Nous remercions le Dr Jeffrey Caplan pour sa formation et des conseils sur l&#39;imagerie confocale. Nous remercions également le Keck microscopie électronique Lab et le Dr Chaoying Ni d&#39;assistance SEM. Ce travail est financé par les Instituts nationaux de la santé (NIDCD, R01DC008965 et R01DC011377). ABZ reconnaît NSF enseignement universitaire intégré et (IGERT) programme de financement la recherche de stage.

Assemblée bioréacteur 1. (Vidéo 1) <ol><li> Faire un moule en aluminium (de filière circulaire + entretoise broches) avec des dimensions intérieures et extérieures prédéterminées (Figure 1). </li><li> Utilisation du moule à partir de l&#39;étape 1.1, la fabrication d&#39;une membrane en silicone (diamètre: 42 mm, épaisseur: 1,5 mm, figure 1) avec un manchon retranché (diamètre: 12 mm, épaisseur ~ 0,25 mm, formée par la broche d&#39;écartement sur ​​la figure 1) en le milieu à l&#39;aide d&#39;un kit d&#39;élastomère de silicone disponible dans le commerce. </li><li> Faites une paire de blocs acryliques (Figure 2 (4, 5)) avec une ouverture circulaire (diamètre: 24 mm) au milieu, graver le haut (1,8 cm d&#39;épaisseur) et bas (0,9 cm d&#39;épaisseur) des blocs avec des crêtes correspondant et rainures 10. </li><li> Prendre en sandwich la membrane de silicone entre les blocs acryliques appariés. Fixez l&#39;ensemble avec quatre vis d&#39;angle à l&#39;aide d&#39;un tournevis dynamométrique micro fixé à une force constante (35 cN · m). En conséquence, un étanche à l&#39;eau, 24 mm de large et 18 mm chambre de vibration profonde est créée (figure 2C). </li><li> Monter une &quot;gamme étendue mini-woofer 3 (figure 2D, 8 Ω/20 W) sous la chambre de vibration par un autre jeu de vis de coin sur le bloc acrylique fond. A ce point, un module de vibration individuelle est assemblé. </li><li> Répliquer sept modules de vibration supplémentaires. Apposer quatre d&#39;entre eux à l&#39;une des deux barres d&#39;aluminium fixes (40 cm x 10 cm x 2,5 cm) en plaçant les bases d&#39;enceintes dans des trous circulaires régulièrement espacées (diamètre: 7 cm, épaisseur: 2 cm) découpées dans les bars. Stabiliser chaque haut-parleur en insérant une vis dans le côté de la barre d&#39;aluminium dans chaque trou circulaire. </li><li> Contrôler individuellement les haut-parleurs par un sélecteur de haut-parleur. Connectez les enceintes individuelles pour le sélecteur de fixer des fils aux entrées positives et négatives sur le corps du haut-parleur, puis aux sorties correspondantes sur le sélecteur. Le sélecteur de haut-parleur permet au signal de til générateur de fonction, après avoir traversé un amplificateur de puissance, pour atteindre les huit haut-parleurs à la fois (figure 2E). </li><li> Placez les deux tableaux de la chambre, le sélecteur de haut-parleur et l&#39;électronique associée à un boîtier anti-humidité. Loger la totalité de l&#39;ensemble dans un incubateur de culture cellulaire du commerce. </li><li> Nourrir les câbles principaux (à travers un tube de PVC de qualité médicale) reliant l&#39;amplificateur de puissance et le sélecteur d&#39;enceinte à travers l&#39;ensemble de filtre à l&#39;arrière de l&#39;incubateur. </li></ol> 2. Fabrication d&#39;échafaudage et caractérisation <ol><li> Dissoudre pastilles PCL dans le chloroforme à une concentration de 15% en poids. Chargez la solution dans une seringue de 10 ml surmonté d&#39;une aiguille émoussée 21 G. </li><li> Verrouiller la seringue sur une pompe à seringue programmable et régler le débit à 1 ml / h. </li><li> Placez le collecteur de papier d&#39;aluminium aluminium en face de l&#39;aiguille horizontalement, avec une aiguille distance pointe-à-collecteur de ~ 18 cm. </li><li> Fixer le alli positifalligator clip pour le milieu de l&#39;aiguille, et la pince crocodile de terre au collecteur d&#39;aluminium, puis réglez la tension de l&#39;alimentation haute tension à 15 kV. ATTENTION: haute tension, maintenir la