Il sostegno finanziario è stato fornito a JMS dal NIH Biomeccanica in Medicina Rigenerativa Training Program T32 (2T32EB003392), per QJ dal Dowd-ICES Fellowship e AWF da New Innovator Award del direttore NIH (1DP2HL117750).

1. Fabbricazione del Maestro Mold Uso Fotolito <ol><li> Le nanofibre proteiche ECM, nanofabrics e nanostrutture per essere fabbricati vengono prima progettati utilizzando Computer Aided Design (CAD). Questo file CAD viene trasferito in una fotomaschera. Il tipo di fotomaschera dipenderà dalla risoluzione delle caratteristiche; con una fotomaschera a base di trasparenza, adeguata per dimensioni caratteristiche fino a ~ 10 micron. Piccole caratteristiche &lt;10 micron richiederà un cromata su fotomaschera vetro. Tutte le nanofibre e nanostrutture qui presentati sono stati fabbricati utilizzando un fotomaschera base di trasparenza, e quindi erano su scala nanometrica in spessore, ma le dimensioni non laterali. Nota: È importante distinguere quali regioni del fotomaschera sarà buio (impedire alla luce UV per passare attraverso) e che sarà trasparente (permettono alla luce di passare attraverso UV) come questo, insieme con il tipo di photoresist (positivo o negativo) , detterà la topografia finale dello stampo master. </li><li> Per iniziare la fabbricazione dello stampo maestro, disidratare un wafer 4 &quot;silicio ponendolo su piastra riscaldante impostato a 150 ° C per 15 min. </li><li> Centrare il wafer sul mandrino vuoto di uno spin-verniciatura. Versare SU8-2015 fotoresist negativo sul centro del wafer e continuare versando in cerchi concentrici fino a circa due terzi del wafer è coperto. Nota: conservare la bottiglia di SU8 vicino al wafer quando si versa per ridurre al minimo la formazione di bolle. </li><li> Programmare il spincoater come segue: <ul><li> Ciclo di diffusione: 500 rpm con una accelerazione di 100 giri / sec per 10 sec. </li><li> Centrifuga: 4.000 rpm con una accelerazione di 100 giri / sec per 30 sec. </li></ul> Nota: Questa ricetta spincoating formerà uno strato di fotoresist che è ~ 10 pm di spessore. Modificando la velocità di centrifuga o la formulazione SU8, lo spessore può essere regolata. </li><li> Morbido cuocere il wafer ponendolo su piastra riscaldante impostato a 95 ° C per 3 min. </li><li> Esporre il wafer con luce UV attraversola fotomaschera per un dosaggio totale di 140 mJ / cm 2. Nota: SU8 è un photoresist negativo dunque regioni in cui la luce UV è in grado di passare attraverso il photomask rimarranno dopo lo sviluppo e diventare funzioni poste sullo stampo master. </li><li> Posta esposizione cuocere il wafer ponendolo su piastra riscaldante impostato a 95 ° C per 4 min. </li><li> Sviluppare il wafer ponendolo in autore SU8 per 3 min. Dopo 3 minuti, risciacquare il wafer con alcol isopropilico. Se un film bianco viene prodotto durante il risciacquo, la cialda non è completamente sviluppato e dovrebbe essere riposto nello sviluppatore per altri 30 sec. Risciacquare con alcol isopropilico. Ripetere questo processo fino a quando un film bianco non si forma durante il risciacquo alcool isopropilico. </li><li> Essiccare il wafer in un flusso di azoto e posto in una capsula di Petri da 150 mm a proteggere dalla polvere. </li></ol> 2. Rendere i PDMS Stamps <ol><li> Preparare il prepolimero PDMS combinando la base elastomero e induritore in un10:01 w rapporto / w. Tipicamente 80 g di base e 8 g di induritore vengono utilizzati per garantire il PDMS sufficienti a coprire lo stampo master in uno strato di 1 cm di spessore. </li><li> Mescolare e degassarla PDMS utilizzando un mixer centripeta impostare la seguente: <ul><li> Mix: 2,000 rpm per 2 minuti </li><li> Degas: 2,000 rpm per 2 min. </li></ul></li><li> Se un mixer non è disponibile mescolare i PDMS a mano per 10 minuti utilizzando un 10 ml pipetta sierologica. Degassarla miscela ponendolo in un essiccatore sotto vuoto per 30 minuti per rimuovere le bolle. </li><li> Versare abbastanza prepolimero PDMS sullo stampo master (modellato wafer di silicio) per formare uno strato di 1 cm di spessore. Curare le PDMS dalla cottura a 65 ° C per 4 ore oa temperatura ambiente per 48 ore. </li><li> Una volta indurito, Le regioni contenenti i modelli possono essere tagliate per formare le PDMS francobolli. Per distinguere il lato funzione dal retro del timbro PDMS, tagliare una tacca su uno degli angoli sul retro del timbro. </li></ol> 3. Microcontatto Printing Patterns ECM <ol><li> Pulire