Ce travail a été soutenu par la subvention de recherche UWS.

1. Chitosan adhésif Préparation <ol><li> La poudre de chitosane est soluble dans une solution d&#39;acide acétique, pour la préparation d&#39;une solution mère d&#39;acide acétique (2% v / v), ajouter 10 ml d&#39;acide acétique glacial à 490 ml d&#39;eau désionisée (DI-H 2 O). </li><li> Pour la préparation d&#39;une solution mère de Rose Bengale (RB) (0,01% p / v) dans de l&#39;acide acétique, pèse 5 mg de RB dans un flacon enveloppé d&#39;une feuille d&#39;aluminium pour éviter photoblanchiment. Ajouter 0,5 ml de DI-H 2 O pour dissoudre la poudre puis ajouter 49,5 ml de la solution mère d&#39;acide acétique (décrit dans la section 1.1). </li><li> Pour la préparation d&#39;une solution de chitosan (1,7% v / v), 0,85 g de poids de poudre de chitosane (MW moyen, le degré d&#39;acétylation de 85%) et ajoute à 50 ml d&#39;une solution d&#39;acide acétique RB. Le contrôle a été préparé par dissolution du chitosane dans une solution d&#39;acide acétique sans RB. </li><li> Ajouter un agitateur magnétique au mélange chitosane et agiter à la température ambiante. Pour assurer la dissolution complète des composants, mélanger le chitosanedans l&#39;acide acétique + RB pour deux semaines et le chitosan dans l&#39;acide acétique pendant 5 jours (témoin). Quand le chitosane est dissous, le pH de la solution augmente de ~ 2,6 à ~ 3,9. Noter que RB est peu soluble à pH acide et il nécessite une augmentation du pH de la solution et une période prolongée sous agitation pour dissoudre. </li><li> Centrifuger les solutions à 3270 xg pendant 1 heure et recueillir le surnageant en utilisant, par exemple, une seringue stérile de 10 ml. Assurez-vous de ne pas perturber le culot impureté. Au cours de cette étape, la solution est nettoyé des impuretés macroscopiques et de la matière insoluble. </li><li> Injecter la solution (centrifugation) chitosane sur une dalle plate Perpex coulé à l&#39;aide d&#39;une seringue stérile. L&#39;épaisseur du film sec est fonction du rapport entre la quantité distribuée de la solution et la surface sur laquelle la solution est étalée. Faire en sorte que la solution est exempte de bulles d&#39;air et également répartis sur la dalle. Pour obtenir des films minces d&#39;une épaisseur ~ um 13, injecter la solution ~ 1 ml de plus de 12 cm 2 de surfacezone (dimensions 3x4 cm ~, figure 1A). </li><li> Couvrir la plaque en plexiglas avec un écran, en feuille d&#39;aluminium, par exemple, pour éviter photoblanchiment de la solution. Faire en sorte que suffisamment d&#39;air de ventilation est assurée à sécher la solution sous l&#39;écran. </li><li> Laisser la solution sécher à la température ambiante (25 ° C) et de pression (1 atm) jusqu&#39;à ce que le film résultant est insoluble dans l&#39;eau et ne gonfle pas macroscopiquement. Ceci est généralement réalisé en 2 semaines. </li><li> Détachez les films avec soin à l&#39;aide d&#39;une spatule mince et plat: ces films peuvent être facilement décollée de la plaque en plexiglas sans les endommager. </li><li> Mesurer l&#39;épaisseur des films à l&#39;aide d&#39;un micromètre. </li><li> Coupez l&#39;adhésif en bandes rectangulaires avec un ciseau tranchant propre et placez-les entre des lames de verre stériles pour préserver leur forme plate. Enveloppez les lames dans du papier aluminium pour protection contre la lumière et les conserver à la température ambiante. Étiqueter les adhésifs avec ou sans RB comme &quot;rose adhésif&quot; ou &quot;ADHES chitosaneive &quot;respectivement. </li><li> La rose des films adhésifs peuvent être stérilisés à l&#39;aide d&#39;éthanol (90%) ou avec un rayonnement gamma (0,1 kGy / h pendant 24 h) 4,5. </li></ol> 2. Adhésif atténuation optique <ol><li> Fixer la colle rose à l&#39;intérieur d&#39;une cuve en quartz. </li><li> Enregistrer le spectre atténué dans la plage de 400 à 800 nm en utilisant un spectrophotomètre à double faisceau 4. </li><li> En supposant que la validité de la loi de Beer, calculer la longueur d&#39;atténuation de l&#39;adhésif comme suit: I = I 0 e-Ax où I 0 est l&#39;intensité du faisceau incident, 1 / A est la longueur d&#39;atténuation, et x est l&#39;épaisseur du film. </li><li> Répétez les étapes 2,1 à 2,3 pour l&#39;adhésif de chitosane pour servir de contrôle. </li></ol> 3 Dans d&#39;application in vitro:. Collage de tissus au laser sur Intestin <ol><li> Intestin de veau récolte immédiatement après l&#39;euthanasie des animaux et conserver à -80 ° C. </li><li> Avant utilisation, décongeler et hydrater la tiDITION en l&#39;immergeant dans DI-H 2 O pendant 10 min. </li><li> Bissecter le tissu en sections (2 x 1 cm) à l&#39;aide d&#39;une lame n ° 10 sous microscope opératoire (x10). Maintenir l&#39;humidité sections en utilisant DI-H 2 O. </li><li> Apportez les souches incision rapprochés (bout à bout). Essuyez l&#39;excès d&#39;eau sur la surface à l&#39;aide des conseils du coton. </li><li> Placer une bande adhésive rose (10 x 6 mm) à travers l&#39;incision sur la couche séreuse avec microforceps assurant le contact complet avec l&#39;intestin (Figure 2). </li><li> L&#39;adhérence de l&#39;adhésif tissu rose est activé par un pompage par diode laser à l&#39;état solide, qui est couplé à une fibre optique multimode (diamètre du noyau 200 um) 4. </li><li> Réglez le niveau de puissance du laser à 180 mW et d&#39;irradier l&#39;adhésif pour ~ 6 min avec une taille de spot lumineux de ~ 5 mm, en utilisant les paramètres indiqués dans le tableau 1. Place irradier l&#39;adhésif en sorte que chaque spot est irradié pendant environ 5 secondes avant de déplacer le faisceau à l&#39;endroit adjacent. Tson procédé garantit que le faisceau laser balaie toute la surface de l&#39;adhésif plusieurs fois. </li><li> Pour évaluer la force de collage de tissus, serrer un échantillon à un tensiomètre seule colonne calibrée (Instron, MA, USA) en utilisant poignées mécaniques. Déplacez les poignées à 22 mm / min jusqu&#39;à ce que les deux