distance de l&#39;aiguille. </li><li> Activer de façon séquentielle sur la pompe à seringue et l&#39;alimentation électrique; nettoyer rapidement / retirer de la solution de polymère résiduel entourant la pointe de l&#39;aiguille à l&#39;aide d&#39;une serviette en papier avant jets de fibres stable et Taylor cône 19 sont formés. </li><li> Laisser les fibres de s&#39;accumuler sur le collecteur Al jusqu&#39;à une épaisseur de 250 à 300 um ~ (~ 7 h dans les conditions de filage de courant). Stocker les échafaudages obtenus dans un dessiccateur sous vide pendant 1 à 2 jours pour éliminer tout solvant résiduel. </li><li> Image, les échafaudages, applique par pulvérisation avec de l&#39;or, à l&#39;aide d&#39;un microscope électronique à balayage pour montrer cohérent morphologie de la fibre. 10 </li></ol> 3. Assemblée bioréacteur et caractérisation <ol><li> Percez un disque cylindrique (diamètre: 8 mm) avec quatre bras (longueur: 2mm) sur le tapis PCL à l&#39;état filé (figure 2A) en utilisant d&#39;abord un diamètre de 12 mm poinçon de biopsie pour couper le diamètre extérieur du disque. Ensuite, utilisez une deuxième, 8 mm biopsie punch pour faire quatre 2 mm de long encoches espacées uniformément autour de la lame circulaire de marquer où les armes sont à couper. Après avoir marqué avec le poinçon de 8 mm, utiliser une lame de scalpel pour couper les bords des bras vers l&#39;extérieur. Insérer l&#39;échafaudage dans la rainure de la membrane en silicone par l&#39;intermédiaire des bras étendus (vidéo 1). Aplatir l&#39;échafaud inséré en appuyant doucement sur la surface en utilisant des pinces à tête plate. </li><li> Attacher un petit morceau de feuille mince d&#39;Al (8 mm x 2 mm, forme orthogonale, figure 2B) à l&#39;échafaud PCL pour aider à la réflexion laser. </li><li> Bloquer la membrane silicone / PCL échafaudage assemblée (comme indiqué dans l&#39;étape 1.4) dans la chambre de vibration. Ajouter 1,5 ml d&#39;eau dans la chambre pour hydrater l&#39;échafaud PCL avant vibrations. </li><li> Utilisation du générateur de fonction, introduire le signe de vibrationsal (par exemple, 200 Hz ondes sinusoïdales avec une tension crête-à-crête, Vpp, de 0,1 V) à la chambre acrylique prise en sandwich. Utilisez un voltmètre pour mesurer avec précision la tension à chaque entrée du haut-parleur. Remarque: la lecture Vcc du générateur de fonction sera différente de la tension éventuelle livré à l&#39;orateur. </li><li> Assemblez le seul point LDV et sécuriser le laser tête de capteur à fibre optique sur un trépied à tête pan-tilt. Angle de la tête de capteur de sorte qu&#39;il est orienté perpendiculairement à la table. Raccorder la tête de capteur de LDV au module d&#39;acquisition de données par l&#39;intermédiaire d&#39;un câble coaxial ensuite le module à l&#39;ordinateur portable via USB. </li><li> Focaliser le faisceau laser perpendiculairement à différents points prédéterminés sur la membrane en silicone (figure 2B et la figure 3). </li><li> Utilisation du logiciel d&#39;acquisition de données, enregistrer le déplacement mi-membrane. Cliquez sur &quot;Paramètres d&#39;acquisition&quot; dans le menu &quot;Options&quot;; puis changer le mode de mesure à &quot;FFT221;. Ensuite, cliquez sur le &quot;mesure continue&quot; dans la barre d&#39;outils principale puis cliquez sur le pic qui se forme à la fréquence choisie (Figure 6D) pour enregistrer le déplacement. </li><li> Tracer le déplacement mi-membrane normal (w 0) en fonction de la position relative entre le substrat. Construire une palette de couleurs 3D du profil vibratoire de surface à l&#39;aide Origine logiciel d&#39;analyse de données 8.5. </li></ol> 4. Vibrante culture cellulaire <ol><li> MSC dérivées de moelle osseuse sous-culture T150 humain dans des flacons de culture de tissu avec une densité d&#39;ensemencement initiale de 4000-5000 cellules / cm 2 dans les milieux d&#39;entretien MSC. </li><li> Après 7-8 