coprioggetto 25 millimetri di vetro del diametro di sonicazione in etanolo al 95% per 1 ora e poi asciugare in forno a 65 ° C. </li><li> Preparare la soluzione sciogliendo PIPAAm PIPAAm in 1-butanolo ad una concentrazione del 10% (w / v, tipicamente 1 g in 10 ml). </li><li> Centrare vetrino coprioggetti sulla morsa a vuoto dei spincoater e pipetta 200 microlitri della soluzione PIPAAm modo che l&#39;intera superficie del vetro è coperta. </li><li> Spincoat il vetrino a 6.000 rpm per 1 min. </li><li> Pulire i PDMS Stamp mediante sonicazione in etanolo al 50% per 30 min e quindi asciugare sotto flusso di azoto. Nota: Asciugatura e passi successivi devono essere eseguite in un armadio biosicurezza per mantenere la sterilità per le applicazioni in cui saranno utilizzati i nanostrutture ECM con le cellule. </li><li> Ricoprire la superficie modellata di ogni timbro PDMS con 200 ml di soluzione proteica, tipicamente 50 mg / ml in acqua distillata sterile per FN. Incubare per 1 ora a temperatura ambiente. Nota: Thè rivestimento volume è un timbro PDMS 1,5 centimetri 2 e dovrà essere regolato a seconda delle dimensioni del timbro PDMS, la proteina ECM utilizzato e la concentrazione della proteina ECM in soluzione. </li><li> Lavare i PDMS francobolli in acqua distillata per rimuovere l&#39;eccesso di proteine ​​e asciugare accuratamente sotto un flusso di azoto. Nota: L&#39;acqua a sinistra sul timbro attiverà lo scioglimento prematuro del rivestimento PIPAAm sul vetrino ed impedire il corretto trasferimento delle proteine. </li><li> Per la fabbricazione sterile, posizionare i coprioggetti PIPAAm rivestite all&#39;interno di una scatola di Petri chiusa e sterilizzare con l&#39;esposizione ai raggi UV, 45 minuti sotto la luce UV in un armadio biosicurezza è sufficiente. Se non è richiesta la sterilità questo passaggio può essere omesso. </li><li> Eseguire la stampa microcontact posizionando il lato caratteristica del timbro PDMS in contatto con il coprioggetto PIPAAm rivestite. Se necessario, utilizzare pinze per toccare leggermente sul retro dei francobolli per rimuovere eventuali bolle d&#39;aria e assicurare il contatto uniforme. </li><li> Dopo 5 kmn, staccare il timbro PDMS dal vetrino. </li><li> In questa fase, proteine ​​ECM aggiuntivi possono essere configurate in modo da creare strutture più complesse e multicomponenti. Fino a 3 stampe sono state verificate per lavorare con questo processo, e più può essere fattibile. </li></ol> 4. Rilascio di ECM nanofibre e Nanostrutture <ol><li> Posizionare il vetrino rivestito PIPAAm modellato in una capsula di Petri 35 millimetri e controllare la fedeltà del modello usando la microscopia a contrasto di fase. A seconda del modello, una camera CCD può essere necessario risolvere le caratteristiche del modello. Microscopia a fluorescenza può anche essere utilizzato per controllare il pattern ottenute le proteine ​​ECM sono fluorescente. </li><li> Aggiungere 3 ml di acqua distillata a 40 ° C per capsula di Petri e consentire all&#39;acqua di raffreddare gradualmente. </li><li> La dissoluzione dello strato PIPAAm e il rilascio dei modelli proteici ECM possono essere monitorati con microscopio a contrasto di fase. Se l&#39;applicazione non consente l&#39;utilizzo di tec otticahniques, il rilascio può essere monitorato misurando la temperatura della soluzione. Tipicamente, l&#39;acqua viene raffreddata a temperatura ambiente, ben al di sotto di LCST PIPAAm (32 ° C), per garantire le nanostrutture proteiche ECM sono stati rilasciati. </li><li> Dopo il rilascio, le nanofibre, nanofabrics e altre nanostrutture sono galleggianti in acqua. Per usarli per ulteriori applicazioni hanno bisogno di essere manipolato. L&#39;approccio esatto dipenderà dell&#39;obiettivo sperimentale e può includere passaggi come immobilizzare su un&#39;altra superficie, muovendosi con un sistema micromanipolatore o incorporare in un idrogel. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Il metodo SIA presentato qui imita assemblaggio matrice cellulo-mediata e consente la progettazione di ECM nanofibre proteiche e nanostrutture con dimensioni sintonizzabile, l&#39;organizzazione e la composizione. Anche se non identica alla ECM cellule generate, SIA crea ECM composto da nanoscala fibrille della proteina 20 che subiscono piegatura reversibili / spiegamento durante sollecitazioni meccaniche 21 e possono legarsi cellule 20. Ciò fornisce una capacità unica di costruire ECM...

