Les auteurs tiennent à remercier Gabriel Arom et Michael Yeranosian pour leurs contributions techniques aux itérations précédentes de ce projet. Ce projet a été financé par le clinicien-chercheur Grant formation OREF (NL), HH Lee Surgical Research Grant (NL), Veterans Administration BLR &amp; D examen du mérite une I01 BX00012601 (DM) et l&#39;appareil locomoteur transplantation Fondation Prix du jeune chercheur (FP).

NOTE: Toutes les chirurgies animales ont été approuvés par le personnel vétérinaire et animale comité de l&#39;utilisation locale avant de commencer les expériences. 1. Préparation de électrofilées polycaprolactone échafaudages <ol><li> Peser et dissoudre l&#39;ester de qualité médicale à terminaison poly (ε-caprolactone) sous forme de granulés dans le 1,1,1,3,3,3-hexafluoro-2-propanol pour créer un 10% p / p de la solution polymère PCL. Laissez le bruit de solution en utilisant une plaque d&#39;agitation pendant au moins 3 heures pour assurer une solution homogène. </li><li> Electrospin la solution PCL pour créer un brassard de fibres PCL très alignés pour échafaudage fabrication. <ol><li> Préparer la configuration d&#39;électrofilage dans une hotte chimique avec le ventilateur en tout temps. Il se agit d&#39;une grande boîte acrylique qui servira de support à vide isolé pour le procédé d&#39;électrofilage et présente des points d&#39;entrée pour la solution source de PCL entraîné par une source de tension, le mandrin collecte de motorisation, et un orifice de vide. Nettoyez le Acryliboîte de c soigneusement avec de l&#39;éthanol et de couvrir toutes les surfaces avec des feuilles de Parafilm pour éliminer toutes les impuretés qui peuvent compromettre la qualité du produit électrofilé. </li><li> Charge environ 3 ml de la solution ci-dessus dans une seringue de 10 ml avec une extrémité franche 18 G, une aiguille de ½ pouce. Retirer les bulles d&#39;air en poussant la seringue jusqu&#39;à. Solution de verrouillage dans une pompe à seringue programmable. Insérer l&#39;aiguille à travers un petit trou dans la surface acrylique tout en laissant environ ½ pouce de l&#39;aiguille à l&#39;extérieur de la boîte acrylique pour la fixation du fil de source de tension. </li><li> Utilisez un 30 mm tournant tour mandrin le collecteur pour les fibres PCL très alignés; couvrir le mandrin hermétiquement avec une mince bande de papier d&#39;aluminium. Verrouiller le mandrin dans le moteur sur le côté opposé de la boîte d&#39;environ 15 cm de l&#39;aiguille de la seringue. </li><li> Insérer le tuyau en plastique dans l&#39;orifice de vide et se connecter à la source de vide de la hotte. Allumez la source de vide et couvrir la boîte en acrylique avec un couvercle. </li><li> Set le débit de la pompe de seringue programmable à 2,5 ml / h perfusion. Allumez le moteur pour faire fonctionner le mandrin à 3450 rpm et fixez le fil positif de la source de tension à la pointe de l&#39;aiguille à l&#39;extérieur de la boîte en utilisant une pince crocodile. </li><li> Une fois la perfusion de la solution PCL a commencé, tourner sur la source de tension et réglé à une tension de fonctionnement de 20 kV. </li><li> Infuser la solution pendant 12 min à créer une manchette homogène à partir de 0,5 ml de la solution PCL. NOTE: En moyenne, chaque revers a assez de matériel électrofilé pour créer deux feuilles de six bandes, qui peuvent être utilisés pour créer un total de trois à quatre couches échafaudages. </li></ol></li><li> Laser couper le brassard PCL pour former de multiples petites feuilles sur un VersaLaser Cutter 2.3 tourne à faible vide, 10,0 keV tension d&#39;atterrissage, 6,4 mm distance de travail, et le diamètre de la sonde de 3,0. NOTE: Dans cet exemple, une conception assistée par ordinateur a été utilisé pour charger le coupe pour obtenir plusieurs feuilles de 1,5 mm x 35 mm x 150 um échafaudages avecrépartie uniformément 150 um de diamètre aux trous 15% de la surface des pores. </li><li> Gravure plasma échafaudages PCL utilisant un nettoyeur à plasma pour induire une hydrophilie de la surface PCL avec une accélération d&#39;ions. Réglez le vide à 450 mTorr et le traitement des échafaudages pendant 30 secondes à puissance élevée (29,6 W). </li><li> Baigner les échafaudages à 70% d&#39;éthanol