Vorremmo ringraziare l&#39;American Heart Association Mid-Atlantic di affiliazione (0555407U, GLB) per il finanziamento.

<ol><li> Prima di electrospinning, collagene, da 6 mesi corion bovina, deve essere estratto con un processo a base di acido acetico. Questo collagene estratto viene poi purificato attraverso una serie successiva di dissoluzioni, precipitazioni e dialisi. Una volta che il collagene è liofilizzato, il processo di electrospinning può iniziare. </li><li> Tre materiali sono ammassati: policaprolattone (PCL), elastina (ELAS), e collagene (COL). Questi materiali saranno separatamente sciolto in 1,1,1,3,3,3 esafluoro-2-propanolo (HFP, TCI America) a concentrazioni di 100 mg / ml, 200 mg / ml, e 70 mg / ml, rispettivamente. </li><li> I materiali sono miscelati in cinque fiale di scintillazione diversi rapporti volumetrici di 98-2-0 PCL-ELAS-COL (intima), 45-45-10, 55-35-10, 65-25-10 e PCL-ELAS-COL (media), e 70-0-30 PCL-ELAS-COL (avventizia). </li><li> Per le prove di trazione uniassiale e singoli test di conformità di livelli, ciascuno dei materiali è stato singolarmente elettrofilate, mentre innesti vascolari multistrato per scoppiare, la ritenzione di sutura, ei test di conformità sono stati elettrofilate da una configurazione 3 a 1. Prove di trazione monoassiale ponteggi erano piatti e rettangolari (2,5 cm di larghezza x 10,2 cm di lunghezza x 0,3 cm di spessore) in quanto la mandrini da 2 mm di diametro utilizzato per electrospin singoli livelli e multilivello innesti erano troppo piccoli per la punzonatura dei campioni di prova. </li><li> Per entrambi elettrofilatura singoli e multi-layer, le soluzioni polimeriche sono stati caricati in una siringa da 3 ml di plastica Becton Dickinson con un brusco punta dell&#39;ago-18 calibro, e collocato su una pompa a siringa ad un tasso dispensario set. I singoli tassi di electrospinning sono stati fissati a 4 ml / h, mentre a più livelli i tassi electrospinning sono state variate in funzione di transizione e di livello. Portate e volumi sono stati come tale: l&#39;intima era elettrofilate ad una velocità di 4 ml / ora e un volume di 0,5 ml seguito da una transizione combinando siringhe intimale e mediale per 0,2 ml a 2 ml / h ciascuno. La siringa intimale è stato poi spento e lo strato mediale è stato permesso di girare per 0,6 ml a 4 ml / h seguito da un passaggio tra media e avventizia per 0,2 ml di soluzione di polimero a 2 ml / ora ciascuno. Infine, il supporto è stato fermato e l&#39;avventizia è stato permesso di girare per 0,4 ml a 4 ml / ora. </li><li> Tutti i campioni sono stati reticolato in 50 mM EDC per 18 ore prima di ogni test 1. </li><li> Prove di trazione uniassiale è stato eseguito su sei campioni per un foglio elettrofilate idratata in PBS per 24 ore. &quot;Dog-osso&quot; a forma di campioni sono state tagliate da stuoie elettrofilate e testato su un sistema Bionix MTS 200 test con una cella di carico di 100 N e un tasso di estensione di 10,0 mm / min. Tensione di picco, modulo, e la tensione a rottura sono stati calcolati utilizzando la versione TestWorks 4. </li><li> Test di ritenzione sutura è stato eseguito su sei campioni di 2 mm di diametro interno tubolare da sei innesti elettrofilate diversi, immersi in PBS per 24 ore a 37 ° C, su un Bionix MTS 200 sistema di test con una cella di 50 N di carico (MTS Systems Corp.) e un tasso di estensione di 150,0 millimetri / min secondo il diritto attraverso procedura descritta nella American National Standards Institute 2. 5-0 commerciale PDS II sutura monofilamento viola è stato posto 2 mm dalla fine del campione e prorogato fino alla sutura aveva tirato attraverso l&#39;innesto. Carico di picco è stato registrato in grammi-forza TestWorks utilizzando la versione 4. </li><li> Forza di prova scoppio è stato eseguito su sei campioni provenienti da sei diversi innesti elettrofilate. Tubi di scoppio, 2-3 cm di lunghezza, sono stati idratati in PBS, dotato oltre 1,5 mm di diametro capezzoli collegare al dispositivo, e fissato con 2-0 sutura seta. Air è stato introdotto nel sistema, aumentando la pressione a una velocità di 5 mmHg / s fino a scoppiare i tubi 2. I risultati sono riportati come la pressione in mmHg in cui tubi rotti. </li><li> Compliance dinamica è stata determinata per sei innesti 2 mm di diametro interno tubolare preso da sei innesti diversi elettrofilate ad una lunghezza di 3 cm sotto simulate condizioni fisiologiche. Entrambi i singoli livelli e costruisce tubi multistrato sono stati elettrofilate e testato nelle stesse condizioni. I campioni sono stati testati in un Tissue Engineering intelligente tramite stimolazione meccanica (articoli) bioreattore sviluppato da tecnologie per la crescita del tessuto riempito con PBS a 37 ° C. Il bioreattore fornito un 1Hz variazione ciclica della pressione all&#39;interno del trapianto, dove sono stati indagati tre livelli diversi di pressione 90/50, 120/80 e 150/110 mmHg sistolica / diastolica 2, 3. </li></ol> Rappresentante Risultati Quando i protocolli electrospinning sono svolte correttamente, il prodotto finale dovrebbe essere un dolce, tubi senza saldatura senza segni iniziali di delaminazione tra gli strati. Quando le prove di trazione uniassiale, test resistenza allo scoppio, test di ritenzione di sutura, e test di conformità vengono eseguiti, i risultati devono indicano che la rigidità strato mediale è aumentato, con una quantità ridotta di elastina, le proprietà meccaniche associate dovrebbero dimostrare un tubo rigido.

