Questo lavoro è stato sostenuto dal Research Grant UWS.

1. Chitosano adesivo Preparazione <ol><li> La polvere chitosano è solubile in soluzione di acido acetico, per la preparazione di una soluzione stock di acido acetico (2% v / v), aggiungere 10 ml di acido acetico glaciale a 490 ml di acqua deionizzata (DI-H 2 O). </li><li> Per la preparazione di una soluzione stock di Rosa Bengala (RB) (0,01% w / v) in acido acetico, pesare 5 mg di RB in un flaconcino avvolto con un foglio di alluminio per evitare fotodecolorazione. Aggiungere 0,5 ml di DI-H 2 O per sciogliere la polvere poi aggiungere 49,5 ml di soluzione di acido acetico magazzino (descritte nella sezione 1.1). </li><li> Per la preparazione della soluzione di chitosano (1,7% w / v), pesare 0,85 g di polvere di chitosano (MW medio, 85% grado di acetilazione) e aggiungere 50 ml di soluzione di acido acetico RB. Il controllo è stata preparata sciogliendo il chitosano in soluzione di acido acetico senza RB. </li><li> Aggiungere un agitatore magnetico alla miscela chitosano ed agitare a temperatura ambiente. Per garantire la completa dissoluzione dei componenti, mescolare il chitosanoin acido acetico + RB per due settimane e il chitosano in acido acetico per 5 giorni (controllo). Quando chitosano è dissolto, il pH della soluzione aumenta da ~ 2.6 a ~ 3.9. Notare che RB è scarsamente solubile in ambiente acido e richiede un aumento del pH della soluzione e un tempo esteso agitazione per scioglierli. </li><li> Centrifugare le soluzioni a 3270 xg per 1 ora e raccogliere il supernatante utilizzando, per esempio, una siringa da 10 ml sterile. Fare attenzione a non disturbare il pellet impurità. Durante questa fase, la soluzione viene pulito da impurità macroscopiche e materia insolubile. </li><li> Iniettare l&#39;(centrifugato) soluzione di chitosano su una lastra di perpex piatto utilizzando una siringa sterile. Lo spessore del film essiccato dipende dal rapporto tra la quantità di soluzione erogata e la superficie su cui si sviluppa la soluzione. Assicurarsi che la soluzione è privo di bolle d&#39;aria e distribuito uniformemente sulla lastra. Per ottenere film sottili con ~ 13 micron di spessore, iniettare ~ 1 ml di soluzione di oltre 12 cm 2 di superficiezona (dimensioni ~ 3x4 centimetri, Figura 1A). </li><li> Coprire la lastra Perspex con uno schermo, in alluminio, ad esempio, per evitare fotodecolorazione della soluzione. Assicurarsi che la ventilazione è garantita abbastanza aria per asciugare la soluzione sotto lo schermo. </li><li> La soluzione viene lasciata asciugare a temperatura ambiente (25 ° C) e pressione (1 atm) finché la pellicola risultante è insolubile in acqua e non si gonfiano macroscopicamente. Questo è in genere raggiunto in 2 settimane. </li><li> Staccare le pellicole con cura usando una spatola sottile e piatto: questi film può essere facilmente staccata la lastra di perspex senza danneggiarli. </li><li> Misurare lo spessore dei film utilizzando un micrometro. </li><li> Tagliare l&#39;adesivo in strisce rettangolari con una forbice affilata pulito e metterli tra le diapositive di vetro sterili per conservare la loro forma piatta. Avvolgere i vetrini in un foglio di alluminio per la luce schermatura e conservarli a temperatura ambiente. Etichettare gli adesivi con o senza RB come &quot;rosa adesivo&quot; o &quot;adhes chitosanoive &quot;rispettivamente. </li><li> La rosa pellicole adesive possono essere sterilizzati con etanolo (90%) o con raggi gamma (0,1 KGy / h per 24 ore) 4,5. </li></ol> 2. Adesivo Attenuazione ottica <ol><li> Fissare l&#39;adesivo rosa all&#39;interno di una cuvetta di quarzo. </li><li> Registrare il suo spettro attenuato