Gli autori sono grati per il sostegno di premi NIH di regia Innovator Nuovi (1DP2 OD006499-01 AA e 1DP2 OD006460-01 a AJE), e King Abdulaziz City per la Scienza e la Tecnologia (UC San Diego Centro di Eccellenza in nanomedicina). Vorremmo ringraziare la signora Jessica Moore per i suoi commenti critici sul manoscritto.

1. Sintesi di marcato in modo fluorescente Acryloyl-PEG-Rgds <ol><li> Reagiscono con il peptide Rgds acriloil-PEG-succinimidil carbossimetil estere (APEG-SCM, PEG MW: 3400 g / mol) e NN diisopropiletilammina (DIPEA) a 1.2:1:2 rapporti molari in dimetilsolfossido (DMSO) sotto argon a temperatura ambiente durante la notte. </li><li> Confermare coniugazione da matrix-assisted laser desorbimento / ionizzazione tempo di volo (MALDI-TOF), spettrometria di massa. Aggiungere 1 ml di APEG-Rgds soluzione di reazione in un punto campione sul target MALDI e asciutto. Preparare una soluzione satura di universale MALDI Matrix in tetraidrofurano (THF) e vortex per 1 min. ~ Aggiungere 1 ml di questa soluzione a punto del campione stesso. Ripetere la procedura per APEG-SCM per il confronto. Caricare e analizzare. Il peso molecolare di APEG-Rgds dovrebbe essere maggiore di APEG-SCM (Figura 2). </li><li> Di coniugare il fluoroforo, aggiungere una quantità equimolare di Alexa Fluor 350 carbossilico (succinimydyl estere), discioltoin un volume minimo di DMSO, al APEG-Rgds soluzione di reazione da 1,1 e reagire sotto argon a temperatura ambiente per una notte. </li><li> Purificare APEG-Rgds-350 da dialisi (MW 3500 Da) contro il DI-H 2 O a 4 ° C per 48 ore a 1.000:1 rapporto volumetrico, lo scambio di dialisato almeno due volte al giorno. </li><li> Congelare asciugare la purificato APEG-Rgds-350 in Freezone Labconco Plus o Blocco del sistema equivalente a secco e conservare a -20 ° C. </li></ol> 2. Preparazione di uno stampo 2D e Realizzazione di un gel PEG 2D con alternata Rgds-350 Livelli <ol><li> Preparare vetrini idrofobiche. Posizionare vetrini puliti in un piatto di vetro in un forno a vuoto. Riscaldare a 80 ° C per 30 minuti per asciugare completamente le superfici. Luogo piatto con le diapositive in una cappa aspirante e aggiungere 250 microlitri Sigmacote a ogni diapositiva, delicatamente a dondolo per 30 sec alla superficie cappotto intero. Sciacquare i vetrini rivestiti con 100% metanolo, seguita da lavaggio con acqua distillata, ammollo due volte per 5 min in a least 10 ml. </li><li> Tagliare distanziali in silicone (0,8 mm di spessore) con 10 mm pugni biopsia. </li><li> Autoclave i distanziali in silicone e Sigmacote trattati con vetrini. </li><li> Formulare soluzioni per ciascun rispettivo strato in provette per microcentrifuga singoli mescolando PEG diacrilato (PEGda) precursore (concentrazione finale 15% w / v) con diverse quantità di iodixanolo (soluzione stock 60% in acqua) per produrre concentrazioni finali diverse (ad esempio il 40%, 30%, 20% e 10%), che completa il volume residuo di tampone fosfato (PBS) per ottenere soluzioni di densità differenziate. In modo simile, per strati alternati, mix APEG-Rgds-350 (concentrazione finale 8 mm) con iodixanolo e PBS per produrre concentrazioni diverse (ad esempio 35%, 25%, e 15%). </li><li> Aggiungi fotoiniziatore (2-idrossi-4&#39;-(2-idrossietossi)-2-metilpropiofenone, magazzino 333mg/ml in N-vinil pirrolidone) per ogni soluzione per vari strati (10 pl di soluzione madre per ml di ciascuna soluzione di strato). Fotoiniziatore