jours de culture cellulaire (à ~ 85% de confluence), trypsiniser les cellules avec un réactif de dissociation cellulaire tel que Accutase, compter l&#39;aide d&#39;un hématimètre, centrifugeuse (440 g pendant 5 min), et remettre en suspension le culot cellulaire dans milieu d&#39;entretien frais de MSC à une concentration de 4,5 x 10 6 cellules / ml. </li><li> Plonger la Scaffo PCLld éthanol à 70% O / N. Après que le solvant est évaporé, exposer les deux côtés de l&#39;échafaudage à la lumière UV germicide (254 nm) pour 5-8 minutes. </li><li> Faire tremper l&#39;échafaud PCL dans une solution de fibronectine 20 pg / ml à 37 ° C pendant 1 heure. Insérer l&#39;échafaudage de fibronectine dans la membrane en silicone. Assemblez le bioréacteur tel que détaillé à l&#39;étape 1. </li><li> Distribuer 40 ul de la suspension cellulaire uniformément sur l&#39;échafaud PCL sécurisé. Permettent aux cellules de se fixer pour 1 à 1,5 h avant d&#39;ajouter encore 1,5 ml de milieu frais à la chambre de vibration. </li><li> Culture de l&#39;échafaud PCL MSC chargé statiquement pendant 3 jours et rafraîchir les médias à la fin de la culture statique. </li><li> Imposer des régimes de vibration sélectionnés pour les constructions cellulaires. Remarque: Par exemple, les cellules sont soumises à un 1-hr-sur-1-h-off (DE) vibrations à 200 Hz avec aw 0 de ~ 40 um pendant 12 heures par jour pour un maximum de 7 jours. Constructions soumises à des stimulations vibratoires sont désignés comme échantillons Vib et les culturouge statique dans des chambres de vibrations identiques servent de témoins statiques (STAT). </li></ol> 5. Évaluations biologiques <ol><li> Recueillir 200 ul de milieux de culture cellulaire à partir de chaque chambre tous les deux jours (jours 1, 3, 5 et 7) et en commun des fractions aliquotes d&#39;un même échantillon en même temps (800 ul chacun). </li><li> Quantifier la production cellulaire de la matrice métalloprotéinase-1 (MMP1) et HA en utilisant un kit ELISA MMP1 développement et un kit ELISA de compétition de l&#39;acide hyaluronique, respectivement, à la suite de la procédure des fabricants. Pendant ce temps, le dosage de la production de précurseur de l&#39;élastine soluble suivant la procédure ELISA précédemment rapporté. Huit </li><li> À la fin du dernier cycle de vibration au jour 7, supprimer rapidement les constructions cellulaires des chambres de vibrations à l&#39;aide de pinces acérées et brièvement les rincer avec de phosphate froid (PBS, 4 ° C). </li><li> Pour la coloration en direct / mort, incuber les constructions avec l&#39;iodure de propidium (1:2000 dans du PBS) etSyto-13 (1:1000 dans du PBS) en même temps pendant 5 min à température ambiante. Image Les constructions colorées avec un microscope confocal multiphotonique. </li><li> Séparément, enfichable geler les constructions cellulaires PBS rincés sur glace sèche et d&#39;extraire l&#39;ARN cellulaire total en suivant un protocole indiqué précédemment pour l&#39;analyse génétique. 9 </li><li> Vérifiez la quantité et la qualité de l&#39;ARN extrait à l&#39;aide d&#39;un spectrophotomètre UV-Vis. échantillons d&#39;ARN avec des ratios de 1,8-2,2 A260/A280 et A260/A230 sont utilisées pour l&#39;analyse qPCR ultérieure. </li><li> N º de transcrire l&#39;ARN (500 ng / échantillon) en ADNc en utilisant un kit de transcription inverse disponible dans le commerce. </li><li> Effectuer la réaction de PCR sur un système de détection de séquence en temps réel en utilisant un mélange maître PCR disponible dans le commerce en suivant la procédure détaillée précédemment. 8 </li><li> Analyser les résultats de qPCR en utilisant un logiciel d&#39;analyse de données qPCR commerciale. Pour assurer la fiabilité de l&#39;analyse de données, plusieurs gènes de référence (YWHAZ, BPT, PPIA) sont utilisés comme intcontrôles ernal, et la variance de l&#39;efficacité des amorces spécifiques sont pris en compte. 8 </li></ol>

