multipotenten Zellen vermitteln. Es ist allgemein anerkannt, dass Gewebe-oder Organstrukturen spiegeln die Funktionen, die sie benötigt, um durchzuführen. Für 22 Stimmlippengewebe werden die hochfrequenten Schwingungen, die bei der Lautbildung auftreten vorgeschlagen wichtig für die Gewebereifung zu sein. In unserer Studie sind PCL Ge...

Der menschliche Stimmlippen, einer epithelialen Schicht, die Lamina propria (LP) und der M. vocalis, ist eine spezialisierte Weichgewebe die den Luftstrom aus der Lunge in akustische Wellen für die Klangerzeugung umwandelt. 1 Stimmlippen schwingen regelmäßig während der normalen Stimmbildung, bei Grundfrequenzen im Bereich aufweisen Stämme von bis zu 30% 100-300 Hz. 2 Erwachsene Stimmlippen LP ist eine Gradienten-Struktur einer oberflächlichen (SLP) zusammengesetzt ist, ein Zwischenprodukt (ILP) und eine Tiefe (DLP)-Schicht. Weitere Klassifikationsgruppen das Epithel und die SLP als Schleimhaut-Schicht und verbindet die ILP-und DLP in die Stimmbandes. 3 Die SLP Schicht enthält in erster Linie eine amorphe Matrix mit spärlich verteilt kollagenen Fasern, während das Band mit reifen Kollagen und Elastin-Fasern angereichert um eine ausreichende Festigkeit verfügen. 4. Die Struktur und Mechanik des Neugeborenen Stimmlippen deutlich von ihrer reifen Kollegen. Obwohl Mechanismuss Regelstimmlippen Entwicklung und Reifung sind noch nicht vollständig verstanden, experimentelle Beweise für die Definition von Rollen der Vokalisation abgeleitete mechanische Belastung hingewiesen. Mehrere medizinische Bedingungen, einschließlich Sprachmissbrauch, Infektionen, chemische Reizstoffe und chirurgische Verfahren, können die Stimmlippe beschädigen. Stimmlippe Störungen betreffen schätzungsweise 3-9% der US-Bevölkerung. Aktuelle Behandlungsmethoden für Erkrankungen der Stimmlippen sind begrenzt, 5 und eine Stammzellen-basierten Tissue-Engineering-Ansatz hat sich als vielversprechende Strategie zur Wiederherstellung der Stimmlippenfunktion entstanden. Mesenchymalen Stammzellen (MSC) sind eine geeignete Alternative zu den primären Stimmlippen Stimmlippen Fibroblasten für Tissue Engineering. 6-9 Stammzell Schicksal Spezifikation und anschließender Gewebeentwicklung werden durch die spezifische Nische sie wohnen, von denen der mechanische Zustand vermittelt ein entscheidender Faktor. 10. Mechanische Kräfte sind wesentliche Regulatoren der Morphogenese einnd Homöostase, vor allem für Gewebe, die routinemäßig von der Last. ausgesetzt sind 11 Aus einer Perspektive Tissue Engineering hat es sich gezeigt, dass die Exposition gegenüber physiologisch relevanten mechanischen Reize fördert die Stammzelldifferenzierung und gewebespezifischen Matrixumbau. 12-15 Gewebekulturbioreaktoren sind entworfen, um die gewünschte physiologische Umgebung für Zell-oder Gewebewachstum in vitro zu simulieren. Für Stimmlippen Tissue Engineering, ist es besonders wichtig, die mechanische Umgebung der phonating Stimmlippen neu zu erstellen. Eine ideale Stimmlippen Bioreaktor sollte Vibrations effektiv zu liefern Hinweise zur Kultivierung von Zellen, so dass eine leichte Kontrolle über die Frequenz, Amplitude und Dauer der Erschütterungen. Titze und Mitarbeiter entwickelten eine Stimmlippen Bioreaktor (T1 Bioreaktor) 16, die statische Strecke mit hoher Frequenz (20-200 Hz) Schwingungen, die zelluläre Produktion von Matrixproteinen stimulieren kombiniert. Das Arbeg dieser Bioreaktor, Webb und Kollegen 17 suchten die Auswirkungen der 10-Tage, 100-Hz-Schwingungen auf den dermalen Fibroblasten in einer Hyaluronsäure (HA)-basierte Hydrogel kultiviert. Konstrukte Vibrationen ausgesetzt zeigten eine erhöhte Expression von HA-Synthase-2 (HAS2), Decorin, Fibromodulin und Matrix-Metalloproteinase-1 (MMP1), bezogen auf die statische Kontrollen. Die stimulierende Wirkung wurde festgestellt, zeitabhängig sein. Vor kurzem, unsere Gruppe 18 stellte ein Stimmlippen Bioreaktor (J1 Bioreaktor) mit einem Leistungsverstärker, einen Funktionsgenerator, einen geschlossenen Lautsprecher und eine Umfangs verankert Silikonmembran, die die schwingende Luft auf die beigefügten Zellen überträgt. Neonatale Vorhautfibroblasten in der J1-Bioreaktor kultiviert wurden 1 h Vibration bei 60, 110 oder 300 Hz unterzogen wird, mit einer Dehnung in der Ebene von bis zu 0,05%. Die qPCR Ergebnisse zeigten, dass die Expression einiger Gene ECM mäßig in Abhängigkeit von der unterschiedlichen Schwingungsfrequenzen verändertund Amplituden. Diese Bioreaktor entwickelt, während faszinierend, haben mehrere Einschränkungen. Zum Beispiel erfordert die T1-System eine große Anzahl von Verbindungsstangen und für mechanische Kopplung, die Begrenzung der Maximalfrequenzen erreichbar. Darüber hinaus können Zellen, um unerwünschte mechanische Bewegung und Flüssigkeits Störung, die die Datenauswertung erschweren unterworfen werden. Die J1-Bioreaktor, auf der anderen Seite, zeigt relativ geringe Wirkungsgrad der Energieumwandlung und ist nicht benutzerfreundlich. Darüber hinaus löst Vibrationen häufig die Zellbeladenen Konstrukte aus der zugrunde liegenden Silikonmembran. Der J2 Stimmlippen Bioreaktor hier berichtet, basierend auf dem gleichen Prinzip wie die J1-Version entworfen, wird die Konsistenz und Reproduzierbarkeit optimiert. Die Stimmbildung ähnlicher Vibrationen sind aerodynamisch in individuell angepassten Vibrationskammern, in denen MSC besiedelten Faser Poly (ε-Caprolacton) (PCL) Gerüste sind effektiver erzeugtEly gesichert. Laservibrometrie (LDV) ermöglicht dem Benutzer, den Vibrationsprofils der Membran / Gerüstanordnung zu überprüfen. In unserer Demonstration werden MSCs 200-Hz-Sinusschwingungen mit einer 1-Stunden-on-1-h-aus (OF) Muster für insgesamt 12 Stunden täglich für 7 Tage ausgesetzt. Zelluläre Antworten auf die verhängte Vibrations Hinweise werden systematisch untersucht. Insgesamt bietet die J2 Stimmlippen die benutzerfreundlichen Funktionen, so dass dynamische Zellkulturstudien, in einem hohen Durchsatz und reproduzierbar durchgeführt werden.

