Les auteurs remercient la Texas A &amp; M University ingénierie et d&#39;expérimenter la gare (TEES) pour le soutien financier de cette recherche. Lindsay Nail remercie le soutien de la Texas A &amp; M University Louis Stokes Alliance pour la participation de la minorité (LSAMP) et la National Science Foundation (NSF) Programme de bourses de recherche des cycles supérieurs (GRFP). Zhang Dawei grâce du Texas A &amp; M University Dissertation Fellowship.

1. synthèse PCL-DA Macromère <ol><li> Exécutez la réaction d&#39;acrylation. <ol><li> Peser 20 g de PCL-diol (M n = ~ 10 000 g / mol) dans 250 ml de ballon à fond rond équipé d&#39;un barreau d&#39;agitation magnétique téflon recouvert. </li><li> Dissoudre le PCL-diol dans du DCM. <ol><li> Ajouter 120 mLlof DCM dans le ballon (concentration = 0,17 g / ml). </li><li> Placer un septum en caoutchouc de façon lâche dans le col de la fiole de façon à éviter l&#39;accumulation de pression tout en empêchant l&#39;évaporation du DCM. </li><li> Agiter la solution pendant environ 30 min à ~ 250 tours par minute pour dissoudre complètement le polymère. </li></ol></li><li> Ajouter ~ 6,6 mg de 4-diméthylaminopyridine (DMAP) à la solution et dissoudre sous agitation. </li><li> Placer un septum en caoutchouc solidement dans le col du flacon. Laisser la solution pour continuer l&#39;agitation. </li><li> A travers le septum en caoutchouc, purge doucement le ballon avec N2 pendant ~ 3 min en utilisant une arrivée de N2 de l&#39;aiguille de pression positive et une aiguille ouvertecomme un exutoire. </li><li> Retirer l&#39;entrée N 2 et la sortie. </li><li> Ajouter 0,56 ml (4,0 mmoles) de triéthylamine (Et3N) goutte à goutte via une seringue en verre équipé d&#39;une aiguille insérée à travers le septum en caoutchouc. </li><li> Ajouter 0,65 ml (8,0 mmoles) de chlorure d&#39;acryloyle goutte à goutte via une seringue en verre équipé d&#39;une aiguille insérée à travers le septum en caoutchouc. </li><li> L&#39;orifice d&#39;entrée de retour N 2 dans le ballon et permettent le contenu sous agitation sous pression positive de N2 pendant environ 30 min. </li><li> Préchauffer un bain d&#39;huile à 55 ° C. </li><li> Après la alloué ~ 30 min, retirez l&#39;entrée N 2 et remplacer le septum avec un condenseur. </li><li> Immerger le ballon dans le bain d&#39;huile préchauffé. </li><li> Laisser le contenu du ballon sous agitation pendant 20 h. </li><li> Après la alloué 20 h, enlever le ballon de bain d&#39;huile et laisser le contenu refroidir à température ambiante. </li><li> En utilisant un évaporateur rotatif, éliminer le solvant de la fiole DCM. </li></ol></li><li> Purify le produit PCL-DA brut. <ol><li> Dans le ballon, ajouter ~ 135 ml d&#39;acétate d&#39;éthyle et on dissout le brut PCL-DA. </li><li> Gravity filtrer la solution à travers un papier filtre dans un propres de 250 ml flacon à fond rond. (Remarque: Solution peut épaissir sur le papier filtre, pas facilement en passant par l&#39;affirmative, appliquez soigneusement chaleur douce avec un pistolet de chauffage..) </li><li> En utilisant un évaporateur rotatif, l&#39;acétate d&#39;éthyle enlever le solvant de la fiole. </li><li> Pour le flacon, ajouter ~ 140 ml de DCM et de dissoudre le brut PCL-DA. </li><li> Transférer le contenu à un 500 ml ampoule à décanter. </li><li> Pour