<ol>
	<li>McClure, M. J., Sell, S. A., Barnes, C. P., Bowen, W. C., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+Electrospun+Polydioxanone-Soluble+Elastin+Blends:+Material+Characterization.">Cross-linking Electrospun Polydioxanone-Soluble Elastin Blends: Material Characterization.</a> Journal of Engineered Fibers and Fabrics. 3, 1-10 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiovascular implants - Vascular graft protheses. VP20, (1994).</li><li>McClure, M. J., Sell, S. A., Simpson, D. G., Walpoth, B. H., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-layered+electrospun+matrix+to+mimic+native+arterial+architecture+using+polycaprolactone,+elastin,+and+collagen:+A+preliminary+study.">A three-layered electrospun matrix to mimic native arterial architecture using polycaprolactone, elastin, and collagen: A preliminary study.</a> Acta Biomater. , (2010).</li></ol>

Diversi autori hanno negli Stati Uniti e tecnologia in attesa di brevetto internazionale in materia presentati in questo manoscritto, e questa tecnologia è stato concesso in licenza a Organogenesi, Inc.

La parte più importante di questo studio è il processo electrospinning. Quando si utilizza un 3-1 input-output ugello, inarcando e la perdita di carica si possono verificare. Se ciò si verifica, la tensione associata al polimero che è in rotazione diminuirà provocando saldati, &quot;umido&quot; fibre e la creazione di delaminazione tra ciascuno dei tre strati. Pertanto, in coerenza potenziali elettrici sono essenziali per ottenere un ideale multistrato del tubo. Mancata corrispondenza d...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Polycaprolactone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Elastin</td>
<td>&nbsp;</td>
<td>Elastin Products Company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Acid</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TRIS</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 L Avanti J-HC Centrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1,1,1,3,3,3 hexafluoro-2-propanol</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3 ml Becton Dickinson Syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CZE1000R Rack Mount Power Supply</td>
<td>&nbsp;</td>
<td>Spellman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>High-Pressure Syringe Pump</td>
<td>&nbsp;</td>
<td>Cole Parmer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>70 % ethanol</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ImageTool 3.0 software</td>
<td>&nbsp;</td>
<td>Shareware provided by UTHSCSA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scanning Electron Microscope</td>
<td>&nbsp;</td>
<td>Zeiss EV050</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MTS Bionix 200 testing system</td>
<td>&nbsp;</td>
<td>MTS Systems Corp.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TestWorks version 4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Intelligent Tissue Engineering via Mechanical Stimulation (ITEMS) Bioreactor</td>
<td>&nbsp;</td>
<td>Tissue Growth Technologies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

