Les auteurs sont reconnaissants pour le soutien de bourses NIH Directeur de Innovator Nouvelles (1DP2 OD006499-01 à AA et 1DP2 OD006460-01 à AJE), et King Abdulaziz City pour la science et la technologie (UC San Diego Centre d&#39;excellence en nanomédecine). Nous tenons à remercier Mme Jessica Moore pour ses commentaires critiques sur le manuscrit.

1. Synthèse de l&#39;marquées par fluorescence Acryloyl-PEG-RGDS <ol><li> Faire réagir le peptide RGDS avec acryloyl-PEG-succinimidyl ester carboxyméthylique (aPEG-SMC, PEG MW: 3400 g / mol) et NN diisopropyléthylamine (DIPEA) dans des rapports molaires 1.2:1:2 dans le diméthylsulfoxyde (DMSO), sous argon, à température ambiante pendant une nuit. </li><li> Confirmer la conjugaison par laser assistée par matrice de désorption / ionisation de temps de vol (MALDI-TOF) par spectrométrie de masse. Ajouter 1 ml de solution réaction aPEG-RGDS à une tache d&#39;échantillon sur la cible MALDI et sec. Préparer une solution saturée de Universal MALDI Matrix dans le tétrahydrofurane (THF) et le vortex pendant 1 min. Ajouter environ 1 ul de cette solution à l&#39;endroit même échantillon. Répétez la procédure pour aPEG-SCM pour la comparaison. Charger et analyser. Le poids moléculaire de aPEG-RGDS doit être supérieure à aPEG-SCM (Figure 2). </li><li> À conjuguer le fluorophore, ajouter une quantité équimolaire d&#39;Alexa Fluor 350 acide carboxylique (ester succinimydyl), dissousdans un volume minimal de DMSO, à la solution réactionnelle aPEG-RGDS de 1,1 et à réagir sous atmosphère d&#39;argon à température ambiante pendant une nuit. </li><li> Purifier aPEG-RGDS-350 par dialyse (MW 3500 Da) contre DI-H 2 O à 4 ° C pendant 48 heures à rapport volumétrique 1000:1, l&#39;échange de dialysat au moins deux fois par jour. </li><li> Congelez sécher le purifiée aPEG-RGDS-350 dans un Labconco Freezone plus ou le système de gel équivalent sec et conserver à -20 ° C. </li></ol> 2. Préparation d&#39;un moule de fabrication 2D et d&#39;un gel de PEG 2D avec alternance Rgds-350 Couches <ol><li> Préparer des lames de verre hydrophobes. Placez lames de verre propres dans un plat en verre dans un four sous vide. La chaleur à 80 ° C pendant 30 min pour sécher complètement les surfaces. Lieu plat avec les diapositives d&#39;une hotte et ajouter 250 ul Sigmacote à chaque diapositive, balançant doucement pendant 30 secondes à la surface de manteau tout entier. Bien rincer les lames recouvertes avec du méthanol à 100%, suivi d&#39;un lavage à l&#39;eau distillée, le trempage deux fois pendant 5 min dans au LEAt 10 ml. </li><li> Couper entretoises en silicone (0,8 mm d&#39;épaisseur) avec 10 poinçons de biopsie mm. </li><li> Autoclaver les entretoises en silicone et des lames de verre Sigmacote traités. </li><li> Formuler des solutions pour chaque couche respective dans des microtubes individuels par mélange PEG diacrylate (PEGDA) précurseur (concentration finale 15% v / v) avec différentes quantités de l&#39;iodixanol (solution mère à 60% dans l&#39;eau) pour obtenir des concentrations finales variant (par exemple 40%, 30%, 20% et 10%), complétant le volume restant avec du tampon phosphate salin (PBS) pour obtenir des solutions de densité à gradient. De la même manière, pour les couches alternées, mélange aPEG-RGDS-350 (concentration finale de 8 mM) avec iodixanol et PBS pour obtenir des concentrations différentes (par exemple, 35%, 25% et 15%). </li><li> Ajouter photo-initiateur (2-hydroxy-4&#39;-(2-hydroxyéthoxy)-2-méthylpropiophénone, stock 333mg/ml en N-vinylpyrrolidone) à chaque solution pour différentes couches (10 ul de solution mère par ml de chaque solution de couche). Photoinitiateur is ajouté en dernier pour éviter la polymérisation avant la