dans un environnement stérile. </li><li> Enduire les échafaudages individuels avec du collagène pour faciliter l&#39;adhérence et la prolifération cellulaire in vivo. <ol><li> Créer une solution de revêtement de collagène par dilution d&#39;une 8: solution stérile de 2,5 Purecol collagène de 3 mg / ml de solution standard, 10x PBS et 0,1 N de NaOH 1: 9 à 1 1 x PBS à 4 ° C. Mélanger soigneusement pour assurer l&#39;homogénéité solution. </li><li> Manteau individuelle 1,5 mm x 35 mm x 150 um échafaudages avec un film en couche mince de la solution de collagène ci-dessus. Laisser sécher pendant 24 heures dans un environnement stérile. </li></ol></li><li> Utilisation 5-0 sutures Vicryl, empiler et apposer quatre individuelle 1,5 mm x 35 mm x 150 um échafaudagesutilisant un Krackow le point de créer un 0,6 mm d&#39;épaisseur finales multicouche, échafaudage, revêtue de collagène qui est prêt pour l&#39;implantation. </li></ol> 2. Protocole de Chirurgie Rat <ol><li> Induire une anesthésie en plaçant rat dans la chambre d&#39;inhalation et offrant 2% d&#39;isoflurane avec 2 L / min d&#39;oxygène. Confirmez le rat est anesthésié correctement en appliquant une pression à Hind pied et d&#39;évaluation pour toute réponse. </li><li> Sur la table de retour non stérile, injecter sous-cutanée de 25 mg / kg ampicilline et 0,03 mg / kg de buprénorphine. </li><li> Appliquer une pommade ophtalmique pour les yeux. Clip de la fourrure de la patte postérieure opératoire et prép le site chirurgical avec trois gommages alternées de chlorhexidine et 70% d&#39;éthanol. </li><li> Transférer le rat à la table d&#39;opération, sur un bloc chauffé pour éviter l&#39;hypothermie. Nez sécurisé cône, et de maintenir l&#39;anesthésie par la procédure avec 2% d&#39;isoflurane dans 2 L / min d&#39;oxygène, livré cône de nez. Drapé de manière stérile, en laissant la branche opératoire exposé. </li><li&gt; Avant de commencer l&#39;opération, d&#39;abord confirmer anesthésie adéquate par le manque d&#39;une réponse à la pression du pied de derrière. </li><li> Faire une incision verticale médiane 2 cm de long pour le genou, centrée au niveau de la rotule. Rentrer la peau latéralement jusqu&#39;à l&#39;incision est centrée sur le genou. </li><li> Utiliser un scalpel pour faire une arthrotomie parapatellaire interne en coupant simplement en dedans de la rotule et se étendant de manière proximale au niveau de la jonction musculo du quadriceps et de manière distale au niveau de l&#39;insertion du tendon rotulien sur le tubercule tibial. Prenez soin de ne pas couper les tendons rotuliens ou quadriceps. </li><li> rotule de sortie latéralement en faisant une incision verticale de 1 cm à travers la capsule du genou juste en dehors du tendon rotulien. </li><li> Se assurer que le genou est en extension. Prenez une paire de ciseaux fins et passer sous la rotule de dehors en dedans. Étaler les ciseaux à quelques reprises afin que le mécanisme extenseur peut être traduit de chaque côté. </li><li> Bien que la flexion til genou, la rotule traduire latéralement pour exposer l&#39;intérieur de l&#39;articulation du genou. Assurer une visualisation claire de l&#39;échancrure intercondylienne et condyle fémoral. Avec un scalpel, sectionner l&#39;ACL et PCL dans l&#39;encoche. </li><li> Charge perceuse avec un k-fil 1,6 mm. Placez la pointe k-fil sur l&#39;origine ACL dans l&#39;échancrure intercondylienne. Percer superolaterally et de visualiser le point de sortie sur l&#39;aspect externe du fémur, en supprimant toute tissus mous que nécessaire avec un scalpel. Passez le k-fil et à quelques reprises pour assurer le passage clair pour la greffe. </li><li> Placez le k-fil sur l&#39;empreinte ACL sur le plateau tibial. Percer antéro-externe et visualiser point de sortie sur le tibia proximal antérolatérale. Utilisez scalpel pour effacer tissus mous que nécessaire pour que le point où le k-fil sort du tibia est totalement visible. </li><li> Passez une aiguille Keith raccourcie (idéalement pas plus de deux pouces de long) à travers le tunnel de fémur. Faire passer les deux extrémités de suture de l&#39;une des extrémités de la greffe dans le chas de la Kaiguille de eith. Utilisation de l&#39;aiguille pour tirer une extrémité du greffon dans