Vorremmo ringraziare John Wirth dei giardini Olbrich per gentilmente fornire i campioni utilizzati in questo progetto. Questo lavoro è supportato in parte dal cuore nazionale, polmone e l'Istituto del sangue (R01HL115282 a G.R.G) National Science Foundation (DGE1144804 a J.R.G e G.R.G) e il fondo di Alumni (H.D.L) e Università di Wisconsin Dipartimento di chirurgia. Quest'opera è stata anche sostenuta in parte l'Environmental Protection Agency (grant STAR No. 83573701), il National Institutes of Health (R01HL093282-01A1 e UH3TR000506) e la National Science Foundation (IGERT DGE1144804).

1. decellularizzazione di tessuto vegetale utilizzando l'approccio basato su detergente
<ol><li>
Utilizzare fresche o congelate F. hispida, campioni di foglia. Congelare i campioni freschi inutilizzati in un congelatore a-20 ° C e conservare per uso futuro (fino ad un anno). Nota: Uso di staminali o foglia di quasi qualsiasi impianto desiderato. Tempi di conservazione prolungati possono causare danni ai tessuti.<ol>
<li>Determinare la dimensione e la forma dei campioni da elaborare sulla base della destinazione d'uso del campione (cioè campioni tagliati a strisce sono adatti per applicazioni di test meccaniche, nel frattempo 8 mm disco campioni sono utili nelle applicazioni multi-pozzetto di coltura ). Tagliare la foglia in dischi di 8 mm con una biopsia nette pugno mentre sommersa sotto temperatura ambiente (20-25 ° C) deionizzata H2O. Nota: Questo protocollo può essere usato su steli e foglie intere. Tuttavia, i campioni più piccoli saranno decellularize più velocemente.</li>
		<li>Incubare i campioni per 5-10 min a temperatura ambiente (20-25 ° C) deionizzata H2O su un frullato settato a un'impostazione bassa velocità per lavare e/o scioglierli. Utilizzare abbastanza deionizzata H2O affinché che tutti i campioni sono completamente bagnati.</li>
	</ol>
</li>
	<li>
Preparare una soluzione di 10% (p/v) sodio dodecil solfato (SDS) in deionizzata H2O. posto i campioni in un contenitore adatto (un vetro o plastica piatto è l'ideale) e aggiungere la soluzione di SDS per coprire completamente i campioni. Incubare i campioni per 5 giorni a temperatura ambiente (20-25 ° C) su un shake piastra insieme ad una bassa velocità per evitare di danneggiare i campioni. Nota: Non sovraffollamento del contenitore, poiché ciò può rallentare il processo di decellularizzazione e condurre al trattamento irregolare di SDS. Durante questo passaggio, campioni dovrebbero acquisire una tonalità marrone.<ol>
<li>Dopo 5 giorni, sostituire la soluzione di SDS con deionizzata H2O. Incubare i campioni per altri 10-15 min sulla piastra di agitare a risciacquare accuratamente qualsiasi soluzione residua di SDS.</li>
	</ol>
</li>
	<li>Preparare un tensioattivo non ionico di 1% (v/v) in soluzione di candeggina al 10% (v/v). Per fare una soluzione di 500 mL, mescolare 5 mL del tensioattivo non ionico con 50 mL di candeggina poi aggiungere 445 mL di deionizzata H2O. Submerge campioni in soluzione appena preparata. Nota: La soluzione di tensioattivo non ionico/candeggina non ha una lunga shelf life, pertanto, deve essere utilizzato entro 48 ore di preparazione.</li>