nel range di 400 - 800 nm usando un spettrofotometro a doppio raggio 4. </li><li> Assumendo la validità della legge di Beer, calcolare la lunghezza di attenuazione del collante nel modo seguente: I = I 0 e-Ax dove 0 è l&#39;intensità del fascio incidente, 1 / A è la lunghezza di attenuazione, ed x è lo spessore del film. </li><li> Ripetere i passaggi 2,1-2,3 per l&#39;adesivo chitosano per servire come controllo. </li></ol> 3 In applicazione in vitro:. Tessuto Bonding laser su Intestino <ol><li> Intestino di vitello raccolta subito dopo l&#39;eutanasia degli animali e conservare a -80 ° C. </li><li> Prima dell&#39;uso, sbrinamento e idratare la tiUMERO immergendolo in DI-H 2 O per 10 min. </li><li> Bisecare il tessuto in sezioni (2 x 1 cm) usando una lama di 10 ° sotto un microscopio operativo (x10). Mantenere l&#39;umido sezioni con DI-H 2 O. </li><li> Portare i monconi incisione vicini (end to end). Rimuovere l&#39;acqua in eccesso dalla superficie con punte di cotone. </li><li> Posizionare una rosa nastro adesivo (10 x 6 mm) attraverso l&#39;incisione sullo strato sierosa con microforceps assicurando pieno contatto con l&#39;intestino (Figura 2). </li><li> L&#39;adesione del tessuto adesivo rosa è attivato da un diodo-pompato laser a stato solido, che è accoppiato ad una fibra ottica multimodale (diametro del nucleo 200 micron) 4. </li><li> Impostare il livello di potenza del laser a 180 mW e irradiare l&#39;adesivo per ~ 6 min con un fascio di punto di ~ 5 mm, utilizzando i parametri elencati nella Tabella 1. Spot-irradiate l&#39;adesivo garantire che ogni spot viene irradiata per ~ 5 secondi prima di spostare il fascio nel punto adiacente. Tla procedura garantisce che il raggio laser scansiona l&#39;intera area superficiale dei tempi diversi adesivi. </li><li> Per valutare la resistenza di legame tessuto, fissare un campione a un tensiometro calibrato singola colonna (Instron, MA, USA) utilizzando i grip meccanico. Spostare i punti di presa a 22 mm / min fino a quando i due monconi del tessuto separati 4. </li><li> Posizionare un adesivo rosa sul tessuto, come descritto nel passaggio 3,5. Non irradiare il campione e misurare la resistenza della giunzione del tessuto come descritto al punto 3.8. Questo esempio serve da controllo. </li></ol> 4 In applicazione Vivo:. Anastomosi nervo tibiale <ol><li> Sedare il ratto Wistar con isoflurano / miscela O 2 (4% durante l&#39;induzione, 2% in seguito) 5. Per via sottocutanea iniettare Buprenex (0,03 mg / kg) per fornire intra-operatoria attività analgesica e alleviare il dolore o disagio possibile. </li><li> La seguente procedura chirurgica deve essere effettuata con un microscopio operatorio (x10). Fai una pelle obliquaincisione di circa 3 cm di lunghezza nella parte dorso-laterale della coscia destra ed esporre il nervo tibiale con un muscolo-splitting approccio attraverso i muscoli glutei 2. </li><li> Parzialmente sezionare e tagliare l&#39;avventizia del nervo tibiale con rette micro-forbici e assorbire l&#39;acqua in eccesso con garza sterile o batuffoli di cotone. </li><li> Tagliare il nervo tibiale con micro-forbici. </li><li> Posizionare una striscia sterile chitosano (dimensioni 5 x 4 mm) al di sotto del nervo tibiale utilizzando micro-pinza. </li><li> Approssimativo i monconi nervosi end-to-end con micro-pinza sulla striscia adesiva rosa. </li><li> La striscia aderisce completamente al nervo formando un collare, che può aiutare con la rotazione del nervo durante l&#39;irradiazione laser della striscia. </li><li> Attivare la rosa adesivo con il laser, come descritto nei punti 3.6) e 3.7). </li><li> Tagliare l&#39;adesivo ridondante alzò dal collare del nervo