is ultimo aggiunto per prevenire la polimerizzazione prima stratificazione dei gel nello stampo, in quanto è sensibile alla luce. </li><li> I passi successivi di questo protocollo sarà effettuata in un armadio biosicurezza per garantire la sterilità. </li><li> Filtro-sterilizzare ogni soluzione sterile utilizzando una siringa da 1 ml e 0,2 micron. Assemblare l&#39;impostazione stampo intramezzando il distanziatore tra due Sigmacore trattati vetrini e sicure con fascette ponendo come illustrato in Figura 1. </li><li> Gettare i gel strati aggiungendo la soluzione più densa (es. PEGda con il 40% iodixanolo) prima, seguita da una soluzione meno densa (es APEG-Rgds-350 con 35% iodixanolo). Ripetere la stratificazione alternate per ottenere diversi strati di composizione desiderata e densità come mostrato in Figura 1. </li><li> Irradiare lo stampo con la luce 365 nm per 3 minuti con un portatile UVR-9000 lampada. Consentire i gel polimerizzati per curare per 5 min. Rimuovere i morsetti, quindi sollevare delicatamente il vetro superiorediapositiva e lo stampo, i gel DGMP stratificate rimarrà nelle diapositive. Utilizzando una spatola sterile, posizionare accuratamente il gel in una provetta da 50 ml contenente PBS sterile o mezzo di coltura per il lavaggio. </li><li> Lavare il gel polimerizzato in PBS a 1.000:1 rapporto volumetrico, lo scambio di buffer di almeno due volte al giorno per rimuovere il modificatore densità, fotoiniziatore, e polimero che non ha reagito. In alternativa, PBS può essere scambiato con il terreno di crescita. Conservare i gel DGMP in PBS o terreno di coltura per l&#39;esperimento di coltura cellulare indicato al punto 3. </li><li> Per visualizzare strati alternati, organizzare gel DGMP (questi gel non saranno utilizzabili per colture cellulari) lungo un righello sul vassoio campione del meccanismo di documentazione gel VersaDoc. Esporre i gel a 350 nm, il tempo di esposizione varia a seconda della concentrazione del fluoroforo. Alternando bande scure e blu nell&#39;idrogel DGMP dimostrano formazione di strati distinti di composizione chimica diversa (Figura 3). </li></ol><ol><li> Per i gel che incorporano peptide Rgds, usare le cellule di adesione dipendenti, ad esempio mioblasti C2C12. </li><li> Inserire delicatamente gel DGMP (memorizzati in PBS) nei pozzetti di piastre di coltura cellulare a 48 pozzetti utilizzando un raschietto sterile cella in un armadio biosicurezza. </li><li> Pre-riscaldare terreno di crescita (terreno Eagle modificato da Dulbecco o DMEM supplementato con 10% v / v di siero fetale bovino e 1% v / v 100x soluzione di penicillina-streptomicina) e PBS in un bagno d&#39;acqua a 37 ° C. </li><li> Lavare una piastra 60% confluenti (10 mm) di C2C12 tre volte con PBS. Aspirare off PBS e cellule raccolto aggiungendo 1 ml di 0,25% tripsina-EDTA e incubando a 37 ° C per 2 min. Risospendere le cellule in mezzo di crescita e cellule di conteggio. Il seme DGMP gel-coltura cellulare contenente bene con mioblasti C2C12 (20.000 cellule / cm 2). Incubare le cellule a 37 ° C in 5% CO 2/95% di umidità relativa. Delicatamente mezzo di scambio, dopo 4 ore, fare attenzione a non rispostare leggermente cellule aderenti. </li><li> Dopo 24 ore, il sequestro conservativo di mioblasti C2C12 sui Rgds contenenti strati di gel DGMP può essere confermata da epifluorescenza e microscopia a contrasto di fase (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>