	<li>
Sostituire la soluzione di tensioattivo non ionico/candeggina ogni 24 ore fino a quando i campioni sono completamente cancellati (vedere Figura 1A per un confronto visivo). Incubare il campione in acqua deionizzata su una zolla di agitare per 2 min per risciacquare la soluzione in eccesso tensioattivo non ionico/candeggina. Mettere la piastra di agitare a un'impostazione bassa per evitare di danneggiare i campioni. Nota: Il tempo necessario per cancellare i campioni utilizzati varia, a seconda del tipo e specie della pianta. La decolorazione completa dei campioni è indicativo di decellularizzazione completo (eseguire la quantificazione del DNA per garantire completa compensazione).<ol>
<li>Lyophilize i campioni per conservarli fino a un anno (congelamento con azoto liquido flash è comodo, congelare i campioni a-80 ° C è anche accettabile) e conservarle a temperatura ambiente (20-25 ° C) a bassa umidità.</li>
		<li>Ricostituire i campioni in Tris-HCl (10 mM, pH 8.5) utilizza abbastanza ai campioni di cappotto. Sciacquare i campioni 2 - 3 volte in media senza siero delicatamente con una micropipetta prima dell'uso (cioè uso 150-300 µ l per risciacquo, in una piastra ben 24 standard). Nota: Tampone Tris-HCl e terreni privi di siero (cioè DMEM, ma qualsiasi cellula fondamentale cultura media può essere sostituito) sono più efficaci nel ridurre l'impatto di attuabilità delle cellule di trattamento con SDS campioni rispetto a quelli trattati con deionizzata H2O da soli.</li>
	</ol>
</li>
</ol>2. preparazione dei campioni utilizzando l'approccio di decellularizzazione senza detersivo
Nota: I primi passi di questa procedura coincidono con passaggi 1.1-1.1.2 (Vedi sopra).
<ol><li>Preparare una soluzione di sodio bicarbonato (NaHCO3) di 3% (p/v) e candeggiante al 5% (v/v) (NaClO). Riscaldare la soluzione in una cappa a 60-70 ° C per i campioni di foglia F. hispida tagliato in dischi di 8 mm mentre si mescolano su un piatto caldo. Nota: Il bicarbonato di sodio può essere sostituito con carbonato di sodio (Na2CO3) o idrossido di sodio (NaOH). La temperatura varia notevolmente (da temperatura ambiente a 90 ° C) e deve essere regolata secondo le proprietà dei campioni utilizzati.</li>
	<li>Una volta che la soluzione non raggiunge la temperatura desiderata, immergere i campioni e ridurre la velocità di agitazione per evitare di danneggiarli. Dopo i campioni vengono visibilmente cancellati (vedere Figura 1B per un confronto visivo), rimuovere con cautela dal bagno. Incubare i campioni una volta nel deionizzata di H2O per 1-2 min rimuovere la soluzione di candeggina in eccesso. Nota: Il tempo necessario per cancellare i campioni possa variare ampiamente. Ad esempio, prezzemolo tagliato può essere cancellato in 10-15 minuti, mentre più spessi e/o più grandi campioni come intero foglie o steli possono richiedere ore nei bagni ad alta temperatura per cancellare completamente.</li>
	<li>Lyophilize i campioni e conservarle a temperatura ambiente (20-25 ° C) a bassa umidità.</li>
</ol>

