Questo lavoro è stato sostenuto in parte dai fondi di avvio della University of Nebraska Medical Center e National Institute of Health (codice di autorizzazione 1R15 AR063901-01).

1. Preparazione della soluzione <ol><li> Preparare una soluzione di poli (ε-caprolattone) (PCL) (M w = 80,000 g / mol) ad una concentrazione approssimativa di 100 mg / ml. Sciogliere PCL in una miscela di diclorometano (DCM) e N, N-dimethlyformamide (DMF) con un rapporto di 4: 1 (v / v) con una concentrazione del 10% (w / v). </li><li> Porre la soluzione in un tubo di vetro da 20 ml per la miscelazione. Mettere tubo di vetro in ultrasuoni per 30 minuti, o fino a quando la soluzione è trasparente. </li></ol> 2. Apparecchiatura Preparazione <ol><li> Aggiungere la soluzione PCL preparata in una siringa da 5 ml con un ago senza punta 21 gauge collegato. </li><li> Inserire pompa a siringa in posizione electrospinning verticale di figura 1. </li><li> Utilizzare un collettore in acciaio inox-gap con uno spazio aperto di 2 cm x 5 cm per il substrato di fibre allineate. Posizionare il collettore 12 centimetri dalla punta dell&#39;ago. </li><li> Collegare la corrente continua (DC) ad alta tensione di alimentazione alago e terra il collettore. Assicurarsi che il collettore è individualmente a terra senza contatto per le altre apparecchiature di laboratorio. </li></ol> 3. Electrospinning <ol><li> Set pompa a siringa a 1.50 ml / h, fino a quando le goccioline che formano sulla punta dell&#39;ago vengono immediatamente sostituiti quando viene rimosso. Quindi, impostare la portata di 0,50 ml / h. </li><li> Girare l&#39;operatore tensione di 12 kV. </li><li> Electrospin fino a quando le fibre uniaxially allineate coprire completamente il gap sul collettore. </li><li> Applicare la colla ai bordi di una piccola lastra di vetro e trasferire le fibre dal collettore gap alla lastra di vetro. Posizionare la lastra di vetro nella parte superiore di un pezzo di foglio di alluminio che ha la stessa dimensione della lastra di vetro ed è messo a terra. </li><li> Posizionare il secondo collettore siringa secondo la figura 3. </li><li> Fissare la maschera di plastica per la seconda pompa siringa e posizione it 2 mm sopra il collettore. </li><li> Aggiungi cumarina 6 a 1% (w / w) per il PCL soluzione se nanoencapsulation è desired- e mescolare fino a quando la soluzione è trasparente. </li><li> Caricare il PCL o PCL / cumarina 6 soluzione in un ago 5 ml con un ottuso ago 21 gauge. Impostare pompa verticale a 0,50 ml / hr e orizzontale per tirare 9 ml / hr o ad una velocità di circa 1 mm / min. </li><li> Electrospin finché la maschera è spostato quasi completamente fuori dal collettore, ma con la zona di bordo ancora sotto la maschera. </li></ol> 4. Fiber Caratterizzazione <ol><li> I campioni con nastro adesivo conduttivo biadesivo al perno metallico e il cappotto con platino per 40 sec con una verniciatura polverizzazione a 40 mA. <ol><li> Esaminare fibre attraverso microscopio elettronico a scansione (SEM) secondo i nostri studi precedenti 17,18. </li><li> Raccogliere immagini ad una tensione di accelerazione di 15 kV. </li></ol></li><li> Eseguire l&#39;analisi rapida di Fourier (FFT) su un campione di fibra separato per misurare l&#39;allineamento fibra. Le informazioni dettagliate su allineamento delle fibre di misura da FFT può riferirsi to studi precedenti 19,20. </li></ol> 5. Cellule staminali Seeding. <ol><li> Sterilizzare i campioni di fibre immergendola in una soluzione di etanolo al 70% per 2 ore. Quindi lavare le fibre con acqua distillata per rimuovere eventuali contaminanti. </li><li> Ottenere ADSCs umane e cellule di coltura in una beuta da 25 cm 2 a 37 ° C in atmosfera di 95% aria / 5% CO 2. Modificare il terreno di coltura cellulare a giorni 10. </li><li> Trypsinize cellule e contare il numero di cellule. Specificamente, rimuovere tutto terreno di coltura dal pallone di coltura cellulare e