l&#39;entonnoir, ajouter 13,5 ml de 2 M de carbonate de potassium (K 2 CO 3). </li><li> Cap l&#39;entonnoir. Mélanger délicatement les deux couches en inversant l&#39;entonnoir et remuant doucement une ou deux fois, en prenant soin de relâcher la pression par l&#39;intermédiaire du robinet. Répétez 3 fois. </li><li> Remplacer le bouchon d&#39;une couche de Parafilm et laisser le mélange se séparer O / N (~ 12 h). </li><li> Recueillir le fond, oucouche Ganic dans un Erlenmeyer de 250 ml. </li><li> Ajouter ~ 5 g de sulfate de magnésium anhydre (MgSÛ4) dans le ballon et agiter doucement. </li><li> Masse filtrer le mélange à travers un papier-filtre qualitatif dans un récipient propre et 250 ml ballon à fond rond. </li><li> En utilisant un évaporateur rotatif, éliminer le solvant de la fiole DCM. </li><li> Sec sous vide poussé pour éliminer le DCM résiduel. (Remarque: PCL-DA doit être stockée à l&#39;abri de la lumière.) </li><li> Confirmez acrylation avec 1 H RMN 22,23. </li></ol></li></ol> 2. Préparation de la SMP Échafaudages (Figure 1) <ol><li> Préparer le modèle de sel fondu. <ol><li> Utilisation d&#39;un tamis de 425 um pour obtenir le chlorure de sodium (NaCl) 460 ± particules ~ 70 um de diamètre. (Note: La taille moyenne de particule peut être confirmée à partir de microscopie électronique à balayage [SEM] images avec le logiciel ImageJ.) 14 </li><li> Dans un ballon en verre de 3 ml (ID = 12,9 mm), ajouter 1,8 g de NaCl précédemment tamisée. </li><li> Ajouter lentement, en quatre portions, 7,5% en poids (par rapport au poids de sel) de l&#39;eau déminéralisée (0,146 g) à la fiole. Mélanger avec une spatule en métal après l&#39;addition de chaque portion d&#39;eau. </li><li> Boucher le flacon, l&#39;envelopper dans un tissu et le placer verticalement dans un tube de centrifugeuse. Centrifugeuse pendant 15 min à 3220 x g. </li><li> Retirez le bouchon et laisser sécher à l&#39;air O / N (~ 12 h). </li></ol></li><li> Dans un nouveau flacon en verre, de préparer une &quot;solution macromère&quot; en combinant 0,15 g de PCL-DA par ml de DCM. (Note:. Pour un échafaudage, ~ 1 ml de solution doit être préparée) Cap et mélanger la solution à haute vitesse sur un vortex pour ~ 1 min. </li><li> Dans un nouveau flacon de 3 ml en verre, de préparer une &quot;solution de photo-initiateur&quot; sur la base de 10% en poids de 2,2-diméthoxy-2-phényl acétophénone (DMP) dans du 1-vinyl-2-pyrrolidinone (NVP). Mélanger 0,115 g de DMP dans 1 ml de NVP. (Remarque: Pour un échafaudage, ~ 180 pi est requis.) Cap, envelopper dans du papier d&#39;aluminium (pour bloquer la lumière) et mélanger la solution à haute vitesse sur un vortexpour ~ 1 min. (Remarque: Si préparé à l&#39;avance, la solution doit être réfrigéré et protégé de la lumière.) </li><li> Envelopper le flacon contenant la solution de macromère (à l&#39;exclusion du bouchon) d&#39;une feuille d&#39;aluminium (pour bloquer la lumière) et ajouter par pipette 15% en volume (sur la base du volume total de solution de macromère) de la solution de photoinitiateur. Cap et mélanger la solution à haute vitesse sur un vortex pour ~ 1 min. </li><li> Envelopper le flacon contenant le modèle de sel fondu (à l&#39;exclusion du bouchon) avec une feuille d&#39;aluminium (pour bloquer la lumière) et ajouter la solution via une pipette macromère / photo-initiateur préalablement préparé (~ 0,6 ml ou jusqu&#39;à ce que le modèle est complètement recouverte). </li><li> Boucher le flacon, l&#39;envelopper dans un tissu et le placer verticalement dans un tube de centrifugeuse. Centrifuger pendant 10 min à 1260 x g pour distribuer la solution de macromère tout au long de la matrice. </li><li> Retirer la feuille d&#39;aluminium, décapsuler le flacon et d&#39;exposer à la lumière UV (365 nm, 25 W) pendant 3 min. O-Air sec / N. </li><li> Retirez le &quot;scaffol contenant du selD &quot;de la fiole avec des pincettes après avoir marqué et fracture de la partie supérieure de la fiole de verre. </li><li> Dans un bécher de 400 ml, ~ préparer 200 ml d&#39;un solvant eau / éthanol (1: 1 en volume: volume). </li><li> Maintenir l&#39;échafaud immergé dans le solvant pendant 4 jours avec des changements quotidiens de solvant eau / éthanol. </li><li> Retirer l&#39;échafaud du solvant et sécher à l&#39;air O / N. </li></ol> 3. Appliquer Polydopamine Coating SMP Échafaudages (Figure 1) <ol><li> Dans bécher de 400 ml équipé d&#39;un barreau d&#39;agitation en Teflon-couverte, préparer ~ 200 ml d&#39;une solution de chlorhydrate de dopamine (2 mg / ml dans du tampon Tris 10 mM, pH = 8,5, 25 ° C). Agiter à ~ 150 rpm. </li><li> Placez une aiguille jetable (longueur = 40 mm; jauge = 20) dans l&#39;échafaudage, ~ moitié de la distance grâce à l&#39;échafaud. Enrouler un fil autour du moyeu de l&#39;aiguille. </li><li> Immerger l&#39;échafaud (avec le moyeu de l&#39;aiguille au-dessus de la surface de la solution) dans la solution sous agitation de la dopamine par le fil d&#39;ancrage sur le bord du gobelet. </li><li> Dégazer l&#39;échafaud en plaçant une seringue dans le moyeu de l&#39;aiguille et de l&#39;utiliser pour aspirer l&#39;air sur l&#39;échafaud. (Note: Le dégazage est terminé lorsqu&#39;il n&#39;y a plus d&#39;air peut être retiré et la solution a complètement infiltré le échafaud). </li><li> Maintenir l&#39;échafaud immergé dans la solution de dopamine agitation pendant 16 heures. </li><li> Retirer l&#39;échafaud de la solution et enlever l&#39;aiguille. Rincer à l&#39;eau DI et sec dans un four sous vide à température ambiante pendant 24 h. </li><li> Placez l&#39;échafaud dans un four à 85 ºC pendant 1 heure. </li><li> Laisser l&#39;échafaud refroidir à température ambiante. L&#39;échafaudage cylindrique finale sera ~ 6 mm de diamètre x ~ 5 mm de hauteur. </li></ol> 4. évaluer le comportement &quot;d&#39;auto-ajustement&quot; <ol><li> Préparer un «modèle irrégulier de défaut CMF&quot; à l&#39;aide d&#39;une feuille de plastique rigide dont l&#39;épaisseur est de ~ 5 mm. Utilisation d&#39;une perceuse pour créer un vide à l&#39;intérieur de la feuille de matière plastique ayant un diamètre moyen un peu moins de ~ 6 mm, comme illustré dans la figure 2A. </li><li> Dans abEaker, de l&#39;eau DI à la chaleur (ce qui représente l&#39;utilisation clinique de sérum physiologique) à une température de 60 ° C ~. </li><li> Placez l&#39;échafaud dans le bécher de ~ 60 ° C de l&#39;eau. Utilisez des pinces pour pousser l&#39;échafaud en dessous de la surface de l&#39;eau, exposant tous les domaines de l&#39;eau. Continuer pendant environ 2 min ou jusqu&#39;à ce que l&#39;échafaudage est sensiblement adoucie (figure 2B). </li><li> Retirer