le tunnel fémoral. </li><li> Répéter l&#39;étape précédente pour passer l&#39;autre extrémité du greffon dans le tunnel tibial. </li><li> Utilisez 4-0 Vicryl suture pour fixer la fin fémorale de la greffe de périoste entourant ou autre tissu mou avec un point figure-de-huit. Manuellement la tension de la greffe avec le genou en extension. Apposer la fin tibial de la greffe de périoste entourant ou autre tissu mou avec un point figure-de-huit. </li><li> Utilisez une paire de ciseaux pour couper l&#39;excès de greffe sur les deux extrémités, laissant 1-2 mm à chaque extrémité passé le point figure-de-huit. </li><li> Étendre le genou et réduire la rotule. Utilisation 4-0 vicryl, placer un seul point figure-de-huit pour fermer la capsule articulaire médial, empêchant subluxation latérale de la rotule. </li><li> Fermez la peau avec un subcuticulaire 5-0 Monocryl ou Vicryl surjet, avec soin de ne pas suturer le muscle sous-jacent ou avoir une suture visible une fois la peau est fermée. </li><li> Injerats ct voie sous-cutanée avec la buprénorphine tous les 12 h pour un total de trois jours après l&#39;opération. Vérifiez site chirurgical pour tout drainage ou déhiscence de la plaie au moment de l&#39;injection. Boiteux et peu d&#39;enflure sont normaux dans les premiers jours après la chirurgie, mais rapidement répondre aux préoccupations postopératoires en collaboration avec le personnel vétérinaire. L&#39;animal peut être revenir à un logement social à deux semaines après l&#39;opération, lorsque les incisions chirurgicales sont complètement guéries. </li></ol> 3. Protocol Data Collection <ol><li> Au moment du sacrifice, asphyxier rats individuellement dans une enceinte fermée de CO 2 suivie d&#39;une thoracotomie. </li><li> Récolter deux membres chirurgicalement reconstruits et controlatérale en séparant à l&#39;articulation de la hanche. <ol><li> Pour les membres reconstruits, enlever tous les tissus mous, y compris le ligament croisé postérieur et les restes de la LCA natif chirurgicalement perturbé, par dissection fine pour isoler seulement le fémur, le tibia et la greffe. </li> &lt;li&gt; Pour les membres controlatéraux, enlever tous les tissus mous à l&#39;exception du LCA natif ainsi que le fémur et le tibia par dissection fine. </li><li> Utilisation d&#39;un outil rotatif tel que Dremel, pour supprimer tout sauf ¾ de 1 cm de l&#39;os à partir de chaque extrémité de l&#39;ensemble fémur-tibia par greffage. </li><li> Au cours de ce processus et tout au long tests biomécaniques, régulièrement et fréquemment pulvériser les régions ligamentaires avec du sérum physiologique pour éviter la dessiccation du genou récoltée qui peuvent modifier les résultats faussement. </li></ol></li><li> Pour l&#39;analyse histologique, fixer chaque genou individuellement dans une solution de paraformaldehyde à 4% à 25 ° C pendant 48 heures. Ensuite, plonger le genou dans une solution de réactif pour Immunocal décalcification complète; ce processus dépend du contenu calcifiés de l&#39;échantillon et peut prendre jusqu&#39;à cinq jours. Vérifiez échantillons individuels par jour pour évaluer les progrès que décalcification incomplète peut réduire la qualité de l&#39;échantillon. Une fois terminé, effectuez la coupe, montage à glissière, et la coloration comme souhaité. </li><li> Perform tests biomécaniques pour évaluer la capacité fonctionnelle du ligament de l&#39;ingénierie tissulaire. <ol><li> Fixez le fémur et le tibia en enveloppant 28 G fil d&#39;acier galvanisé autour de l&#39;épiphyse de chaque os séparément. Ce est pour éviter que les données de tests biomécaniques inexactes de rupture en traction prématurée de l&#39;échantillon à l&#39;os plutôt qu&#39;à le ligament d&#39;intérêt. </li><li> Pot fémur dans un mélange de ciment polyméthylméthacrylate (PMMA) de l&#39;os. Pour ce faire, mélanger les deux composants du ciment et d&#39;utiliser immédiatement le mélange visqueux pour sécuriser le fémur dans le métal, recouvrant complètement la diaphyse de l&#39;os dans le pot cimentée avec l&#39;épiphyse et le ligament fixé librement saillante. Laisser polymérisation radicalaire spontanée de transformer progressivement les composants mixtes visqueux à un matériau pâteux et finalement dans une matrice durcie