La matrice extracellulare (ECM) nei tessuti è composto di proteine ​​multifunzionali coinvolte nella regolazione chimici e fisici di processi cellulari multiple comprendenti adesione, proliferazione, differenziazione e apoptosi 1-3. L&#39;ECM è sintetizzato, assemblato, e organizzato dalle cellule e le fibrille proteiche costituenti hanno composizioni uniche, dimensioni delle fibre, geometrie e architetture interconnesse che variano con il tipo di tessuto e stadio di sviluppo. Studi recenti hanno dimostrato che l&#39;ECM può fornire indicazioni istruttive per guidare le cellule a formare tessuti ingegnerizzati 4, suggerendo che ricapitolando l&#39;ECM in termini di composizione e struttura potrebbe consentire lo sviluppo di materiali biomimetici per applicazioni di ingegneria tissutale e delle biotecnologie. Un certo numero di metodi di fabbricazione sono stati sviluppati per ingegnerizzare supporti polimerici che possono mimare aspetti della ECM nei tessuti. Ad esempio, elettrospinning e fase SEPARzione hanno entrambi dimostrato la capacità di formare matrici porose di fibre con diametri da decine di micrometri fino a decine di nanometri 5-7. Entrambe le tecniche hanno anche dimostrato che matrici altamente porosi di nanofibre possono supportare l&#39;adesione cellulare e l&#39;infiltrazione nel impalcatura 8. Tuttavia, questi approcci sono limitati nelle possibili geometrie fibre, orientamenti e 3D architetture che possono essere creati. Electrospinning produce tipicamente ponteggi con fibre orientate sia casuale o altamente allineati, mentre la separazione di fase produce ponteggi a fibre orientate in modo casuale. Vi sono inoltre limitazioni sui materiali, con i ricercatori in genere utilizzando polimeri sintetici, come poli (ε-caprolattone) 8 e acido poli (lattico-co-glicolico) 9, che vengono successivamente rivestite con proteine ​​ECM per promuovere l&#39;adesione cellulare. Biopolimeri naturali sono anche utilizzati, tra cui collagene di tipo I 10, gelatina 11, fibrinogeno 12,chitosano 13, 14 e seta, ma rappresentano solo un piccolo sottoinsieme di proteine ​​trovate in tessuto nativo. La maggior parte dei tessuti contengono un grande ambiente di proteine ​​ECM e polisaccaridi inclusi fibronectina (FN), laminina (LN), collagene di tipo IV e acido ialuronico che sono difficili o impossibili da realizzare nanofibre con metodi esistenti. Per affrontare questa sfida, abbiamo concentrato i nostri sforzi di ricerca sul imitando il modo in cui le cellule sintetizzano, assemblare e organizzare le fibrille della proteina ECM nel loro ambiente. Mentre il processo fibrillogenesi specifico varia per differenti proteine ​​ECM, tipicamente un cambiamento conformazionale nella molecola proteica ECM è attivata da un enzimatica o interazione recettore-mediata, che espone siti autoassemblaggio criptici. Qui usiamo FN come sistema modello per comprendere meglio il processo di fibrillogenesi. Brevemente, omodimeri FN legano ai recettori di integrina sulla superficie cellulare tramite la sequenza amminoacidica RGD nel 10 tipo IIRipeto unità. Una volta legato, le integrine si allontanano via actomyosin contrazione e si sviluppano i dimeri FN per esporre siti di self-assembly criptici. L&#39;esposizione di questi siti di legame FN-FN permette ai dimeri FN per assemblare in un fibrille insolubili proprio sulla superficie cellulare 15. Lavori in sistemi cell-free ha dimostrato che criptici siti di legame FN-FN possono essere rivelati tramite dispiegarsi con denaturanti 16 o tensione superficiale a un-liquido-solido aria interfaccia 17-19. Tuttavia, le fibre FN creata di queste tecniche sono limitate a dimensioni e geometrie fibre specifiche e sono tipicamente legati ad una superficie. Qui si descrive un chiamato assieme superficie avviato approccio (SIA) 20 che supera queste limitazioni utilizzando interazioni proteina-superficie per creare posa libera nanofibre insolubili, nanofabrics (fogli 2D) e altre nanostrutture composte da proteine ​​ECM singoli o multipli (Figura 1 ). In questo process, proteine ​​ECM vengono assorbiti da un compatto, conformazione globulare in soluzione e parzialmente denaturato (piegato) su una fantasia, polidimetilsilossano idrofobo (PDMS) timbro. Le proteine ​​ECM vengono poi trasferiti in questo stato su un poli termoreattiva (N-isopropylacrylamide) (PIPAAm) superficie attraverso microcontact stampa 22. Quando idratato con acqua 40 ° C la PIPAAm rimane un solido, ma quando raffreddato a 32 ° C che passa attraverso una temperatura di soluzione critica inferiore (LCST) dove diventa idrofila, si gonfia con acqua e poi si dissolve, rilasciando le nanostrutture ECM assemblati off di la superficie. Il metodo SIA consente di controllare le dimensioni con precisione su scala nanometrica. Controllando parametri chiave quali la composizione, geometria fibra, e l&#39;architettura, è possibile ricapitolare molte proprietà della ECM trovati in vivo e di sviluppare scaffold avanzate per applicazioni di ingegneria tissutale e biotecnologia.