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L&#39;ingénierie des tissus succès des cordes vocales fonctionnels in vitro nécessite la recréation d&#39;un pli comme micro vocal à la médiation des comportements de cellules multipotentes. Il est généralement admis que les structures de tissus ou d&#39;organes reflètent les fonctions qu&#39;ils sont appelés à exécuter. 22 Pour les tissus des cordes vocales, les vibrations de haute fréquence qui se produisent pendant la phonation sont proposées pour être important pour la maturation d...

Le pli vocal humain, composé d&#39;une couche épithéliale, la lamina propria (LP) et le muscle vocalis, est un tissu mou spécialisé qui convertit le flux d&#39;air dans les poumons en ondes acoustiques pour produire des sons. 1 Cordes vocales oscillent régulièrement pendant la phonation normale, présentant des souches de jusqu&#39;à 30% à des fréquences fondamentales allant de 100-300 Hz. 2 adultes cordes vocales LP est une structure composée d&#39;un gradient superficiel (SLP), un intermédiaire (PLI) et un (DLP) couche profonde. D&#39;autres groupes de classification la épithélium et le SLP que la couche de muqueuse, et combine l&#39;ILP et DLP dans le ligament vocal. 3 La couche de SLP contient essentiellement une matrice amorphe avec des fibres de collagène peu dispersés, alors que le ligament est enrichie en collagène mature et les fibres d&#39;élastine pour offrir une résistance suffisante. 4 La structure et la mécanique des cordes vocales nouveau-nés varient considérablement de leurs homologues adultes. Bien que le mécanismes régulant le développement de cordes vocales et de la maturation ne sont pas encore entièrement compris, la preuve expérimentale a souligné les rôles qui définissent des contraintes mécaniques vocalisation dérivés. Plusieurs conditions médicales, y compris l&#39;abus de la voix, des infections, des produits chimiques irritants et des interventions chirurgicales, peuvent endommager la corde vocale. Troubles des cordes vocales affectent environ 3-9% de la population américaine. Méthodes actuelles de traitement pour les troubles des cordes vocales sont limitées 5 et un tissu approche d&#39;ingénierie à base de cellules souches est apparue comme une stratégie prometteuse pour la restauration de la fonction de cordes vocales. cellules souches mésenchymateuses (CSM) sont une alternative appropriée aux primaires fibroblastes des cordes vocales pour voix génie pli de tissu. 6-9 spécification du destin des cellules souches et le développement du tissu ultérieure sont médiés par le créneau spécifique qu&#39;ils résident dans, dont l&#39;état mécanique est un facteur essentiel. 10 forces mécaniques sont des régulateurs essentiels du tissu morphogenèse unnd homéostasie, en particulier pour les tissus qui sont couramment soumis à une charge. 11 Du point de vue de l&#39;ingénierie tissulaire, il a été démontré que l&#39;exposition à des stimulations mécaniques physiologiquement pertinentes favorise la différenciation des cellules souches et spécifique d&#39;un tissu remodelage de la matrice 12 à 15. bioréacteurs de culture de tissus sont conçus pour simuler l&#39;environnement physiologique désirée pour la croissance des cellules ou des tissus in vitro. Pour vocal génie pli de tissu, il est particulièrement important de recréer l&#39;environnement mécanique des cordes vocales phonating. Une corde vocale bioréacteur idéal devrait fournir efficacement des signaux vibratoires de cellules en culture, ce qui permet un contrôle facile sur la fréquence, l&#39;amplitude et la durée des vibrations. Et ses collègues ont conçu titze un pli bioréacteur vocal (T1 bioréacteur) 16 qui combine étirement statique à haute fréquence (20 à 200 Hz) des oscillations de stimuler la production cellulaire de protéines de la matrice. Using ce bioréacteur, Webb et ses collègues 17 ont étudié les effets de 10 jours, les vibrations de 100 Hz sur les fibroblastes dermiques cultivées dans un acide hyaluronique (HA) à base d&#39;hydrogel. Les constructions soumises à des vibrations présentaient une expression élevée de HA synthase-2 (HAS2), la décorine, la fibromoduline et la métalloprotéinase matricielle-1 (MMP1), par rapport aux témoins statiques. Les effets stimulants sont révélés être dépendants du temps. Plus récemment, notre groupe de 18 a réuni une fois bioréacteur vocal (J1 bioréacteur) en utilisant un amplificateur de puissance, un générateur de fonctions, un haut-parleur clos et une membrane de silicone circonférence ancrée qui transfère l&#39;air oscillant aux cellules attachées. Des fibroblastes de prépuce néonatal cultivées dans le bioréacteur à J1 ont été soumis à 1 h de vibration à 60, 110 ou 300 Hz, avec une déformation dans le plan d&#39;au plus 0,05%. Les résultats de qPCR ont suggéré que l&#39;expression de certains gènes de la MEC a été légèrement modifiée en réponse à des fréquences vibratoires variéeset amplitudes. Ces conceptions de bioréacteurs, tandis intrigante, présentent plusieurs limites. Par exemple, le système T1 nécessite un grand nombre de connecteurs et de barres de couplage mécanique, ce qui limite les fréquences maximales réalisables. De plus, les cellules peuvent être soumises à une agitation mécanique indésirable et fluide perturbation qui complique l&#39;interprétation des données. Le bioréacteur à J1, d&#39;autre part, présente une efficacité relativement faible de conversion d&#39;énergie et n&#39;est pas facile à utiliser. En outre, les vibrations se détache souvent les constructions cellulaires chargées de la membrane de silicone sous-jacent. Le J2 cordes vocales bioréacteur rapporté ici, conçu sur la base du même principe que la version J1, est optimisée pour l&#39;uniformité et la reproductibilité. Les vibrations de la phonation imitant sont générés aérodynamique dans des chambres de vibrations équipés individuellement où poly fibreux MSC-peuplé (ε-caprolactone) (PCL) échafaudages sont effectively fixé. Laser Doppler Vibrométrie (LDV) permet à l&#39;utilisateur de vérifier le profil vibratoire de l&#39;ensemble membrane / échafaud. Dans notre démonstration, les CSM sont exposés à 200 Hz vibrations sinusoïdales avec un (DE) modèle 1-hr-sur-1-h-off pour un total de 12 heures par jour pendant 7 jours. Les réponses cellulaires aux signaux vibratoires imposées sont étudiées systématiquement. Dans l&#39;ensemble, la corde vocale J2 fournit la plupart des fonctionnalités conviviales, permettant des études de culture cellulaire dynamique à être menées dans un débit élevé et de façon reproductible.