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Qui, vengono descritti due metodi per decellularize tessuti vegetali. I risultati presentati qui, accoppiato con i risultati di studi precedenti25, suggeriscono che i protocolli messi avanti siano probabili applicabile ad un ampio spettro di specie vegetali e può essere eseguita su entrambi i gambi e foglie. Queste procedure sono semplici e non richiedono attrezzature specializzate, quindi pianta decellularizzazione può essere effettuata nella maggior parte dei laboratori. È interessante nota c...

Ingegneria tissutale è emerso nel 1980 per creare tessuto vivente sostituti e, potenzialmente, indirizzo significativo dell'organo e del tessuto carenze1. Una strategia è utilizzato Ponteggi per stimolare e guidare il corpo per rigenerare tessuti o organi mancante. Anche se avanzato fabbricazione approcci quali stampa 3-d hanno prodotto ponteggi con proprietà fisiche uniche, la capacità di produrre impalcature con una vasta gamma di proprietà fisiche e biologiche realizzabili rimane una sfida2 , 3. Inoltre, a causa di una mancanza di una rete vascolare funzionale, queste tecniche sono state limitate a rigenerare tessuti 3-dimensionale. L'uso di decellularizzazione tessuti animali ed umani come scaffold ha aiutato a eludere questo problema4,5,6,7. Tuttavia, costo elevato, lotto variabilità e disponibilità limitata può limitare l'uso molto diffuso di decellularizzazione animale ponteggi8. Ci sono inoltre preoccupazioni circa la potenziale trasmissione di malattie ai pazienti e reazione immunologica per alcuni tessuti di mammiferi decellularizzati9.
Cellulosa, derivata dalla pianta e fonti batteriche, è stato ampiamente utilizzato per generare biomateriali per una vasta gamma di applicazioni nella medicina rigenerativa. Alcuni esempi includono: osso10,11, cartilagine12,13,14 e15di guarigione della ferita. Impalcature che sono composti di cellulosa hanno un ulteriore vantaggio, in quanto sono durevoli e resistenti per essere ripartiti dalle cellule di mammiferi. Questo è dovuto al fatto che le cellule di mammiferi non producono gli enzimi necessari per abbattere le molecole di cellulosa. In confronto, scaffolds impiegando macromolecole della matrice extracellulare, come il collagene, sono prontamente ripartiti16 e potrebbero non essere idonei per applicazioni a lungo termine. Scaffold di collagene può essere stabilizzata tramite cross-linking chimico. Tuttavia, esiste un compromesso a causa della tossicità intrinseca dei linkers che influenzano la biocompatibilità delle impalcature17. Al contrario, cellulosa ha il potenziale per rimanere presenti presso il sito dell'impianto per periodi prolungati di tempo perché è impermeabile alla degradazione enzimatica di cellule di mammifero18,19,20. Questo può essere modificato regolando la velocità di degradazione attraverso il pretrattamento idrolisi e co-consegna di impalcature con cellulasi21. La biocompatibilità di cellulosa pianta-derivati decellularizzati impalcature in vivo è stata dimostrata anche in uno studio condotto su topi22.
Attraverso centinaia di milioni di anni di evoluzione, piante hanno perfezionato la loro struttura e composizione per aumentare l'efficienza di trasporto di fluidi e di ritenzione. Pianta vascolare vasi minimizza la resistenza idraulica di ramificazione in vasi più piccoli, simili al sistema vascolare dei mammiferi secondo legge23 di Murray. Dopo decellularizzazione, complessa rete dell'impianto di vasi e pori interconnessi è mantenuto. Considerando il vasto numero di specie di piante distinte prontamente disponibili, impalcature pianta-derivati hanno il potenziale per superare i limiti di progettazione attualmente interessano impalcature in tessuto ingegneria24,25. Per esempio, Modulevsky et al hanno dimostrato che l'angiogenesi e migrazione cellulare si è verificato quando apple decellularizzati tessuto è stato impiantato sottocute sul retro di un mouse22. Allo stesso modo, Gershlak et al ha mostrato che le cellule endoteliali possono essere coltivate all'interno del vasculature di decellularizzazione foglie24. In un esperimento separato, Gershlak et al sono stati anche in grado di mostrare che cardiomiociti potevano essere coltivati sulla superficie delle foglie e sono stati in grado di contrarre24.
Piante includono anche organizzazione complessa da cellulare alla scala macroscopica, che è difficile da raggiungere anche con le più avanzate tecniche di produzione sviluppate fino ad oggi. Il complesso disegno gerarchico dei tessuti vegetali li rende più forti rispetto alla somma dei loro costituenti26. Piante possiedono una pletora di diverse proprietà meccaniche che vanno dai componenti rigidi e duri come steli, a quelli molto più flessibile e duttile come foglie27. Foglie variano a seconda della specie in termini di dimensioni, forma, rompono la resistenza, il grado di vascolarizzazione e possono portare a diversi gradi di idrofilia. Nel complesso, queste proprietà pianta suggeriscono che piante decellularizzati possono servire come dispositivi medici unici e altamente funzionali, tra cui come impalcature di ingegneria tissutale.
Questo protocollo si concentra su due metodi per decellularize tessuti vegetali, come foglie e steli, per uso come impalcature in ingegneria tissutale. Il primo metodo è una detergente a base di tecnica che utilizza una serie di bagni per rimuovere il DNA e materia cellulare, che è stato adattato da una tecnica ampiamente utilizzata per decellularize dei mammiferi e vegetali tessuti6,22,25 ,28,29,30. Il secondo metodo è privo di detersivo ed è adattato da un protocollo di "scheletrizzazione" generalmente utilizzato per rimuovere i tessuti molli delle foglie31. Lavoro precedente ha mostrato che simmering foglie in una soluzione di candeggina e bicarbonato di sodio ha facilitato la separazione del vasculature dai tessuti molli circostanti31. Questa tecnica può essere citata torna a esperimenti svolti in 17th e 18 secoli delth , come il lavoro di Albertus Seba32 ed Edward Parrish33. Questi esperimenti centrati intorno lasciando la materia vegetale, come foglie e frutta, immersa nell'acqua per lunghi periodi di tempo (settimane o mesi) e permettendo i tessuti più morbidi a distanza decadere naturalmente. Qui l'approccio di "scheletrizzazione" è adattato per utilizzare condizioni più miti, come ad esempio i tempi di incubazione più lunghi a temperature più basse, per rimuovere i residui cellulari e per evitare di alterare significativamente la struttura dei tessuti molli. Per gli esperimenti dettagliati nel presente documento, sono stati utilizzati tre tipi di pianta: Ficus hispida, Pachira aquatica e una specie di Garcinia. Risultati di quantificazione del DNA, prove meccaniche e impatto sull'attività metabolica cellulare da entrambi i metodi sono descritti.