l&#39;échafaud du bêcher et appuyez immédiatement (à la main) dans le défaut de modèle. </li><li> Laisser refroidir à température ambiante (~ 5-10 min) (Figure 2C). </li><li> Retirer du défaut d&#39;observer la nouvelle, forme temporaire fixe et le retour de l&#39;Etat relativement plus rigide (figure 2D). </li></ol> 5. Test de la mémoire de façonner le comportement <ol><li> Utiliser un analyseur mécanique dynamique (DMA; par exemple, un TA Instruments Q800 comme utilisé ici), lancer un cyclique thermique test de compression mécanique souche contrôlée sur un échafaudage sur deux cycles (N) pour déterminer la forme de fixité (R f) etfaçonner récupération (R r) (Figure 3). <ol><li> Équilibrer à 60 ° C (T élevé) pendant 5 min. </li><li> Compresser à une souche maximale (ε m = 50%) à 50% / min. </li><li> Tenir à ε m (5 min). </li><li> Refroidir à 25 ° C (T bas) et maintenir pendant 10 minutes pour fixer la forme temporaire. </li><li> Retirer la charge. </li><li> Mesurer la déformation à la rupture dans l&#39;état sans stress (ε u). </li><li> Réchauffer à 60 ° C (T haute) et maintenir pendant 10 min pour récupérer la forme permanente. </li><li> Mesurer la souche récupéré (ε p). </li><li> Alors qu&#39;il était encore à 60 ° C (T élevé), démarrer le cycle de Nd 2 (N = 2) par compression de l&#39;échafaudage à 50% de la hauteur récupérée après la 1 ère cycles (N = 1). </li><li> Répétez 5.1.3-5.1.8 Pour N = 2. </li><li> Calculer R f et R r pour N = 1 et 2 en utilisant les équations suivantes: R f (N) = [ε u (n) / ε m] r R (N) = [ε m - ε p (N)] / [ε m - ε p (N -1)] </li></ol></li></ol> 6. Visualisation taille des pores et des pores interconnectivité <ol><li> En utilisant la microscopie électronique à balayage (SEM; par exemple, une FEI Quanta SEM tel qu&#39;utilisé ici), observer la taille des pores et des pores d&#39;interconnexion. <ol><li> En utilisant des pinces pour maintenir l&#39;échafaud de SMP, plonger dans de l&#39;azote liquide 2 pendant 1 min. </li><li> Retirer du N2 liquide et fracture le long du milieu de l&#39;échafaudage avec une lame de rasoir propre. </li><li> Avec du ruban de carbone, apposer l&#39;une des moitiés SMP d&#39;échafaudage sur l&#39;échantillonscène avec la surface fracturée vers le haut. </li><li> Pulvérisation manteau avec Au-Pt (~ 4 nm). </li><li> Capturez l&#39;image SEM à une tension d&#39;accélération recommandée de 10-15 kV (figure 4A). </li></ol></li></ol> 7. Test de la bioactivité in vitro <ol><li> Dans un tube centrifuge de 50 ml, ajouter 30 ml de ~ 1X SBF 24. </li><li> Obtenir un échafaudage dans sa forme permanente originale, cylindrique moulé. Couper l&#39;échafaud de moitié (à travers le bord circulaire) en utilisant une lame propre. </li><li> Placez une demi-personne d&#39;échafaudage dans le tube de centrifugeuse préparé et bouchon. </li><li> Maintenir le tube à 37 ° C dans un bain-marie dans des conditions statiques sans changement SBF. </li><li> Après 14 jours, retirez l&#39;échafaud du SBF et l&#39;air sec pendant 24 heures. </li><li> Avec du ruban de carbone, apposer l&#39;échafaud sur la scène de l&#39;échantillon avec la surface fracturée vers le haut. </li><li> Pulvérisation manteau avec Au-Pt (~ 4 nm). </li><li> Capturez l&#39;image MEB à un refélicité tension d&#39;accélération de 10-15 kV (figure 4B). </li></ol>