superposition des gels dans le moule, car elle est sensible à la lumière. </li><li> Les étapes suivantes de ce protocole sera effectué dans une enceinte de biosécurité pour garantir la stérilité. </li><li> Filtrez-stériliser chaque solution à l&#39;aide d&#39;une seringue de 1 ml stérile et filtre de 0,2 um. Assembler le réglage de moule en prenant en sandwich l&#39;élément d&#39;espacement entre deux lames de verre imprégnées et Sigmacore avec des colliers de placer comme illustré à la figure 1. </li><li> Jeter les gels couches en ajoutant la solution la plus dense (par exemple PEGDA avec iodixanol 40%) en premier, suivi par une solution moins dense (par exemple aPEG-RGDS-350 avec iodixanol 35%). Répétez la réalisation alternative de couches pour atteindre plusieurs couches de composition souhaitée et la densité comme le montre la figure 1. </li><li> Irradier le moule avec de la lumière à 365 nm pendant 3 min en utilisant un portable UVR-9000 lampe. Laisser les gels polymérisés durcir pendant 5 min. Retirer les pinces, puis soulevez doucement le dessus en verretoboggan et le moule, les gels DGPM stratifiés restera sur les diapositives. En utilisant une spatule stérile, placer soigneusement les gels dans un tube de 50 ml contenant du PBS stérile ou milieu de culture pour le lavage. </li><li> Laver les gels polymérisés dans du PBS à 1,000:1 rapport volumétrique, l&#39;échange de tampon au moins deux fois par jour pour éliminer le modificateur de densité, photo-initiateur, et le polymère qui n&#39;a pas réagi. En variante, PBS peut être échangé avec un milieu de croissance cellulaire. Stocker les gels DGPM dans du PBS ou un milieu de croissance pour l&#39;expérience de culture cellulaire décrit à l&#39;étape 3. </li><li> Pour visualiser des couches alternées, organiser gel DGMP (ces gels ne seront pas utilisables pour la culture cellulaire) le long d&#39;une règle sur le plateau d&#39;échantillons d&#39;une unité VersaDoc documentation de gel. Exposer les gels à 350 nm, le temps d&#39;exposition varie en fonction de la concentration du fluorophore. Une alternance de bandes sombres et bleu dans l&#39;hydrogel DGMP démontrer la formation de couches discrètes de composition chimique distincte (figure 3). </li></ol><ol><li> Pour les gels incorporant peptide RGDS, utiliser des cellules d&#39;adhérence dépendants, tels que myoblastes C2C12. </li><li> Insérez doucement gels DGPM (stocké dans du PBS) dans les puits de plaques à 48 puits de culture cellulaire en utilisant un grattoir à cellules stérile dans une enceinte de sécurité biologique. </li><li> Milieu de croissance pré-chaud (milieu de Dulbecco modifié Eagle ou DMEM supplémenté avec 10% v / v de sérum de veau fœtal et 1% v / v 100x solution de pénicilline-streptomycine) et de PBS dans un bain-marie réglé à 37 ° C. </li><li> Laver une plaque 60% de confluence (10 mm) de cellules C2C12 trois fois avec du PBS. Aspirer PBS et les cellules de récolte en ajoutant 1 ml de 0,25% de trypsine-EDTA et incuber à 37 ° C pendant 2 min. Remettre en suspension les cellules dans un milieu de croissance et des cellules de comptage. Ensemencer la DGMP gel contenant de la culture cellulaire et avec des myoblastes C2C12 (20.000 cellules / cm 2). Incuber les cellules à 37 ° C dans 5% de CO 2/95% d&#39;humidité relative. Moyen d&#39;échange doucement au bout de 4 h, attention à ne pas redéplacer légèrement cellules adhérentes. </li><li> Après 24 h, la fixation des myoblastes C2C12 sur les couches RGDS contenant des gels DGPM peut être confirmé par épifluorescence et microscopie à contraste de phase (Zeiss Axiovert 200). </li></ol>

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

Les auteurs n&#39;ont pas de conflits d&#39;intérêts à déclarer.