<table border="0" cellpadding="0" cellspacing="0" style="width:771px;" width="771">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Sodium dodecyl sulfate</td>
			<td style="width:111px;">Sigma Life Science</td>
			<td style="width:136px;">75746-1KG</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">Triton X-100</td>
			<td style="width:111px;">MP Biomedicals, LLC</td>
			<td style="width:136px;">807426</td>
			<td style="width:277px;">Non-ionic surfactant referenced in paper. Very viscous reagent, can help to cut end of pipette tip when drawing it up.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Concentrated bleach (8.25% sodium hypochlorite)</td>
			<td style="width:111px;">Clorox</td>
			<td style="width:136px;">Item #: 31009</td>
			<td style="width:277px;">Standard concentrated bleach.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Sodium bicarbonate</td>
			<td style="width:111px;">Acros Organics</td>
			<td style="width:136px;">217120010</td>
			<td style="width:277px;">Can be substituted with sodium hydroxide or sodium carbonate.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">8 mm Biopunch</td>
			<td style="width:111px;">HealthLink</td>
			<td style="width:136px;">15111-80</td>
			<td style="width:277px;">Cuts samples that fit well in 24 well plate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Belly Dancer-Shake table</td>
			<td style="width:111px;">Stovall Life Sciences</td>
			<td style="width:136px;">BDRAA115S</td>
			<td style="width:277px;">Use low speeds to not damage tissues. Can use any model/brand of shake table.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Isotemp hot/stir plate</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;"></td>
			<td style="width:277px;">Can use any style/brand of hot/stir plate.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Beaker</td>
			<td style="width:111px;">Any</td>
			<td style="width:136px;"></td>
			<td style="width:277px;">Can use any size beaker as long as it will fit your samples and not overcrowd them.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Tris Hydrochloride</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">BP153-500</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DMEM</td>
			<td style="width:111px;">Corning</td>
			<td style="width:136px;">MT50003PC</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Quant-iT Picogreen dsDNA assay</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:136px;">P11496</td>
			<td style="width:277px;">Can use any dsDNA quantification mehtod on hand.</td>
		</tr>
	</tbody>
</table>

two methods for decellularization of plant tissues for tissue engineering applications