souches de tissus séparés 4. </li><li> Placez un adhésif rose sur le tissu comme décrit à l&#39;étape 3.5. Ne pas irradier l&#39;échantillon et à mesurer la résistance de liaison du tissu comme décrit dans l&#39;étape 3,8. Cet échantillon sert de témoin. </li></ol> 4 Dans demande Vivo:. Anastomose nerf tibial <ol><li> Calmer le rat Wistar avec de l&#39;isoflurane / O 2 mélange (4% pendant l&#39;induction, 2% par la suite) 5. Voie sous-cutanée injecter Buprenex (0,03 mg / kg) pour fournir intra-opératoire activité analgésique et soulager la douleur ou d&#39;inconfort possible. </li><li> La procédure suivante chirurgicale doit être réalisée sous microscope opératoire (x10). Faire une peau obliqueincision d&#39;environ 3 cm de long dans la partie dorso-latérale de la cuisse droite et exposer le nerf tibial avec une approche de division des muscles dans les muscles fessiers 2. </li><li> Partiellement décortiquer et couper l&#39;adventice du nerf tibial à l&#39;aide de micro-ciseaux droits et absorber l&#39;excès d&#39;eau avec une gaze stérile ou des conseils en coton. </li><li> Couper le nerf tibial avec des micro-ciseaux. </li><li> Placez une solution stérile de chitosan bande (dimensions 5 x 4 mm) en dessous du nerf tibial à l&#39;aide de micro-pince. </li><li> Rapprochant les souches nerveuses de bout en bout avec les micro-pince sur la bande adhésive rose. </li><li> La bande adhère pleinement à la formation d&#39;une collerette nerveuse, qui peut aider à la rotation du nerf lors de l&#39;irradiation laser de la bande. </li><li> Activer la rose adhésif avec le laser comme décrit dans les étapes 3.6) et 3.7). </li><li> Coupez l&#39;adhésif double emploi a augmenté depuis le collier le nerf opéré. </li><li> Fermez les muscles et la peau avec cinq points de suture 3-0 5. </li&gt; <li> Les procédures standards de récupération post-opératoires doivent être observées, y compris l&#39;inspection quotidienne de déhiscence ou la formation de pus et de l&#39;administration d&#39;analgésiques, si nécessaire. </li></ol> 5. Les résultats représentatifs Les films obtenus sont rose vif en couleur, mince et avoir une surface lisse (Figure 1). Ils sont également plus souples et peuvent être roulées dans des tubes de petit diamètre sans larmes qui causent ou tout autre dommage apparent (figure 1B). L&#39;adhésif rose a deux pics d&#39;absorption à 530 et 562 nm; le laser vert est donc fortement absorbée par l&#39;adhésif et la longueur d&#39;atténuation correspondant à 532 nm est de ~ 12 um (figure 3). En revanche, le chitosan films sans RB atténue faiblement le laser (1 / A 162 ~ um), probablement en raison de la diffusion. Il ressort de l&#39;intrigue spectres qu&#39;aucune agrégation significative de RB se produit dans les films. La rose des liaisons adhésives fermementà l&#39;intestin lors d&#39;une irradiation par laser (figure 2) la réalisation d&#39;une caractéristique de charge maximale à la rupture de 0,9 ± 0,4 N (n = 30). La force d&#39;adhérence a été estimé que la charge maximale divisée par la surface adhésive, à savoir ± 15 6 kPa (n = 30). L&#39;adhésif non irradié rose collé beaucoup moins de tissu (2 ± 2 kPa, n = 30, test t non apparié p &lt;10 6). L&#39;anastomose in vivo du nerf tibial a été réalisée avec succès après l&#39;application et le collage de l&#39;adhésif rose en liaison avec le laser vert. Une semaine après l&#39;opération, le nerf opéré était dans la continuité et la solidité de réparation était de 17 ± 9 kPa (n = 10). <img alt="Figure 1" src="/files/ftp_upload/4158/4158fig1.jpg" /> Figure 1. A) La solution RB-chitosane est coulé sur une dalle plate en plexiglas (épaisseur de la dalle ~ 5 mm). Papier millimétré est placé sous la dalle de faciliter l&#39;étape sèche-cast. Le f insoluble dans l&#39;eauilm est obtenue de deux semaines après la coulée. B) L&#39;adhésif rose est flexible et peut être roulé dans un cylindre de petite taille. <img alt="Figure 2" src="/files/ftp_upload/4158/4158fig2.jpg" /> Figure 2. L&#39;adhésif est collé à la rose intestin de veau après l&#39;irradiation laser. Irradiation uniforme est appliquée sur l&#39;adhésif pendant RB liaison tissulaire; toutefois taches sélectionnés sont présentés dans cette image pour illustrer l&#39;effet photo-blanchiment du laser sur l&#39;adhésif. <img alt="Figure 3" src="/files/ftp_upload/4158/4158fig3.jpg" /> Figure 3. Le spectre d&#39;absorption (en unités arbitraires) de l&#39;adhésif rose montre deux pics à 530 et 562 nm. Le laser vert (λ = 532 nm) est donc fortement absorbée par l&#39;adhésif lors de l&#39;exposition au laser. L&#39;adhésif sans chitosane RB absorbe mal du faisceau laser. <table border="1"><tbody><tr><td></td><td> n </td><td><strong&gt; Domaine (mm 2) </td><td> Puissance (W) </td><td> Temps (s) </td><td> Fluence (J / cm 2) </td><td> I (W / cm 2) </td><td> Charge Max / zone (kPa) </td></tr><tr><td> Adhésif + 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> Adhésif </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> Tableau 1. Légende. n, le nombre d&#39;échantillons; Area, surface de l&#39;adhésif rose (moyenne ± erreur maximum); Puissance, la puissance du laser (moyenne ± erreur maximum); Time, temps d&#39;irradiation (moyenne ± erreur maximum); Fluence, la fluence laser moyenne, moi, estimée irradiance; maxi / zone, la charge maximale à la rupture du tissu réparé divisée par la surface adhésive (moyenne ± 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+singlet+oxygen+on+the+extracellular+matrix+protein+collagen:+oxidation+of+the+collagen+crosslink+histidinohydroxylysinonorleucine+and+histidine.">Effects of singlet oxygen on the extracellular matrix protein collagen: oxidation of the collagen crosslink histidinohydroxylysinonorleucine and histidine.</a> Arch. Biochem. Biophys. 384, 133-142 (2000).</li><li>Encinas, M. V., Rufs, A. 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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Aucun conflit d&#39;intérêt déclaré.