lavare le cellule con tampone fosfato salino (PBS) due volte. Poi aggiungere 1 m l di una soluzione 0,25% tripsina-EDTA (pre-caldo in bagno d&#39;acqua a 37 ° C) per coprire il monostrato cellulare e incubare la beuta nell&#39;incubatrice coltura cellulare per 2-3 minuti. Aggiungere 4 ml terreno di coltura e lavare tutte le cellule dalla superficie pipettando il mezzo su tutta la superficie. Centrifugare la sospensione cellulare e ri-disperse esimoe pellet di cellule nel mezzo di coltura. Contare le celle tramite emocitometro. Seed circa 1 × 10 4 celle al patibolo nanofibre collocato in un piatto -Cultura 35 millimetri e incubare per 3 e 7 giorni. </li><li> Cellule macchia con fluoresceina diacetato (5 mg / ml in acetone) (FDA) allora l&#39;immagine dei campioni utilizzando un microscopio a fluorescenza. In particolare, aggiungere 100 ml di soluzione FDA al piatto cultura e incubare per 30 min. Lavare le cellule con PBS per tre volte prima di imaging fluorescente. Analizzare l&#39;orientamento delle cellule da un programma personalizzato mat-lab in base alle nostre precedenti studi 10. </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+orientation+at+the+tendon+to+bone+insertion+and+its+influence+on+stress+concentrations.">Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations.</a> J. Biomech. 39 (10), 1842-1851 (2006).</li><li>Thomopoulos, S., Williams, G. R., Gimbel, J. A., Favata, M., Soslowsky, 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=Variation+of+biomechanical,+structural,+and+compositional+properties+along+the+tendon+to+bone+insertion+site.">Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site.</a> J. Orthop. Res. 21 (3), 413-419 (2003).</li><li>Thomopoulos, S., Genin, G. M., Galatz, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+morphogenesis+of+the+tendon-to-bone+insertion+-+What+development+can+teach+us+about+healing.">The development and morphogenesis of the tendon-to-bone insertion - What development can teach us about healing.</a> Musculoskelet Neuronal Interact. 10 (1), 35-45 (2010).</li><li>Li, X., Xie, J., Lipner, J., Yuan, X., Thomopoulos, S., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+scaffolds+with+gradations+in+mineral+content+for+mimicking+the+tendon-to-bone+insertion+site.">Nanofiber scaffolds with gradations in mineral content for mimicking the tendon-to-bone insertion site.</a> Nano Lett. 9 (7), 2763-2768 (2009).</li><li>Kunzler, T. P., Huwiler, C., Drobek, T., V&#246;r&#246;s, J., Spencer, N. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+nanofiber+scaffolds+with+gradations+in+fiber+organization+and+their+potential+applications.">Fabrication of nanofiber scaffolds with gradations in fiber organization and their potential applications.</a> Macromol. Biosci. 12 (10), 1336-1341 (2012).</li><li>James, R., Kumbar, S. G., Laurencin, C. T., Balian, G., Chhabra, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+tissue+engineering:+adipose-derived+stem+cell+and+GDF-5+mediated+regeneration+using+electrospun+matrix+systems.">Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems.</a> Biomed. Mater. 6 (2), 025011 (2011).</li><li>Bodle, J. C., Hanson, A. D., Loboa, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells+in+functional+bone+tissue+engineering:+lessons+from+bone+mechanobiology.">Adipose-derived stem cells in functional bone tissue engineering: lessons from bone mechanobiology.</a> Tissue Eng. Part B Rev. 17 (3), 195-211 (2011).</li><li>Lee, J. H., Rhie, J. W., Oh, D. Y., Ahn, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+human+adipose+tissue-derived+stromal+cells+(hASCs)+in+a+porous+three-dimensional+scaffold.">Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold.</a> Biochem. Biophys. Res. Commun. 370 (3), 456-460 (2008).</li><li>Tapp, H., Hanley, E. N., Patt, J. C., Gruber, H. 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The authors have nothing to disclose.