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Poly(N-isopropylacrylamide) / PIPAAm</td>
			<td style="width:145px;">Polysciences</td>
			<td style="width:123px;">21458-10</td>
			<td style="width:167px;">40,000 Mw</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sylgard 184 Silicone kit (PDMS)</td>
			<td style="width:145px;">Dow Corning</td>
			<td style="width:123px;"></td>
			<td style="width:167px;">Mix 10 parts base with 1 part curing agent.&#160;</td>
		</tr>
		<tr height="21">
			<td height="42" style="height:42px;width:216px;">Butanol</td>
			<td style="width:145px;"></td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Fibronectin</td>
			<td style="width:145px;">BD biosciences</td>
			<td style="width:123px;">354008</td>
			<td style="width:167px;">Human, 1mg</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Laminin</td>
			<td style="width:145px;">BD biosciences</td>
			<td style="width:123px;">354239</td>
			<td style="width:167px;">Ultrapure, mouse, 1mg</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Negative Photoresist</td>
			<td style="width:145px;">Microchem</td>
			<td style="width:123px;">SU8-2015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">SU8 Developer</td>
			<td style="width:145px;">Microchem</td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:216px;">Sonicator Branson M3510</td>
			<td style="width:145px;">Branson Ultrasonic Corporation</td>
			<td style="width:123px;">CPN-952-318</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Thinky ARE-250 Mixer</td>
			<td style="width:145px;">Thinky Corporation</td>
			<td style="width:123px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Spincoater</td>
			<td style="width:145px;">Specialty Coating Systems</td>
			<td style="width:123px;">G3P-8</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glass cover 25mm diameter, No 1.5</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:123px;">12-545-86</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

ecm protein nanofibers and nanostructures engineered using surface-initiated assembly