Le rose adhésif fabrication est basé sur un simple processus de fonte à sec, mais la dissolution de RB à pH acide sous agitation prolongée nécessite de la solution de chitosane. Il est important de laisser la solution sécher jusqu&#39;à ce qu&#39;il devienne un film insoluble dans l&#39;eau. Cela se produit lorsque la teneur en eau du poids est environ 10% dans le film sec 6. Des films insolubles sont généralement obtenus 2 semaines après la coulée à sec dans des conditions standard de températu...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Rose Bengale </td><td> Sigma-Aldrich </td><td> 632-69-9 </td><td></td></tr><tr><td> Chitosan (moyenne MW) </td><td> Sigma-Aldrich </td><td> 10318AJ </td><td></td></tr><tr><td> L&#39;acide acétique glacial </td><td> Sigma-Aldrich </td><td> 08050051 </td><td> 2% v / v dans l&#39;eau DI </td></tr><tr><td> Agitateur magnétique </td><td> Heidolph </td><td> MR Hei-Mix S </td><td></td></tr><tr><td> Centrifuger </td><td> Beckman Coulter </td><td> Allegra X-12R </td><td></td></tr><tr><td> Spectrophotomètre </td><td> Varian </td><td> Cary 4000 UV-Visible </td><td></td></tr><tr><td> Laser </td><td> Laser CNI </td><td> MGL-532 </td><td></td></tr><tr><td> Micromètre </td><td> Mitutoyo </td><td> Série 227 </td><td></td></tr><tr><td> Microscope d&#39;opération </td><td> Zeiss </td><td> OPMI </td><td></td></tr></tbody></table>