<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>
		</tr>
		<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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		<tr height="16">
			<td height="16" style="height:17px;">chloroform</td>
			<td>Sigma Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr height="16">
			<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>
		</tr>
		<tr height="33">
			<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>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Accutase cell dissociation reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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.

Les échafaudages PCL fabriqués par électrofilage contiennent des pores interstitiels de taille micronique et des fibres enchevêtrées de manière aléatoire ayant un diamètre moyen de 4,7 pm (Figure 4A). A plus fort grossissement, rainures et pores nanométriques sont visibles sur fibres individuelles (figure 4B). Revêtement des échafaudages avec la fibronectine améliore le caractère hydrophile et facilite l&#39;adhérence cellulaire initiale / épandage sur l&#39;échafaudage ...

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.

Construction et caractérisation d&#39;un pli bioréacteur roman Vocal

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 vues.  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.

Vidéo: 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.

Greffes d&#39;ingénierie biologique vasculaire basé sur l&#39;aide d&#39;un bioréacteur pulsatile

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

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Bio-ingénierie

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.

Synthétique, Multi-Layer, auto-oscillation de fabrication Vocal Modèle Fold

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.

Conception d&#39;un bioréacteur de chargement mécanique biaxial pour l&#39;ingénierie tissulaire

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.

Fonctionnement d&#39;un bioréacteur de laboratoire

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.

Étude de la séparation d’écoulement tridimensionnelle induite par un polype de pli vocal modèle

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.

Caractérisation de leuco-plaquettaire Rich fibrine, un roman biomatériaux

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.

Un bioréacteur pour Novel Haute Culture de la densité des communautés microbiennes diverses

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.

Construction et mise en place d&#39;un bioréacteur photosynthétique Algal à échelle de banc avec surveillance de la température, de la lumière et du pH pour les tests de croissance cinétique

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.

Préparation et caractérisation de nouvelles nanoparticules imitant HDL pour l&#39;encapsulation du facteur de croissance nerveuse

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