<table border="0" cellpadding="0" cellspacing="0" width="853">
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			<td height="16" style="height:17px;width:248px;">silicone elastomer kit</td>
			<td style="width:188px;">Dow Corning</td>
			<td style="width:124px;">Sylgard 184</td>
			<td style="width:293px;">cure the membrane at 100 C for 2 hr</td>
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		<tr height="16">
			<td height="16" style="height:17px;">PCL</td>
			<td>Sigma Aldrich</td>
			<td>440744-500G</td>
			<td>Mn ~ 80 kDa, dissolve overnight</td>
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			<td height="16" style="height:17px;">chloroform</td>
			<td>Sigma Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
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			<td height="16" style="height:17px;">human bone marrow-derived MSCs</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td>received with passage 2</td>
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			<td height="33" style="height:33px;">MSC maintenance media</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td style="width:293px;">10% FBS in basal media supplemented 
			with L-glutamine, gentamicin and amphotericin</td>
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		<tr height="16">
			<td height="16" style="height:17px;">Accutase cell dissociation reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
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			<td height="16" style="height:17px;">ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
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		<tr height="16">
			<td height="16" style="height:17px;">fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2006-1MG</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">MMP1 DuoSet ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY901</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HA ELISA kit</td>
			<td>Echelon Biosciences</td>
			<td>&#160;K-1200&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
			<td></td>
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		<tr height="16">
			<td height="16" style="height:17px;">propidium iodide&#160;</td>
			<td>Life Technologies</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Syto-13&#160;</td>
			<td>Life Technologies</td>
			<td>S7575</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">QuantiTect reverse transcription kit&#160;</td>
			<td>Qiagen</td>
			<td>205311</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">SYBR Green PCR master mix</td>
			<td>Life Technologies</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">replacement speaker</td>
			<td style="width:188px;">DAYTON audio 
			(via Parts Express)</td>
			<td>DS90-8</td>
			<td>paper cone, full range (80-13000 Hz), 85dB</td>
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		<tr height="16">
			<td height="16" style="height:17px;">Ergo Micro torque screwdriver</td>
			<td>Mountz</td>
			<td># 020377</td>
			<td>torque range: 20-120 cN.m</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">stereo speaker selector</td>
			<td>RadioShack</td>
			<td>40-244</td>
			<td>maximum power handling 50 W</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">function generator&#160;</td>
			<td>Agilent&#160;</td>
			<td>33220A</td>
			<td>frequency range 1 &#181;Hz- 20 MHz</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">power amplifier&#160;</td>
			<td>PYLE audio</td>
			<td>PylePro PT2400</td>
			<td style="width:293px;">frequency response: 10 Hz-50 kHz, two speaker 
			channels</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">cell culture incubator&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Steri-Cult 3307</td>
			<td></td>
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		<tr height="16">
			<td height="16" style="height:17px;">syringe pump&#160;</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
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		<tr height="16">
			<td height="16" style="height:17px;">High voltage power supply</td>
			<td>Spellman</td>
			<td>CZE 1000R</td>
			<td>output voltage: 0-30 kV</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">scanning electron microscope&#160;</td>
			<td>JEOL-USA</td>
			<td>JSM-7400F</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">desk gold sputter coater</td>
			<td>Denton Vacuum</td>
			<td>DSK00V-0013</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Doppler laser vibrometer&#160;</td>
			<td>Polytec</td>
			<td>PDV-100</td>
			<td>non-contact velocity measurement (0-22 kHz)</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCR sequence detection system&#160;</td>
			<td>Applied Biosystems</td>
			<td>ABI7300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">multiphoton confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510Meta NLO</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">UV-VIS Spectrophotometer&#160;</td>
			<td style="width:188px;">NanoDrop Products 
			via Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">VibSoft Data Acquisition Software</td>
			<td>Polytec</td>
			<td></td>
			<td>acquisition bandwidth up to 40 MHz</td>
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		<tr height="16">
			<td height="16" style="height:17px;">Origin 8.5 data analysis software&#160;</td>
			<td>OriginLab</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">qbasePlus qPCR data analysis software&#160;</td>
			<td>Biogazelle</td>
			<td>V2.3</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">aluminium alloy&#160;</td>
			<td>McMaster-Carr</td>
			<td>Alloy 6061</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">acrylic blocks</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
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		<tr height="27">
			<td height="27" style="height:27px;">polycarbonate anti-humidity chamber</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td style="width:293px;">Impact-Resistant&#160;Polycarbonate</td>
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		<tr height="16">
			<td height="16" style="height:17px;">screws&#160;</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">electronic cable/wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">medical grade PVC tubing</td>
			<td>US Plastic Corp.</td>
			<td>Tygon S-50-HL</td>
			<td>clear, biocompatible</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">10 mL syringe&#160;</td>
			<td>Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">21 G blunt ended needle</td>
			<td>Small Parts</td>
			<td>NE-213PL-25</td>
			<td>1-1/2&#34; length</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Alligator clip adapters&#160;</td>
			<td>RadioShack</td>
			<td>270-354</td>
			<td>fully insulated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">8 mm biopsy punch</td>
			<td><a href="http://www.sklarcorp.com/ProductListUS.aspx?DepartmentID=10&amp;CategoryID=96">Sklar&#160;Surgical Instruments</a></td>
			<td>96-1152</td>
			<td>sterile, disposable</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">12 mm biopsy punch</td>
			<td>Acuderm (via Fisher Scientific)</td>
			<td style="width:124px;">NC9998681</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">tissue culture flasks</td>
			<td>Corning</td>
			<td></td>
			<td>cell culture treated</td>
		</tr>
	</tbody>
</table>