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

Soudure tissulaire photochimique (PTB) est une technique de suture pour la réparation des tissus, ce qui est obtenu en appliquant une solution au rose bengale (RB) entre deux bords de tissu 1,2. Ceux-ci sont ensuite irradiés par un laser qui est sélectivement absorbée par le RB. Les réactions photochimiques résultant prétendument réticuler les fibres de collagène dans le tissu avec 3 la production de chaleur minimale. Dans le présent rapport, RB a été incorporé dans des films minces de chitosane pour fabriquer une colle tissulaire roman qui est activée par laser. Des films adhésifs, sur la base de chitosane et contenant ~ 0,1% en poids, RB sont fabriqués et liée à d&#39;intestin de veau et nerfs tibiaux rat par un laser à l&#39;état solide (λ = 532 nm, la fluence ~ 110 J / cm 2, la taille de tache ~ 0,5 cm) . Un tensiomètre à colonne unique, en interface avec un ordinateur personnel, est utilisé pour tester la résistance de liaison. Les obligations RB-chitosane adhésives fermement à l&#39;intestin avec une force de 15 ± 6 kPa, (n = 30). La force d&#39;adhérence diminue à 2 etplusmn; 2 kPa (n = 30) lorsque le laser n&#39;est pas appliquée à l&#39;adhésif. L&#39;anastomose des nerfs tibial peut être également réalisé sans l&#39;utilisation de fils de suture. Un nouveau chitosane adhésif qui a été fabriqué à des liaisons photochimique tissu et ne nécessite pas de sutures.

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

Les sutures sont généralement nécessaires pour réparer les tissus pendant les interventions chirurgicales. Cependant, leur application peut être problématique car elles sont envahissantes et peuvent endommager les tissus. Les procédés de fabrication et l&#39;application d&#39;un adhésif tissulaire roman sont ici rapportés. Ce film adhésif est activé par laser et ne nécessite pas l&#39;utilisation de fils de suture.

Fabrication et application de Rose Bengale-chitosane Films à la réparation des tissus au 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 vues.  University of Western Sydney, NSW Australia. Les sutures sont généralement nécessaires pour réparer les tissus pendant les interventions chirurgicales. Cependant, leur application peut être problématique car elles sont envahissantes et peuvent endommager les tissus. Les procédés de fabrication et l'application d'un adhésif tissulaire roman sont ici rapportés. Ce film adhésif est activé par laser et ne nécessite pas l'utilisation de fils de suture.

Vidéo: 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.

La fabrication des matériaux injectables d&#39;origine biologique pour l&#39;ingénierie tissulaire du myocarde

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

Recherche

Bio-ingénierie

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).

Champ électrique contrôlé par les migrations des Réalisé cellules progénitrices neurales dans des environnements 2D et 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.

Les tissus d&#39;ingénierie muscle squelettique de myoblastes murins cellules progénitrices et application d&#39;une stimulation électrique

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.

Les échafaudages auto-déclaration pour les 3-Dimensional Culture cellulaire

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.

Fabrication de tissus de cartilage humain en utilisant trois dimensions jet d&#39;encre Technologie d&#39;impression

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.

Adapter le processus Électrofilage de fournir trois environnements uniques pour un Tri-couches<em&gt; In Vitro</em&gt; Modèle du mur Airway

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.

Tissu haché en comprimé Collagen: Un Biotransplant contenant des cellules de réparation Reconstructive simple mise en scène

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.

, Fabrication de réseaux microfluidiques vasculaire dérivés à base de laser guidée par l&#39;image

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.

Fabrication et caractérisation des fantômes de tissu optique contenant la Macrostructure

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.

Double imageur photoacoustique à balayage Raster pour visualisation vasculaire

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