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DGMP è una semplice strategia per la preparazione di gel multistrato che non si basano su strumenti costosi. Questo protocollo può essere adattato per creare ponteggi utilizzano altri materiali biocompatibili, quali collagene e acido ialuronico. Piccole molecole bioattive, per esempio cellule promotore di adesione peptide Rgds, possono essere legati alla matrice polimerica per evitare miscelazione di segnali tra gli strati. Proteine ​​possono essere incapsulati in strati distinti senza la necessità di coniugazione chimica come, a seconda della dimensione delle maglie della matrice, sono meno inclini a diffondere attraverso idrogel 10. Qui abbiamo usato iodixanolo (Nycoprep), un modificatore densità inerte, che è stato precedentemente utilizzato per applicazioni di cellule vitali. Modificatori densità altre come saccarosio e destrosio può anche essere usato. Variando il tempo di assestamento (t s), è possibile mettere a punto le interfacce tra i due strati per produrre transizioni morbide o taglienti, se necessario (tempo più lungo assestamento dà transizioni più uniformi) &lt;sup&gt; 10. Per esempio, fluide transizioni tra strati di gel DGMP potrebbe essere utilizzato per generare un gradiente continuo di una stecca biologica per studiare processi cellulari come chemiotassi. L&#39;effetto del modificatore densità sulla rigidità gel è mostrato in Figura 5 per un 15% aPEGda gel; una caratterizzazione più completa di rigidità e di porosità in funzione della concentrazione e PEGda iodixanolo attualmente è sottoposto. Mentre la concentrazione PEGda in questo esempio è relativamente elevato, abbiamo osservato un modulo elastico maggiore del 60% in gel con il 30% rispetto al iodixanolo senza gel. La variazione rigidità gel può essere regolata per modulando la concentrazione macromero o densità di reticolazione. Abbiamo anche applicato la tecnica DGMP creare 3D multistrato utilizzando gel di poliacrilammide e precursori PEG 10. Variando la concentrazione e il grado di reticolazione del prepolimero consente variazione strutturaleponteggi, che possono essere usati per esplorare il comportamento cellulare come la crescita e la migrazione polarizzata in 3D. In sintesi, DGMP è una tecnica adattabile che può essere applicato a fabbricare scaffold 2D e 3D da una varietà di materiali biocompatibili per un&#39;ampia gamma di applicazioni di ricerca biomedica e di base.

<table border="1"><tbody><tr><td> Reagente o dello strumento </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Polietilene glicole succinimydyl carbossimetil (a-PEG-SCM) </td><td> Laysan </td><td> 120-64 </td></tr><tr><td> Polyethelyene glicol diacrilato (PEGda) </td><td> Dajac Labs </td><td> 9359 </td></tr><tr><td> Arginina-Glicina-Acido aspartico-serina (Rgds) </td><td> American Peptide </td><td> 49-01-4 </td></tr><tr><td> N, N - Diisopropiletilammina (DIPEA) </td><td> Sigma </td><td> D125806 </td></tr><tr><td> Dimetilsolfossido (DMSO) </td><td> Sigma </td><td> D2438 </td></tr><tr><td> N, N-dimetilformammide (DMF) </td><td> Pescatore </td><td> D119-4 </td></tr><tr><td> Tetraidrofurano (THF) </td><td> Pescatore </td><td> T397 </td></tr><tr><td> Dialisi cassetta (3500Da) </td><td> Thermo Scientific </td><td> 66330 </td></tr><tr><td> Alexa Fluor 350 carbossilico succinimydyl </td><td> Life Technologies </td><td> A-10.168 </td></tr><tr><td> Sigmacote </td><td> Sigma </td><td> SL2 </td></tr><tr><td> Distanziali in silicone </td><td> Grainger </td><td> 1MWA4 </td></tr><tr><td> Biopsia pugni </td><td> Acuderm </td><td> P1025 (10 mm) P850 (8 mm) </td></tr><tr><td> Fosfato di Dulbecco tamponata salina (DPBS) </td><td> Hyclone </td><td> SH30028 </td></tr><tr><td> Iodixanolo (NycoPrep) </td><td> Pescatore </td><td> NC9388846 </td></tr><tr><td> 2-idrossi-4&#39;-(2-idrossietossi)-2-metilpropiofenone </td><td> Sigma </td><td> 410896 </td></tr><tr><td> Mezzo di Dulbecco Eagle modificato (DMEM) </td><td> Life Technologies </td><td> 11054 </td></tr><tr><td> Siero fetale bovino </td><td> Life Technologies </td><td> 10082 </td></tr><tr><td> Penicillina-streptomicina </td><td> Life Technologies </td><td> 15140 </td></tr><tr><td> Mioblasti 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