Gli innesti autologhi, sintetici e derivati animali attualmente utilizzati come scaffold per la sostituzione del tessuto hanno limitazioni a causa della scarsa disponibilità, scarsa biocompatibilità e costo. Tessuti vegetali hanno caratteristiche favorevoli che li rendono particolarmente adatti per utilizzare come scaffold, quali elevata area superficiale, trasporto d'acqua eccellente e ritenzione, porosità interconnesse, preesistenti reti vascolari e una vasta gamma di meccanica Proprietà. Due metodi di successo di decellularizzazione pianta per applicazioni di ingegneria del tessuto sono descritti qui. Il primo metodo si basa sui bagni detergenti per rimuovere la materia cellulare, che è simile ai metodi stabiliti in precedenza utilizzati per cancellare tessuti di mammiferi. Il secondo è un metodo privo di detersivo, adattato da un protocollo che isola il sistema vascolare foglia e prevede l'utilizzo di una candeggina riscaldata e il bagno di sale per eliminare le foglie e steli. Entrambi i metodi portano a impalcature con proprietà meccaniche comparabili e basso impatto metabolico cellulare, permettendo così all'utente di selezionare il protocollo che meglio si adatta alle loro applicazione prevista.

Entrambi i metodi hanno reso impalcature adatte alla coltura cellulare e applicazioni di ingegneria tissutale. La figura 1 Mostra il flusso di lavoro generale del processo di decellularizzazione utilizzando una foglia intatta per il metodo basato su detergente e tagliati campioni (8 mm di diametro) per il metodo privo di detersivo. Successo decellularizzazione di Ficus hispida tessuti dopo entrambi i metodi hanno reso chiari e intatti campioni (<stro...

Watch this Scientific Journal Video about Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications at JoVE.com

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Due metodi per decellularizzazione di tessuti vegetali per applicazioni di ingegneria del tessuto

Qui vi presentiamo, e contrasto due protocolli utilizzati per decellularize tessuti vegetali: un approccio basato su detergente e un approccio privo di detersivo. Entrambi i metodi si lasciano alle spalle la matrice extracellulare dei tessuti vegetali utilizzati, che quindi può essere utilizzata come scaffold per applicazioni di ingegneria dei tessuti.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

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Research

JoVE Journal

Bioengineering

14.5K Views.  University of Wisconsin-Madison. Qui vi presentiamo, e contrasto due protocolli utilizzati per decellularize tessuti vegetali: un approccio basato su detergente e un approccio privo di detersivo. Entrambi i metodi si lasciano alle spalle la matrice extracellulare dei tessuti vegetali utilizzati, che quindi può essere utilizzata come scaffold per applicazioni di ingegneria dei tessuti.

Video: Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Fabbricazione di derivazione biologica Materiali iniettabili per l&#39;ingegneria tissutale miocardica

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Ricerca

Bioingegneria

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

Decellularization e Recellularization di fegati interi

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

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 ...

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 ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Ingegneria tessuti muscolo scheletrico di cellule murine progenitrici mioblasti e di applicazione di stimolazione elettrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capillare Forza litografia per Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Recupero chirurgico, Isolamento e<em&gt; In vitro</em&gt; Ampliamento anteriore umana crociati Cells Legamento di derivazione per applicazioni di ingegneria tissutale

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparazione di Thermoresponsive Nanostrutturati Superfici Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Capsule Estrazione di Plant-based per applicazioni di microincapsulazione

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Due metodi per decellularizzazione di tessuti vegetali per applicazioni di ingegneria del tessuto

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Fabbricazione di derivazione biologica Materiali iniettabili per l'ingegneria tissutale miocardica