tri-layered electrospinning to mimic native arterial architecture using polycaprolactone, elastin, and collagen: a preliminary study

In tutta l&#39;arteria nativa, collagene ed elastina svolgono un ruolo importante, fornendo un backbone meccanica, impedendo la rottura nave, e la promozione di recupero ai sensi deformazioni pulsatile. L&#39;obiettivo di questo studio era quello di riprodurre la struttura delle arterie native da fabbricare una realtà a più condotto elettrofilate composto di poli (caprolattone) (PCL) con l&#39;aggiunta di elastina e collagene con miscele di 45-45-10, 55-35 - 10 e 65-25-10 PCL-ELAS-COL per dimostrare le proprietà meccaniche indicative di natale tessuto arterioso, pur rimanendo favorevole alla rigenerazione dei tessuti. Innesti interi e singoli strati sono stati analizzati utilizzando test uniassiale di trazione, compliance dinamica, ritenzione di sutura, e la resistenza allo scoppio. I risultati di conformità rivelato che le modifiche al livello medio / mediale cambiato il comportamento complessivo del trapianto con valori di tutto rispetto innesto compresi tra 0,8 - 2,8% / 100 mmHg, mentre i risultati monoassiale dimostrato una gamma media modulo di 2,0-11,8 MPa. Sia i dati modulo e la conformità dei valori visualizzati all&#39;interno della gamma di arteria nativa. Modellistica matematica è stato implementato per mostrare come i cambiamenti nel livello di rigidità influenzano lo stress complessivo parete circonferenziale, e come un aiuto di progettazione per ottenere la migliore combinazione meccanica dei materiali. Nel complesso, i risultati hanno indicato che un innesto può essere progettato per imitare un tri-strato struttura alterando le proprietà dei livelli.

Watch this Scientific Journal Video about Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study at JoVE.com

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Lo scopo di questo studio era quello di imitare il nativo tre architettura a strati della parete arteriosa. Per fare questo, electrospinning è stato impiegato con l&#39;uso di un 3-1 (input-output) ugello e miscele di policaprolattone, elastina e collagene.

Tri-strato Electrospinning di Mimic nativa Architettura arteriosa utilizzando Policaprolactone, elastina e collagene: uno studio preliminare

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

tri-layered-electrospinning-to-mimic-native-arterial-architecture

Research

JoVE Journal

Bioengineering

12.8K Views.  Virginia Commonwealth University. Lo scopo di questo studio era quello di imitare il nativo tre architettura a strati della parete arteriosa. Per fare questo, electrospinning è stato impiegato con l'uso di un 3-1 (input-output) ugello e miscele di policaprolattone, elastina e collagene.

Video: Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Progettazione di un bioreattore per la pressione ciclica Ex Vivo Studio delle valvole cardiache aortica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Ricerca

Bioingegneria

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

In sintesi in vitro di Native, Spaziatura fibrosa lungo e segmentale collagene spaziatura lungo

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Un metodo migliore per la preparazione di tipo I collagene della pelle

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging denaturate collagene fili<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via Photo-triggered ibridazione di Caged collagene Mimetic peptidi

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Combinazione di Microstereolithography e Electrospinning per la Produzione di Membrane Equipaggiato con nicchie per la rigenerazione della cornea

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar gradiente di diffusione del sistema per Indagare Chemiotassi in un 3D collagene Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

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 ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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 ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

Creazione di un modello geometrico strutturalmente realistico agli elementi finiti di un Cardiomyocyte per studiare il ruolo dell'architettura cellulare in biologia dei sistemi del Cardiomyocyte

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...