operato. </li><li> Chiudere i muscoli e la pelle, con cinque punti di sutura 3-0 5. </li&gt; <li> Standard post-operatorie procedure di recupero devono essere seguite con relative ispezioni al giorno per deiscenza o formazione di pus e amministrazione analgesico, se necessario. </li></ol> 5. Risultati rappresentativi I film che si ottengono sono aumentati luminoso a colori, fini e hanno una superficie liscia (Figura 1). Sono anche flessibile e può essere arrotolato in tubi di piccolo diametro senza lacrime causano o altri danni evidenti (Figura 1B). L&#39;adesivo rosa ha due picchi di assorbimento a 530 e 562 nm, il laser verde è pertanto fortemente assorbita dal collante e la lunghezza corrispondente attenuazione a 532 nm è ~ 12 um (Figura 3). In contrasto, chitosano film senza RB attenua debolmente il laser (1 / a ~ 162 micron), probabilmente dovuta alla dispersione. Risulta dalla trama spettri che nessuna aggregazione significativa di RB si verifica nei film. La rosa adesivo si lega saldamentel&#39;intestino upon irradiazione laser (Figura 2) realizzare un tipico carico massimo a rottura di 0,9 ± 0,4 N (n = 30). La forza adesiva è stato stimato come il carico massimo diviso per l&#39;area della superficie adesiva, cioè, 15 ± 6 kPa (n = 30). Il non irradiato adesivo rosa legato significativamente inferiore al tessuto (2 ± 2 kPa, n = 30, t-test non appaiati p &lt;10 6). In vivo anastomosi del nervo tibiale è stato raggiunto con successo dopo l&#39;applicazione ed il legame dell&#39;adesivo rose in combinazione con il laser verde. Una settimana dopo l&#39;intervento, il nervo è stato operato in continuità e la forza di riparazione pari a 17 ± 9 kPa (n = 10). <img alt="Figura 1" src="/files/ftp_upload/4158/4158fig1.jpg" /> Figura 1. A) L&#39;RB-chitosano soluzione viene colata su una lastra piana Perspex (spessore soletta ~ 5 mm). Carta millimetrata è posto sotto la lastra di facilitare l&#39;asciutto-cast passo. L&#39;insolubile in acqua film è ottenuta due settimane dopo la fusione. B) L&#39;adesivo rosa è flessibile e può essere arrotolato in un piccolo cilindro. <img alt="Figura 2" src="/files/ftp_upload/4158/4158fig2.jpg" /> Figura 2. L&#39;adesivo rosa è legato a intestino di vitello dopo irradiazione laser. Irraggiamento uniforme viene applicato l&#39;adesivo durante RB tessuto-bonding; tuttavia spot selezionati sono mostrati in figura per illustrare l&#39;effetto fotometabolismo del laser sull&#39;adesivo. <img alt="Figura 3" src="/files/ftp_upload/4158/4158fig3.jpg" /> Figura 3. Lo spettro di assorbimento (in unità arbitrarie) dell&#39;adesivo rosa mostra due picchi a 530 e 562 nm. Il laser verde (λ = 532 nm) è quindi fortemente assorbita dall&#39;adesivo durante l&#39;esposizione laser. L&#39;adesivo chitosano senza RB assorbe poco il raggio laser. <table border="1"><tbody><tr><td></td><td> n </td><td>2) </td><td> Potenza (W) </td><td> Tempo (s) </td><td> Fluence (J / cm 2) </td><td> I (W / cm 2) </td><td> Carico massimo / Area (kPa) </td></tr><tr><td> Adesivo + Laser </td><td> 30 </td><td> 60 ± 10 </td><td> 0,18 ± 0,03 </td><td> 365 ± 5 </td><td> ~ 110 </td><td> ~ 0,9 </td><td> 15 ± 1 </td></tr><tr><td> Adesivo </td><td> 30 </td><td> 60 ± 10 </td><td> NA </td><td> NA </td><td> NA </td><td> NA </td><td> 2 ± 2 </td></tr></tbody></table> Tabella 1. Legend. n, numero del campione, Area, superficie dell&#39;adesivo rosa (media ± errore massimo), Power, potenza del laser (media ± errore massimo), Tempo, tempo di irradiazione (media ± errore massimo), Fluence, fluenza laser media, io, stimato irradiance; Carico massimo / area, carico massimo a rottura del tessuto riparato divisa per la superficie adesiva, (media ± SE).