DGMP est une stratégie simple pour la préparation de gels multicouches qui ne repose pas sur une instrumentation coûteuse. Ce protocole peut être adapté pour créer des échafaudages en utilisant d&#39;autres matériaux biocompatibles, tels que le collagène et l&#39;acide hyaluronique. Petites molécules bioactives, par exemple de cellules de stimulation d&#39;adhérence peptide RGDS, peut être attaché à la matrice de polymère pour empêcher le mélange de signaux entre les couches. Protéines peuvent être encapsulées dans des couches distinctes sans la nécessité d&#39;une conjugaison chimique car ils en fonction de la taille de maille de matrice, sont moins sujettes à diffuser à travers les hydrogels 10. Ici, nous avons utilisé l&#39;iodixanol (Nycoprep), un modificateur de la densité inerte, ce qui a déjà été utilisé pour des applications de cellules viables. Modificateurs de densité tels que le saccharose et le dextrose peuvent également être utilisés. En faisant varier le temps de décantation (t s), on peut peaufiner les interfaces entre deux couches pour produire des transitions en douceur ou tranchants besoin (temps de décantation donne plus des transitions plus douces) &lt;sup&gt; 10. Par exemple, des transitions plus douces entre les couches de gel DGPM peut être utilisé pour générer un gradient continu d&#39;une information biologique pour l&#39;étude des processus cellulaires tels que la chimiotaxie. L&#39;effet du modificateur de la densité sur la rigidité du gel est illustré à la figure 5 pour un gel de 15% aPEGda; une caractérisation plus complète de la rigidité et de la porosité en fonction de PEGDA et les concentrations iodixanol est actuellement en cours d&#39;évaluation. Alors que la concentration PEGDA dans cet exemple est relativement élevé, nous avons observé un module élastique de 60% supérieure dans des gels avec iodixanol 30% par rapport aux gels sans. La variation de la rigidité du gel peut être réglée par modulation de la concentration ou la densité de réticulation macromère. Nous avons également appliqué la technique DGMP pour créer en 3D en utilisant des gels de polyacrylamide multicouches et des précurseurs de PEG 10. Faire varier la concentration ou le degré de réticulation du prépolymère permet une variation structurelle dans leéchafaudages, qui peuvent être utilisées pour étudier le comportement des cellules telles que la croissance et la migration polarisée en 3D. En résumé, DGMP est une technique adaptable qui peut être appliqué à fabriquer des échafaudages en 2D et 3D à partir d&#39;une variété de matériaux biocompatibles pour une large gamme d&#39;applications de recherche biomédicale et de base.

<table border="1"><tbody><tr><td> Réactif ou instrument </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Polyéthylène glycol succinimydyl carboxyméthylcellulose (a-PEG-SCM) </td><td> Laysan </td><td> 120-64 </td></tr><tr><td> Polyethelyene glycol diacrylate (PEGDA) </td><td> Labs Dajac </td><td> 9359 </td></tr><tr><td> Arginine-glycine-acide aspartique-sérine (RGDS) </td><td> American Peptide </td><td> 49-01-4 </td></tr><tr><td> N, N - diisopropyléthylamine (DIPEA) </td><td> Sigma </td><td> D125806 </td></tr><tr><td> Diméthylsulfoxyde (DMSO) </td><td> Sigma </td><td> D2438 </td></tr><tr><td> N, N-diméthylformamide (DMF) </td><td> Pêcheur </td><td> D119-4 </td></tr><tr><td> Tétrahydrofurane (THF) </td><td> Pêcheur </td><td> T397 </td></tr><tr><td> Cassette de dialyse (3500Da) </td><td> Thermo Scientific </td><td> 66330 </td></tr><tr><td> Alexa Fluor 350 ester d&#39;acide carboxylique succinimydyl </td><td> Life Technologies </td><td> A-10168 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> Entretoises en silicone </td><td> Grainger </td><td> 1MWA4 </td></tr><tr><td> Pinces à biopsie </td><td> Acuderm </td><td> P1025 (10 mm) P850 (8 mm) </td></tr><tr><td> Phosphate de Dulbecco saline tamponnée (DPBS) </td><td> Hyclone </td><td> SH30028 </td></tr><tr><td> Iodixanol (NycoPrep) </td><td> Pêcheur </td><td> NC9388846 </td></tr><tr><td> 2-hydroxy-4&#39;-(2-hydroxyéthoxy)-2-méthylpropiophénone </td><td> Sigma </td><td> 410896 </td></tr><tr><td> Dulbecco modifié Eagle (DMEM) </td><td> Life Technologies </td><td> 11054 </td></tr><tr><td> Sérum de veau fœtal </td><td> Life Technologies </td><td> 10082 </td></tr><tr><td> Pénicilline-streptomycine </td><td> Life Technologies </td><td> 15140 </td></tr><tr><td> Myoblastes C2C12 </td><td> ATCC </td><td> CRL-1772 </td></tr><tr><td> MALDI </td><td> Bruker </td><td> N / A </td></tr><tr><td> UVR-9000 </td><td> Bayco </td><td> UVR-9000 </td></tr><tr><td> VersaDoc </td><td> Bio-Rad </td><td> N / A </td></tr></tbody></table>