Complesse matrici di coltura di tessuti, in cui tipi e concentrazioni di stimoli biologici (ad esempio fattori di crescita, inibitori, o piccole molecole) o struttura a matrice (ad esempio composizione, concentrazione, o rigidità della matrice) variano nello spazio, consentirebbe un&#39;ampia gamma di indagini riguardo a come queste variabili influenzano la differenziazione cellulare, la migrazione, e altri fenomeni. La sfida principale nella creazione di matrici stratificate è mantenere l&#39;integrità strutturale di interfacce strato senza diffusione di singoli componenti da ogni strato 1. Attuali metodologie questo comporta in particolare photopatterning 2-3, litografia 4, functionalization5 sequenziale, liofilizzazione 6, microfluidica 7, 8 o centrifugazione, molti dei quali richiedono strumentazione sofisticata e competenze tecniche. Altri tramite attacco sequenziale dei singoli strati, che può portare alla delaminazione degli strati 9 </sup&gt;. DGMP supera questi problemi utilizzando un modificatore inerte densità come iodixanolo per creare strati di diversa densità 10. Poiché il modificatore densità può essere miscelato con qualsiasi prepolimero o molecola bioattiva, DGMP permette ogni strato scaffold da personalizzare. Semplicemente variando la concentrazione del modificatore densità impedisce miscelazione degli strati adiacenti, mentre rimangono acquosa. Successiva polimerizzazione passo comporta una strutturalmente continua scaffold multistrato, in cui ogni strato ha chimico distinto e proprietà meccaniche. Il modificatore di densità può essere facilmente rimosso con risciacquo sufficiente senza perturbazione dei singoli strati o loro componenti. Questa tecnica è particolarmente indicato per la creazione di idrogel di varie dimensioni, forme e materiali. Un protocollo per la fabbricazione di una 2D-polietilene glicole (PEG) gel, in cui strati alternati incorporano Rgds-350, è descritto di seguito. Usiamo PEG because è biocompatibile e inerte. Rgds, un peptide adesione cellulare 11, è utilizzato per dimostrare restrizione spaziale di una stecca biologica, e la coniugazione di un fluoroforo (Alexa Fluor 350) permette di distinguere visivamente vari strati. Questa procedura può essere adattato per altri materiali (ad esempio collagene, acido ialuronico, ecc) e può essere esteso per fabbricare gel 3D con alcune modifiche 10.