construction and characterization of a novel vocal fold bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

Die PCL-Scaffolds durch Elektrospinning hergestellt enthalten mikrometergroßen Poren und Zwischen zufällig verstrickt Fasern mit einem durchschnittlichen Durchmesser von 4,7 um (Abbildung 4A). Bei einer höheren Vergrößerung, nanoskalige Rillen und Poren auf einzelnen Fasern (4B) sichtbar. Die Beschichtung der Gerüste mit Fibronektin verbessert Hydrophilie und erleichtert die anfängliche Zelladhäsion / Verteilung auf der PCL-Gerüst (unveröffentlichte Beobachtung). <p class=...

Watch this Scientific Journal Video about Construction and Characterization of a Novel Vocal Fold Bioreactor at JoVE.com

Construction and Characterization of a Novel Vocal Fold Bioreactor

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Konstruktion und Charakterisierung eines neuartigen Bioreaktor Stimmlippen

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

construction-and-characterization-of-a-novel-vocal-fold-bioreactor

Research

JoVE Journal

Bioengineering

9.1K Aufrufe.  University of Delaware. A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Serie: Construction and Characterization of a Novel Vocal Fold Bioreactor

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Engineering Biological-Based Gefäßprothesen Mit einem Pulsatile Bioreaktor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Forschung

Biotechnik

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

Synthetische Multi-Layer-, Selbst-Oszillierende Stimmlippen Modell Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Entwurf eines Biaxiale Mechanische Belastung Bioreaktor für Tissue Engineering

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Betrieb einer Tischbioreaktor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Untersuchung der dreidimensionalen Strömungstrennung, die durch ein Modell Vocal Fold Polyp induziert wird

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Charakterisierung von Leukozyten-Thrombozyten-Rich-Fibrin, ein neuartiges Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

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 ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

Aufbau und Aufbau eines handelsüblichen Algen-Photosynthese-Bioreaktors mit Temperatur-, Licht- und pH-Monitoring für kinetische Wachstumstests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Vorbereitung und Charakterisierung von neuartigen HDL-imitierenden Nanopartikeln für die Nervenwachstumsfaktorverkapselung

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...