The most critical part of the protocol is generation of the gradient scaffold. It is imperative that the mask covering the collector moves at a constant velocity so there is a gradual change within the fiber scaffold. The correct preparation of PCL solution is also important to ensure electrospinning success. Checking the fiber morphology prior to electrospinning is recommendable, especially after the encapsulation of Coumarin-6, which may require a higher voltage to electrospin correctly. 
Fu...

Nanofibre sono un&#39;utilità popolare per ingegneria tissutale a causa della loro capacità di mimare la matrice extracellulare nella sua struttura e dimensione relativa 1. Tuttavia, alcune interfacce tessuto nativo, come il sito tendine-osso, contengono fibre collagene, che presentano una struttura organizzativa variabile che aumenta in allineamento verso il tendine e diminuisce al sito osseo 2-5. Così, per la rigenerazione tissutale efficace è necessario per fabbricare un ponteggio che potrebbe efficacemente imitare questo gradiente strutturale. In precedenza, vi è stata una ricerca condotta sui cambiamenti graduali nella composizione delle fibre, in particolare, il contenuto di minerali 6. Tuttavia, ricreando la componente strutturale dei tessuti connettivi rimane in gran parte inesplorato. Uno studio precedente esaminato gradienti morfologiche studiando l&#39;effetto della silice superficie densità di particelle sulla proliferazione degli osteoblasti ratto cranica e trovato un inverrapporto tra la densità se particella di silice e la proliferazione cellulare 7. Ma i cambiamenti morfologici che mediate proliferazione cellulare in lavori precedenti erano per lo più relativi alla rugosità superficiale manca la capacità di mimare fibra cambiamenti organizzativi 7,8. Uno studio recente ha tentato di realizzare un ponteggio che imitava gli orientamenti fibre collagene unici utilizzando un nuovo collettore per electrospinning 9. Anche se questo studio è riuscito a produrre un ponteggio con fibre sia allineate e casuale, non è riuscito a imitare i graduali cambiamenti esposti nei tessuti nativi. Inoltre, nella produzione di componenti separati, con un cambiamento immediato dal allineato all&#39;orientamento casuale, le proprietà biomeccaniche di questa impalcatura è diminuito in modo significativo. Nessun lavoro precedente è stato in grado di produrre applicabili ponteggi nanofibre con continui gradazioni orientamenti fibra di allineato e casuali. Il nostro recente studio ha dimostrato con successo la ricreazione di ponteggi nanofibrecon gradazioni nell&#39;organizzazione fibra che può potenzialmente mimare l&#39;organizzazione collagene nativo all&#39;inserimento di 10 tendine-osso. Questo lavoro si propone di presentare i protocolli utilizzati per la produzione di scaffold nanofibre con una struttura molto simile a quella di organizzazione fibre nell&#39;interfaccia nativa tendine-osso tessuti. Strutture nanofibre gradiente sono potenzialmente di vasta portata applicazioni attraverso una varietà di campi. Ci siamo concentrati sulle applicazioni per l&#39;ingegneria tissutale del sito di tendine-osso combinando le nostre impalcature con le cellule staminali derivate da tessuto adiposo (ADSCs) che sono già utilizzate per la rigenerazione dei tessuti su vari substrati 11-14. Inoltre, ADSCs sono molto simili in natura per le cellule staminali del midollo osseo in termini di multipotenza e la loro risorsa è abbondante che può essere raccolto mediante una semplice procedura di liposuzione 15,16. Semina queste cellule per ponteggi nanofibre sfumati aumenta ulteriormente la loro tiscitare applicazioni ingegneristiche consentendo la distribuzione controllata delle cellule che possono potenzialmente differenziarsi in vari tessuti. Oltre a seminare le cellule staminali, nanofibre possono essere incapsulati con molecole di segnalazione per la regolazione della risposta cellulare. Accoppiamento nanoencapsulation con il gradiente organizzativa di tali ponteggi consente lo studio del comportamento cellulare o possibili disegni implantari e rivestimenti. Incapsulamento di molecole funzionali come proteina morfogenetica 2 (BMP2), che ha dimostrato di indurre la differenziazione degli osteoblasti 15,16, potrebbe migliorare ulteriormente le applicazioni di ingegneria tissutale di questi ponteggi 10.

<table><tbody><tr><td>Polycaprolactone</td><td>Sigma-Aldrich</td><td>440744</td><td></td></tr><tr><td>N,N-Dimethlyformamide</td><td>Fisher Chemical</td><td>D-119-1</td><td></td></tr><tr><td>Dichloromethane</td><td>Fisher Chemical</td><td>AC61093-1000</td><td></td></tr><tr><td>Coumarin 6</td><td>Sigma-Aldrich</td><td>546283</td><td></td></tr><tr><td>Adipose Derived Stem Cells</td><td>Cellular engineering Technologies</td><td>HMSC.AD-100</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>Fluorescein Diacetate</td><td>Sigma-Aldrich</td><td>F7378</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td></td></tr><tr><td>&amp;alpha;-Modified Eagle&#039;s Medium</td><td>Invitrogen</td><td>a10490-01</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>s25120a</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Invitrogen</td><td>10010023</td><td></td></tr><tr><td>Glass Slides</td><td>VWR international, LLC</td><td>101412-842</td><td></td></tr><tr><td>Syringe Pump</td><td>Fisher Scientific</td><td>14-831-200</td><td>Single syringe</td></tr><tr><td>Ultrasonic Cleaner</td><td>Branson</td><td>1510</td><td></td></tr><tr><td>High Voltage DC Power Supply</td><td>Gamma High Voltage Research</td><td>ES30</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>FEI</td><td>Nova 2300</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Zeiss</td><td>Axio Imager 2</td><td></td></tr></tbody></table>

electrospun nanofiber scaffolds with gradations in fiber organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Usando questo protocollo, si è formata una stuoia in fibra con pendenza organizzativa. La figura 3 mostra le immagini SEM scattate in varie posizioni sul patibolo nanofibre. Qualitativamente, si può determinare che esiste una progressione da fibre uniaxially allineate a 0 mm (Figura 3A) ad una fibra assortimento casuale a 6 mm (Figura 3D). La FFT dà un valore quantitativo per l&#39;allineamento delle fibre, specifiche sui processi quantitativi sono dettagliate qui <s...