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

Matrices complexes de culture de tissus, dans lequel les types et les concentrations de stimuli biologiques (par exemple des facteurs de croissance, des inhibiteurs ou des petites molécules) ou de la structure de matrice (par exemple la composition, la concentration ou la rigidité de la matrice) varient dans l&#39;espace, permettrait à un large éventail d&#39;enquêtes concernant la façon dont ces variables influent sur la différenciation cellulaire, la migration et d&#39;autres phénomènes. Le défi majeur dans la création de matrices stratifiés est le maintien de l&#39;intégrité structurale des interfaces des couches sans diffusion de composants individuels de chaque couche 1. Les méthodes actuelles pour atteindre cet objectif comprennent PHOTOSTRUCTURATION 2-3, lithographie 4, functionalization5 séquentiel, lyophilisation 6, microfluidique 7 ou centrifugation 8, dont beaucoup exigent une instrumentation sophistiquée et des compétences techniques. D&#39;autres s&#39;appuient sur ​​la fixation séquentielle de couches individuelles, qui peuvent conduire à la délamination de couches 9 </sup&gt;. DGMP surmonte ces problèmes en utilisant un modificateur de la densité inerte tel que l&#39;iodixanol de créer des couches de différentes densités 10. Depuis le modificateur de densité peut être mélangé à une autre molécule bioactive ou prépolymère, DGMP permet à chaque couche d&#39;échafaudage à être personnalisés. Simplement varier la concentration du modificateur de densité empêche le mélange des couches adjacentes pendant qu&#39;ils restent aqueuse. Subséquente seule étape de polymérisation donne lieu à une structure continue multicouche échafaudage, dans lequel chaque couche a chimique distincte et des propriétés mécaniques. Le modificateur de densité peut être facilement enlevée avec suffisamment de rinçage sans perturbation des couches individuelles ou de leurs composantes. Cette technique est donc bien adapté pour la création d&#39;hydrogels de différentes tailles, formes et matériaux. Un protocole de fabrication d&#39;un 2D-polyéthylène glycol (PEG) du gel, dans lequel des couches alternées incorporer RGDS-350, est décrit ci-dessous. Nous utilisons PEG bomme il est biocompatible et inerte. RGDS, un peptide de 11 l&#39;adhésion cellulaire, est utilisé pour démontrer restriction spatiale d&#39;une information biologique, et la conjugaison d&#39;un fluorophore (Alexa Fluor 350) nous permet de distinguer visuellement les différentes couches. Cette procédure peut être adaptée à d&#39;autres matériaux (par exemple le collagène, l&#39;acide hyaluronique, etc) et peut être étendue à fabriquer des gels 3D avec quelques modifications 10.