MALDI-TOF analisi conferma la coniugazione del peptide ad Rgds acriloil-PEG (Figura 2). Gel di imaging rivela alternata Rgds-350 (blu) dopo la fotopolimerizzazione strati (Figura 3A). Come mostrato in Figura 3A, 2D gel DGMP dimensioni possono variare in base al diametro degli stampi in silicone (10 mm, sinistra, 8 mm, a destra), e pertanto sono facilmente personalizzabile per uso in saggi multipli - in questo caso per adattarsi a 48 pozzetti di piastra di cella (Figura 3B). Epifluorescenza e microscopio a contrasto di fase di mioblasti C2C12 coltivate su un gel DGMP mostra attacco selettivo Rgds-350-strati contenenti PEG (Figura 4), ​​dimostrando compartimentalizzazione del peptide adesione cellulare (Rgds). <img src="/files/ftp_upload/50018/50018fig1.jpg" alt="Figura 1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50018/50018fig1highres.jpg"/> Figura 1. Molecolare Weight analisi mediante MALDI-TOF confronto APEG-SCM-APEG Rgds ottenuti dopo la coniugazione del peptide Rgds. <img src="/files/ftp_upload/50018/50018fig2.jpg" alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/50018/50018fig2highres.jpg"/> Figura 2. Rappresentazione schematica di fabbricazione DGMP gel. Dopo i gradienti sono stratificati, possono essere autorizzati a stabilirsi per periodi di tempo variabili (t s) per creare interfacce laureati, seguita da fotopolimerizzazione. Stratificati DGMP gel può essere facilmente estratto dallo stampo per un ulteriore uso. <a href="https://www.jove.com/files/ftp_upload/50018/50018fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img src="/files/ftp_upload/50018/50018fig3.jpg" alt="Figura 3" /> Figura 3. A) gel 2D multistrato ottenuti dopo la fotopolimerizzazione ripreso con 350 nm e canali bianchi leggeri di VersaDoc gel documentazione della trasmissione. La scala di grigi rivela strati alternati contenenti Rgds in bianco. B) Inserimento di gel DGMP in piatti cellulari a 48 pozzetti. <img src="/files/ftp_upload/50018/50018fig4.jpg" alt="Figura 4" /> Figura 4. Contrasto di fase e l&#39;immagine unita ad epifluorescenza di mioblasti C2C12 coltivate su DGMP gel (scala bar 50 micron). <img src="/files/ftp_upload/50018/50018fig5.jpg" alt="Figura 5" /> Figura 5. Effetto della iodixanolo sull&#39;elasticità gel superficie. Atomic microscopia misure di forza di campioni statici di substrati PEG reticolati con metodi stabiliti in precedenza con un trigger 2 nN forza 12. * P &lt;0,05 e ** 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

Qui si descrive una strategia unica per la creazione di matrici biocompatibili, stratificate con interfacce continui tra strati distinti per l&#39;ingegneria tissutale. Tale scaffold in grado di fornire un ambiente ideale personalizzabile per modulare il comportamento cellulare da vari segnali biologici, chimici o meccanici

Gradiente di densità multistrato polimerizzazione (DGMP): una tecnica innovativa per la creazione di comparti multipli, Ponteggi personalizzabili per l&#39;ingegneria tissutale

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 Views.  University of California, San Diego. Qui si descrive una strategia unica per la creazione di matrici biocompatibili, stratificate con interfacce continui tra strati distinti per l'ingegneria tissutale. Tale scaffold in grado di fornire un ambiente ideale personalizzabile per modulare il comportamento cellulare da vari segnali biologici, chimici o meccanici

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

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 elastomerico Ponteggi in Ingegneria tessuto arterioso

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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Bioingegneria

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.

Elaborazione Postproduzione di fibre elettrofilate per l&#39;ingegneria tissutale

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.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

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 bioreattore Novel per l&#39;alta densità di coltivazione diverse comunità microbiche

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 Rod a base di personalizzabili morbide robotizzati pneumatici Gripper dispositivi per la manipolazione dei tessuti delicati

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.

Neonatale cardiaco Impalcature: Matrici innovativi per gli Studi rigenerativi

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.

Sciogliere elettrofilatura scrittura di scaffold tridimensionale Poly(ε-caprolactone) con morfologie controllabile per applicazioni di ingegneria del tessuto

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.

Multimateriali di cartilagine e pelle tessuto analoghi utilizzando un romanzo passivo unità tecnica di miscelazione per 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 protocollo per Decellularizing Mouse coclee per l'ingegneria dei tessuti dell'orecchio interno

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.

Coltura di cellule di mammifero in scaffold tridimensionale Peptide

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