<ol>
	<li>O'Neill, A. C., Winograd, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anastomosis+using+a+photochemical+tissue+bonding+technique.">Microvascular anastomosis using a photochemical tissue bonding technique.</a> Lasers Surg. Med. 39, 716-722 (2007).</li><li>Yao, M., Yaroslavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxicity+is+not+associated+with+photochemical+tissue+bonding+of+skin.">Phototoxicity is not associated with photochemical tissue bonding of skin.</a> Lasers Surg. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sutureless+nerve+repair+with+laser-activated+chitosan+adhesive:+a+pilot+in+vivo+study.">Sutureless nerve repair with laser-activated chitosan adhesive: a pilot in vivo study.</a> Photomed. Laser Surg. 26, 227-234 (2008).</li><li>Lauto, A., Hook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan+adhesive+for+laser+tissue+repair:+in+vitro+characterization.">Chitosan adhesive for laser tissue repair: in vitro characterization.</a> Lasers Surg. Med. 36, 193-201 (2005).</li><li>Au, V., Madison, S. 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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=Xanthene+dyes/amine+as+photoinitiators+of+radical+polymerization:+A+comparative+and+photochemical+study+in+aqueous+medium.">Xanthene dyes/amine as photoinitiators of radical polymerization: A comparative and photochemical study in aqueous medium.</a> Polymer. 13, 2762-2767 (2009).</li><li>Pini, R., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+closure+of+the+lens+capsule+by+laser+welding.">A new technique for the closure of the lens capsule by laser welding.</a> Ophtalmic Surgery Lasers &#38; Imaging. 39, 260-2626 (2008).</li><li>Wieken, K., Angioi-Duprez, K., Lim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+anastomosis+with+glue:+comparative+histologic+study+of+fibrin+and+cyanoacrylate+glue.">Nerve anastomosis with glue: comparative histologic study of fibrin and cyanoacrylate glue.</a> J. Reconstr. Microsurg. 19, 17-20 (2003).</li><li>Menovsky, T., Beek, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser,+fibrin+glue,+or+suture+repair+of+peripheral+nerves:+a+comparative+functional,+histological,+and+morphometric+study+in+the+rat+sciatic+nerve.">Laser, fibrin glue, or suture repair of peripheral nerves: a comparative functional, histological, and morphometric study in the rat sciatic nerve.</a> J. Neurosurg. 95, 694-699 (2001).</li><li>Garc&#237;a, P., Jorge, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+the+mechanical+behavior+of+a+cyanoacrylate+and+a+bioadhesive.">Comparative study of the mechanical behavior of a cyanoacrylate and a bioadhesive.</a> J. Mater. Sci. Mater. 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La rosa fabbricazione adesivo è basato su un semplice processo a secco-cast, sebbene la dissoluzione di RB a pH acido richiede prolungata agitazione della soluzione di chitosano. È importante lasciare asciugare la soluzione fino a diventare una pellicola insolubile in acqua. Ciò avviene quando il contenuto di acqua peso ~ 10% nel film secco 6. Pellicole insolubili vengono solitamente ottenuti 2 settimane dopo secco colata in condizioni standard di temperatura e pressione (~ 25 ° C e 1 atm ~). Il meccanism...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Rosa Bengala </td><td> Sigma-Aldrich </td><td> 632-69-9 </td><td></td></tr><tr><td> Chitosano (MW medio) </td><td> Sigma-Aldrich </td><td> 10318AJ </td><td></td></tr><tr><td> Acido acetico glaciale </td><td> Sigma-Aldrich </td><td> 08050051 </td><td> 2% v / v in acqua DI </td></tr><tr><td> Agitatore magnetico </td><td> Heidolph </td><td> MR Hei-Mix S </td><td></td></tr><tr><td> Centrifuga </td><td> Beckman Coulter </td><td> Allegra X-12R </td><td></td></tr><tr><td> Spettrofotometro </td><td> Varian </td><td> Cary 4000 UV-Visibile </td><td></td></tr><tr><td> Laser </td><td> CNI laser </td><td> MGL-532 </td><td></td></tr><tr><td> Micrometro </td><td> Mitutoyo </td><td> Serie 227 </td><td></td></tr><tr><td> Microscopio chirurgico </td><td> Zeiss </td><td> OPMI </td><td></td></tr></tbody></table>