MALDI-TOF confirme la conjugaison du peptide RGDS à acryloyl-PEG (figure 2). Gel d&#39;imagerie révèle alternance Rgds-350 (bleu) couches après photopolymérisation (figure 3A). Comme le montre la figure 3A, la taille 2D gel DGMP peut varier en fonction du diamètre des moules en silicone (10 mm, gauche, 8 mm, à droite), et donc sont facilement personnalisables pour une utilisation dans des essais multiples - dans ce cas, pour s&#39;adapter à un 48 plaques à puits culture cellulaire (figure 3B). Épifluorescence et microscopie à contraste de phase de myoblastes C2C12 cultivées sur un gel de DGMP montre une fixation sélective sur RGDS-350-PEG contenant des couches (figure 4), ce qui démontre la compartimentation du peptide d&#39;adhésion cellulaire (RGDS). <img src="/files/ftp_upload/50018/50018fig1.jpg" alt="Figure 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50018/50018fig1highres.jpg"/> Figure 1. Moléculaire Weight analyse par MALDI-TOF comparant aPEG-SCM pour APEG-RGDS obtenus après conjugaison du peptide RGDS. <img src="/files/ftp_upload/50018/50018fig2.jpg" alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50018/50018fig2highres.jpg"/> Figure 2. Représentation schématique de la DGMP gel de fabrication. Après les gradients sont superposés, ils peuvent être autorisés à s&#39;installer pour des périodes de temps (t s) de créer des interfaces graduées, suivis par photopolymérisation. Stratifiés gels DGPM peut être facilement extraite du moule pour une utilisation ultérieure. <a href="https://www.jove.com/files/ftp_upload/50018/50018fig2large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img src="/files/ftp_upload/50018/50018fig3.jpg" alt="Figure 3" /> Figure 3. A) 2D gels multicouches obtenus après photopolymérisation imagée en utilisant 350 nm et blancs canaux de lumière VersaDoc unité de documentation de gel. L&#39;image noir et blanc montre une alternance de couches contenant RGDS en blanc. B) Insertion de gel DGMP dans 48 puits, boîtes de culture cellulaire. <img src="/files/ftp_upload/50018/50018fig4.jpg" alt="Figure 4" /> Figure 4. Contraste de phase et l&#39;image fusionnée à épifluorescence des myoblastes C2C12 cultivées sur des gels DGMP (échelle de bar 50 m). <img src="/files/ftp_upload/50018/50018fig5.jpg" alt="Figure 5" /> Figure 5. Effet de l&#39;iodixanol sur l&#39;élasticité de la surface du gel. Atomic mesures par microscopie à force d&#39;échantillons statiques de substrats PEG réticulées en utilisant des méthodes déjà établies avec un déclencheur 2 nN vigueur 12. * P &lt;0,05 et ** p &lt;0,01.

Watch this Scientific Journal Video about Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering at JoVE.com

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

Nous décrivons ici une stratégie unique pour créer des matrices biocompatibles, en couches avec des interfaces continues entre des couches distinctes pour l&#39;ingénierie tissulaire. Un tel échafaudage pourrait fournir un environnement idéal personnalisable pour moduler le comportement cellulaire par différents indices biologiques, chimiques ou mécaniques

Gradient de densité multicouche de polymérisation (DGMP): une nouvelle technique pour la création multi-compartiment, échafaudages personnalisables pour l&#39;ingénierie tissulaire

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

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

12.4K vues.  University of California, San Diego. Nous décrivons ici une stratégie unique pour créer des matrices biocompatibles, en couches avec des interfaces continues entre des couches distinctes pour l'ingénierie tissulaire. Un tel échafaudage pourrait fournir un environnement idéal personnalisable pour moduler le comportement cellulaire par différents indices biologiques, chimiques ou mécaniques

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Elastomeric PGS Scaffolds in Arterial Tissue Engineering

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

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

PGS élastomère échafaudages en génie tissulaire artérielle

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

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Bio-ingénierie

Postproduction Processing of Electrospun Fibres for Tissue Engineering

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

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

Traitement post-production des fibres électrofilées de génie tissulaire

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

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

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

Les échafaudages auto-déclaration pour les 3-Dimensional Culture cellulaire

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

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

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

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

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 ...

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

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

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

Fabrication à base de Rod personnalisables souples Robotic pneumatiques Dispositifs Gripper pour la manipulation des tissus Délicat

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

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

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

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

Néonatale cardiaque Echafaudages: Matrices nouveaux pour Études régénératives

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

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

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

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

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

Faire fondre électrofilage écrit d’échafaudages Poly(ε-caprolactone) en trois dimensions avec des Morphologies contrôlables pour les Applications de génie tissulaire

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

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

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

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

Bioprinting du Cartilage et peau tissus analogues utilisant un roman passif unité Technique de mélange pour Bioink Precellularization

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

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

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

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

Un protocole pour Decellularizing limaçon de souris pour l’ingénierie tissulaire oreille interne

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

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Culture de cellules mammifères dans les échafaudages de Peptide en trois dimensions

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...