La matrice extracellulare (ECM) nei tessuti è sintetizzato e assemblato da cellule formano un fibrillare 3D, rete proteina strettamente regolata con diametro delle fibre, composizione e organizzazione. Oltre a fornire supporto strutturale, le proprietà fisiche e chimiche del ECM svolgono un ruolo importante in diversi processi cellulari tra cui l&#39;adesione, la differenziazione e apoptosi. In vivo, l&#39;ECM è assemblato esponendo criptico autoassemblaggio (fibrillogenesi) siti all&#39;interno di proteine . Questo processo varia per diverse proteine, ma fibronectina (FN) fibrillogenesi è ben caratterizzato e serve come sistema modello per il montaggio ECM cellulo-mediata. In particolare, le cellule utilizzano i recettori delle integrine sulla membrana cellulare per legare dimeri FN e le forze contrattili actomyosin generati a spiegare ed esporre siti di legame per il montaggio in fibre insolubili. Questo processo mediata dal recettore permette alle cellule di assemblare e organizzare la ECM dal cellulare alla SCA Tissueles. Qui, vi presentiamo un metodo denominato assembly superficie avviato (SIA), che riassume l&#39;assemblaggio matrice cellulo-mediata tramite interazioni proteina-superficie a svolgersi proteine ​​ECM e li assemblano in fibre insolubili. In primo luogo, le proteine ​​ECM sono adsorbiti su un polidimetilsilossano idrofobo (PDMS), superficie dove si parte snaturano (spieghi) ed espongono domini di legame criptici. Le proteine ​​ripiegate vengono poi trasferiti in ben definite micro-e nanostrutture attraverso la stampa microcontact su un poli termoreattiva (N-isopropylacrylamide) (PIPAAm) di superficie. Dissoluzione termicamente attivato il PIPAAm conduce al montaggio finale e il rilascio di nanofibre proteiche insolubili ECM e nanostrutture con geometrie ben definite. Architetture complesse sono possibili ingegneria definiti modelli sulle PDMS timbri utilizzati per la stampa microcontact. Oltre a FN, il processo SIA può essere utilizzato con laminina, fibrinogeno e collagene di tipo I e IV creare multicomponente ECM nanostrucTures. Così, SIA può essere utilizzato per progettare ECM materiali a base di proteine ​​con un preciso controllo della composizione proteica, geometria fibre e architettura scaffold per ricapitolare la struttura e la composizione della ECM in vivo.

SIA è in grado di ingegneria ECM nanofibre proteiche con un controllo preciso sopra le dimensioni di fibre. Per dimostrare questo, matrici di nanofibre FN con dimensioni planari di 50 x 20 micron sono stati modellati su un vetrino PIPAAm rivestita (Figura 2A). Al rilascio, le fibre contratte perché erano sotto un pre-sollecitazione inerente quando modellata sulla superficie PIPAAm (Figura 2B). Analisi delle nanofibre FN ha rivelato che erano monodispersa pre-release, con una lunghezza...

Watch this Scientific Journal Video about ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly at JoVE.com

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

Un metodo per ottenere nanofibre e nanostrutture complesse da proteine ​​singole o multiple della matrice extracellulare è descritto. Questo metodo utilizza interazioni proteina-superficie per creare materiali a base di proteine-free-standing con la composizione sintonizzabile e l&#39;architettura per l&#39;uso in una vasta gamma di applicazioni di ingegneria tissutale e delle biotecnologie.

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 ...

ecm-protein-nanofibers-nanostructures-engineered-using-surface

Research

JoVE Journal

Bioengineering

12.4K Views.  Carnegie Mellon University.  Un metodo per ottenere nanofibre e nanostrutture complesse da proteine ​​singole o multiple della matrice extracellulare è descritto. Questo metodo utilizza interazioni proteina-superficie per creare materiali a base di proteine-free-standing con la composizione sintonizzabile e l'architettura per l'uso in una vasta gamma di applicazioni di ingegneria tissutale e delle biotecnologie. 

Video: ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Visualizzazione del DNA ricombinante e Complessi proteina usando la microscopia a forza atomica

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Ricerca

Bioingegneria

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

L&#39;utilizzo di nanostrutture di cristallo plasmoniche e fotonici per Enhanced manipolazione micro e nanoparticelle

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Sintesi, assemblaggio e caratterizzazione di film monostrato Protetta delle nanoparticelle d&#39;oro per Elettrochimica proteine ​​monostrato

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

Progettato Vascolarizzato muscolare Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning e montaggio dei microvasi 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Scaling di Engineered vascolari innesti Usando 3D Stampato Guide e il metodo di impilamento Anello

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Non distruttive monitoraggio dello sviluppo del vaso sanguigno tessutale basati su Scaffold degradabile usando la tomografia a coerenza ottica

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Innesto ingegnerizzato per la ricostruzione esofagea circonferenza nei ratti

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Auto-assemblaggio di acidi nucleici peptidici modificati gamma in nanostrutture complesse in miscele di solventi organici

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Assemblaggio rapido di costrutti multi-gene utilizzando la clonazione modulare del Golden Gate

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

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

Nanofibre ECM proteine e Nanostrutture Engineered Uso Assembly-Surface avviato