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

The authors have nothing to disclose.

Ce protocole décrit la préparation d&#39;un échafaudage à base de PCL polydopamine revêtue dont l&#39;auto-ajustage comportement, ainsi que ostéoinductivité et la bioactivité, il est d&#39;intérêt pour le traitement de défauts osseux irréguliers FMC. Les aspects du protocole peuvent être modifiés pour modifier diverses caractéristiques d&#39;échafaudage. Le protocole commence par acrylation d&#39;un PCL-diol pour permettre la guérison UV. Dans l&#39;exemple indiqué, la PCL-...

Actuellement considérée comme la norme de cranio-maxillo-faciale (CMF) traitements de défaut osseux or, la transplantation de greffons autologues récoltées est entravé par des procédures complexes de greffage, morbidité du site donneur et la disponibilité limitée 1. Une difficulté particulière se dessine et fixant le autogreffe rigide étroitement dans le défaut afin d&#39;obtenir l&#39;ostéointégration et de prévenir la résorption du greffon. L&#39;ingénierie tissulaire a été étudiée comme une stratégie alternative à autogreffe et substituts osseux synthétiques (par exemple de ciment osseux) 2,3. Essentiel à la réussite d&#39;une approche de l&#39;ingénierie tissulaire est un échafaud avec un ensemble spécifique de propriétés. Tout d&#39;abord, afin de parvenir à l&#39;ostéointégration, l&#39;échafaudage doit former un contact étroit avec le tissu osseux adjacent 4. L&#39;échafaudage doit également être ostéoconducteur, permettant la migration cellulaire, la diffusion des nutriments et 4,5 neotissue dépôt. Ce comportement est généralement réalisé avec sca biodégradableffolds présentant un pore morphologie fortement interconnecté. Enfin, l&#39;échafaudage doit être bioactif de manière à promouvoir l&#39;intégration et la liaison avec le tissu osseux 5 environnante. Ici, nous présentons un protocole pour préparer un échafaudage d&#39;ingénierie tissulaire avec ces propriétés. Surtout, cette échafaudage présente la capacité de &quot;l&#39;auto-ajustement&quot; dans des défauts CMF irrégulières en raison de sa forme le comportement de mémoire 6. Thermosensible polymères à mémoire de forme (SMPS) sont connus pour subir un changement de forme lors de l&#39;exposition à la chaleur 7,8. PMC sont constitués de &quot;NETPoints» (c.-à-reticulations chimiques ou physiques) qui déterminent la forme permanente et des &quot;segments de commutation&quot; qui maintiennent la forme temporaire et retrouver la forme permanente. Les segments de commutation présentent une température de transition thermique (T trans) correspondant soit à la transition vitreuse (T g) ou fondre transition (T m) du polymère. CommeEn conséquence, les PMC peut être déformé de manière séquentielle dans une forme temporaire à T&gt; T trans, fixé dans la forme temporaire à T &lt;T trans, et récupéré à la forme permanente à T&gt; T trans. Ainsi, un échafaudage de SMP pourrait parvenir à &quot;l&#39;auto-ajustement&quot; dans un défaut CMF comme suit 6. Après exposition à réchauffer une solution saline (T&gt; T trans), un échafaudage de SMP deviendrait malléable, permettant un échafaudage cylindrique générique prêt à être dans un défaut irrégulière pressé à la main, avec récupération de forme promouvoir l&#39;expansion de l&#39;échafaudage à la limite de défaut. Lors du refroidissement (T &lt;T trans), l&#39;échafaud reviendrait à son état ​​relativement plus rigide, avec la forme de fixité maintenir sa nouvelle forme temporaire au sein de la défectuosité. Dans ce protocole, un échafaudage de lait écrémé en poudre est préparée à partir de polycaprolactone (PCL), un polymère biodégradable a étudié intensivement pour la régénération des tissus et d&#39;autres applications biomédicales 9-11. Pour mémoire de forme, ee T m de PCL sert de T trans et varie entre 43 et 60 ° C, en fonction du poids moléculaire du 12 PCL. Dans ce protocole, la T trans (c.-T m) de l&#39;échafaudage est de 56,6 ± 0,3 ºC 6. Afin d&#39;atteindre ostéoconductivité, un protocole a été développé pour rendre échafaudages SMP à base de PCL avec des pores fortement interconnectés basés sur une méthode 6,13,14 solvant coulée particules-lixiviation (SCPL). Le diacrylate de polycaprolactone (PCL-DA) (M n = 10 000 ~ g / mol) a été utilisé pour permettre rapide, la reticulation photochimique et a été dissous dans du dichlorométhane (DCM) pour permettre au solvant de coulée sur le modèle de sel. Après la cure photochimique et l&#39;évaporation du solvant, le modèle de sel a été éliminé par lessivage dans l&#39;eau. La taille moyenne de sel régule la taille des pores échafaudage. Fait important, le gabarit de sel a été fusionné avec de l&#39;eau avant le coulage de solvant pour obtenir pore interconnectiviTy. La bioactivité a été conférée à l&#39;échafaudage pour le SMP par la formation in situ d&#39;un revêtement sur ​​polydopamine parois des pores 6. La bioactivité est souvent introduit dans les échafaudages par l&#39;inclusion de verre ou de vitrocéramique de 15 charges. Toutefois, ceux-ci peuvent donner lieu à des propriétés mécaniques fragiles indésirables. La dopamine a été montré pour former une mince couche adhérente de polydopamine sur une variété de substrats 16-19. Dans ce protocole, l&#39;échafaudage pour SMP a été soumis à une solution légèrement basique (pH = 8,5) de la dopamine pour former un revêtement de nanothick de polydopamine sur toutes les surfaces de paroi des pores 6. En plus d&#39;améliorer l&#39;hydrophilie de surface pour améliorer l&#39;adhérence cellulaire et d&#39;étalement, polydopamine a été montré pour être biologiquement actif sur le plan de formation d&#39;hydroxyapatite (HAP) par exposition à un liquide corporel simulé (SBF) 18,20,21. Dans une dernière étape, l&#39;échafaud revêtu est exposé à un traitement thermique à 85 ° C (T&gt; T trans) which conduit à une densification échafaudage. Le traitement thermique a été noté précédemment comme essentiel pour le comportement à mémoire de forme d&#39;échafaudage, peut-être en raison de domaines cristallins PCL se réorganiser pour une plus grande proximité 14. Nous décrivons en outre des méthodes pour caractériser le comportement d&#39;auto-ajustement au sein d&#39;un défaut de modèle irrégulier, façonner le comportement de la mémoire en ce qui concerne les tests de compression mécanique cyclique thermiques déformation contrôlée (par exemple reprise de forme et la forme de fixité), pore morphologie, et dans la bioactivité in vitro. Stratégies pour adapter les propriétés d&#39;échafaudage sont également présentés.