Watch this Scientific Journal Video about Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization at JoVE.com

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Qui, vi presentiamo un protocollo per fabbricare scaffold elettrofilate nanofibre con organizzazione sfumati di fibre e di esplorare le loro applicazioni nella regolazione della morfologia delle cellule / orientamento. Gradienti per quanto riguarda le proprietà fisiche e chimiche dei ponteggi nanofibre offrono un&#39;ampia varietà di applicazioni in campo biomedico.

Elettrofilate nanofibre Ponteggi con gradazioni Organizzazione Fiber

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

electrospun-nanofiber-scaffolds-with-gradations-in-fiber-organization

Research

JoVE Journal

Bioengineering

9.8K Views.  University of Nebraska Medical Center.  Qui, vi presentiamo un protocollo per fabbricare scaffold elettrofilate nanofibre con organizzazione sfumati di fibre e di esplorare le loro applicazioni nella regolazione della morfologia delle cellule / orientamento. Gradienti per quanto riguarda le proprietà fisiche e chimiche dei ponteggi nanofibre offrono un'ampia varietà di applicazioni in campo biomedico. 

Video: Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Fondamenti electrospinning: Soluzione Ottimizzazione e parametri Apparatus

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

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Bioingegneria

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

La cultura del motore principale e neuroni sensoriali in Media definiti in elettrofilate Poli-L-lactide Ponteggi nanofibre

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Neuroscienze

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 ...

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Espansione del due-dimensione elettrofilate nanofibre in tre dimensioni ponteggi

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

Genetica

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 ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

Nanofibre ECM proteine ​​e Nanostrutture Engineered Uso Assembly-Surface avviato

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Electrospinning Growth Factor Releasing Microsfere in Fibrosi Ponteggi

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Elettrofilate Fibrosa Ponteggi di Poli (glicerolo-dodecanedioate) per l&#39;ingegneria neurale tessuti da cellule staminali embrionali di topo

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adattamento del processo Electrospinning per fornire tre ambienti unici per un Tri-livello<em&gt; In Vitro</em&gt; Modello della parete delle vie aeree

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

Electrospinning Ponteggi polimeri fibrosi per l&#39;ingegneria tissutale e colture cellulari

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Elettrofilate nanofibre Ponteggi con gradazioni Organizzazione Fiber

Riepilogo

Esplora Altri Video

Capitoli in questo video

Fondamenti electrospinning: Soluzione Ottimizzazione e parametri Apparatus

La cultura del motore principale e neuroni sensoriali in Media definiti in elettrofilate Poli-L-lactide Ponteggi nanofibre

Elaborazione Postproduzione di fibre elettrofilate per l'ingegneria tissutale

Espansione del due-dimensione elettrofilate nanofibre in tre dimensioni ponteggi

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Nanofibre ECM proteine e Nanostrutture Engineered Uso Assembly-Surface avviato

Electrospinning Growth Factor Releasing Microsfere in Fibrosi Ponteggi

Elettrofilate Fibrosa Ponteggi di Poli (glicerolo-dodecanedioate) per l'ingegneria neurale tessuti da cellule staminali embrionali di topo

Adattamento del processo Electrospinning per fornire tre ambienti unici per un Tri-livello

Electrospinning Ponteggi polimeri fibrosi per l'ingegneria tissutale e colture cellulari

Elettrofilate nanofibre Ponteggi con gradazioni Organizzazione Fiber

Riepilogo

Esplora Altri Video

Capitoli in questo video

Fondamenti electrospinning: Soluzione Ottimizzazione e parametri Apparatus

La cultura del motore principale e neuroni sensoriali in Media definiti in elettrofilate Poli-L-lactide Ponteggi nanofibre

Elaborazione Postproduzione di fibre elettrofilate per l'ingegneria tissutale

Espansione del due-dimensione elettrofilate nanofibre in tre dimensioni ponteggi

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Nanofibre ECM proteine ​​e Nanostrutture Engineered Uso Assembly-Surface avviato

Electrospinning Growth Factor Releasing Microsfere in Fibrosi Ponteggi

Elettrofilate Fibrosa Ponteggi di Poli (glicerolo-dodecanedioate) per l'ingegneria neurale tessuti da cellule staminali embrionali di topo

Adattamento del processo Electrospinning per fornire tre ambienti unici per un Tri-livello

Electrospinning Ponteggi polimeri fibrosi per l'ingegneria tissutale e colture cellulari

Nanofibre ECM proteine e Nanostrutture Engineered Uso Assembly-Surface avviato