fabrication and application of rose bengal-chitosan films in laser tissue repair

Bonding tessuto fotochimica (PTB) è una tecnica di sutura per la riparazione dei tessuti, che si ottiene applicando una soluzione di rosa bengala (RB) tra 1,2 tissue due bordi. Questi sono poi irradiata da un laser che viene selettivamente assorbito dal RB. La reazione risultante fotochimiche presumibilmente reticolare le fibre di collagene nel tessuto con minima produzione di calore 3. In questo rapporto, RB è stato inserito nel film sottili di chitosano per fabbricare un adesivo nuovo tessuto che è laser-attivato. Pellicole adesive, a base di chitosano e contenente RB ~ 0,1% in peso, sono fabbricati e incollate intestino di vitello e nervi tibiale ratto da un laser a stato solido (λ = 532 nm, Fluence ~ 110 J / cm 2, spot size ~ 0,5 cm) . Una colonna singola tensiometro, interfacciato con un personal computer, viene utilizzato per testare la resistenza della giunzione. Le RB-chitosano legami adesivi con fermezza verso l&#39;intestino con una forza di 15 ± 6 kPa, (n = 30). La resistenza di adesione scende a 2 eplusmn; 2 kPa (n = 30), quando il laser non viene applicato l&#39;adesivo. L&#39;anastomosi di nervi tibiali possono essere anche completata senza l&#39;uso di suture. Un romanzo chitosano adesivo è stato fabbricato che legami fotochimicamente al tessuto e non richiede suture.

Watch this Scientific Journal Video about Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair at JoVE.com

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Le suture sono di solito necessari per riparare il tessuto durante gli interventi chirurgici. Tuttavia, la loro applicazione può essere problematico in quanto sono invasivi e possono danneggiare il tessuto. I metodi di fabbricazione e l&#39;applicazione di un adesivo tessuto romanzo sono qui riportati. Questo film adesivo viene laser-attivata e non richiede l&#39;uso di suture.

Fabbricazione e applicazione di Rosa Bengala-chitosano Films in riparazione dei tessuti Laser

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

fabrication-application-rose-bengal-chitosan-films-laser-tissue

Research

JoVE Journal

Bioengineering

12.1K Views.  University of Western Sydney, NSW Australia. Le suture sono di solito necessari per riparare il tessuto durante gli interventi chirurgici. Tuttavia, la loro applicazione può essere problematico in quanto sono invasivi e possono danneggiare il tessuto. I metodi di fabbricazione e l'applicazione di un adesivo tessuto romanzo sono qui riportati. Questo film adesivo viene laser-attivata e non richiede l'uso di suture.

Video: Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

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

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

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

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

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Bioingegneria

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Di campo elettrico controllato migrazione diretta di cellule progenitrici neurali in ambienti 2D e 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

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

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

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

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

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

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 ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Umana Cartilagine Tessuto Fabrication Uso tridimensionale Tecnologia Inkjet Printing

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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 ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tissue tritata in Compressed Collagene: A Biotransplant cellulare contenente per la riparazione singolo-messa in scena Ricostruttiva

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Immagine-guidate, Fabrication laser a base di reti microfluidici vascolare-derivati

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabbricazione e caratterizzazione dei fantasmi di tessuto ottico contenente macrostruttura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Doppia scansione Raster Photoacoustic Small-Animal Imager per la visualizzazione vascolare

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...