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

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

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

L&#39;échafaudage de SMP à base de PCL résultant est capable d&#39;auto-montage dans un modèle CMF défaut (Figure 2). Après une brève exposition à chauffer une solution saline (~ 60 ° C), l&#39;échafaud cylindrique adoucit permettant l&#39;échafaud pour être pressé manuellement dans et d&#39;élargir l&#39;intérieur du défaut de modèle. Après refroidissement à TA, l&#39;échafaudage est fixé dans sa nouvelle forme temporaire qui est retenu lors de l&#39;élimination du défaut. <p...

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

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

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

Fabrication d&#39;une mémoire de forme &quot;auto-ajustement&quot; à base de PCL bioactifs, Polymer Échafaudages

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

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

Research

JoVE Journal

Bioengineering

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

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

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

Shape Memory Polymers for Active Cell Culture

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

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

Polymères à mémoire de forme pour la Culture cellule active

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

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Bio-ingénierie

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

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

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

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

Un Based Chitosan, Laser adhésif activé Thin Film chirurgicaux, «SurgiLux &#39;: Préparation et démonstration

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

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

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

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

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

Test de microtension environnement contrôlé de nanocomposites polymères mécaniquement adaptatifs pour<em&gt; Ex vivo</em&gt; Caractérisation

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

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

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

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

Fabrication microfluidique de fibres polymères et biohybrides avec une taille et une forme redessinées

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

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

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

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

Fabrication robuste préformes à fibres naturelles Utilisant bactérienne Cellulose Binder

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

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

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

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

Fabrication de mécanique accordable et bioactifs Métal échafaudages pour des applications biomédicales

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

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

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

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

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

Fabrication de Inverted colloïdal cristal Poly (éthylène glycol) Échafaudages: Une cellule plate-forme en trois dimensions de la culture pour l&#39;ingénierie tissulaire du foie

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

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

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

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

Fabrication et caractérisation des fantômes de tissu optique contenant la Macrostructure

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

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

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

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

Microdissection de tissu rénal primaire Segments et intégration avec la technologie de nouvelle construction exempte d’échafaudage

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

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

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

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

Fabrication évolutive d’étirable, Dual Channel, puces microfluidiques orgue

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