solide. NOTE: Ce processus prend plusieurs minutes et peut être contrôlée en étudiant manuellement la température d&#39;un bolus fabriqué à partir dele ciment restant; la température doit augmenter de manière transitoire au cours de la réaction de polymérisation exothermique et se abaisser à la température ambiante après la solidification de matériaux. </li><li> Répétez le même processus ci-dessus pour cimenter le tibia, sauf tout en conservant le ligament du genou à 20 ° de flexion pour les essais mécaniques idéale. </li><li> Monter le complexe fémur, tibia cémenté par greffage sur un appareil d&#39;essai de traction, et pour préparer la charge d&#39;enregistrement et de déplacement en fonction du temps à partir du début de la tension à l&#39;échec. Dans cet exemple, nous avons utilisé un modèle Instron 5564 avec une cellule de charge de 1 kN. </li><li> Pré-tension de la greffe à 2 N à un débit de 0,5 N / min rampe et ensuite tester la greffe à l&#39;échec à une vitesse de 0,5 mm / s souche. Pendant le processus, assurez-vous que le ligament ne parvient pas à la mi-substance et que le fémur et le tibia sont osseuse sécurisé et pas prématurément à défaut, qui peut évaluer à tort propriétés biomécaniques du ligament testé. </li><li> Utilisez la charge déplacement générécourbes pour calculer la charge de rupture et la raideur du ligament testé. </li></ol></li></ol>

<ol>
	<li>Fetto, J. F., Marshall, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+natural+history+and+diagnosis+of+anterior+cruciate+ligament+insufficiency.">The natural history and diagnosis of anterior cruciate ligament insufficiency.</a> Clin Orthop Relat Res. (147), 29-38 (1980).</li><li>Kim, Y. M., Lee, C. A., Matava, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+results+of+arthroscopic+single-bundle+transtibial+posterior+cruciate+ligament+reconstruction:+a+systematic+review.">Clinical results of arthroscopic single-bundle transtibial posterior cruciate ligament reconstruction: a systematic review.</a> Am J Sports Med. 39 (2), 425-434 (2011).</li><li>Andersson, C., Odensten, M., Gillquist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knee+function+after+surgical+or+nonsurgical+treatment+of+acute+rupture+of+the+anterior+cruciate+ligament:+a+randomized+study+with+a+long-term+follow-up+period.">Knee function after surgical or nonsurgical treatment of acute rupture of the anterior cruciate ligament: a randomized study with a long-term follow-up period.</a> Clin Orthop Relat Res. (264), 255-263 (1991).</li><li>Klimkiewicz, J. J., Petrie, R. S., Harner, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+treatment+of+combined+injury+to+anterior+cruciate+ligament,+posterior+cruciate+ligament,+and+medial+structures.">Surgical treatment of combined injury to anterior cruciate ligament, posterior cruciate ligament, and medial structures.</a> Clin Sports Med. 19 (3), 479-492 (2000).</li><li>Petrigliano, F. A., McAllister, D. R., Wu, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+for+anterior+cruciate+ligament+reconstruction:+a+review+of+current+strategies.">Tissue engineering for anterior cruciate ligament reconstruction: a review of current strategies.</a> Arthroscopy. 22 (4), 441-451 (2006).</li><li>Groot, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+porous+polyurethanes+for+meniscal+reconstruction+and+meniscal+prostheses.">Use of porous polyurethanes for meniscal reconstruction and meniscal prostheses.</a> Biomaterials. 17 (2), 163-173 (1996).</li><li>Leong, N. L., Petrigliano, F. A., McAllister, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+tissue+engineering+strategies+in+anterior+cruciate+ligament+reconstruction.">Current tissue engineering strategies in anterior cruciate ligament reconstruction.</a> J Biomed Mater Res A. 102 (5), 1614-1624 (2014).</li><li>Duling, R. R., Dupaix, R. B., Katsube, N., Lannutti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+electrospun+polycaprolactone+(PCL):+a+potential+scaffold+for+tissue+engineering.">Mechanical characterization of electrospun polycaprolactone (PCL): a potential scaffold for tissue engineering.</a> J Biomech Eng. 130 (1), 011006 (2008).</li><li>Shao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycaprolactone+electrospun+mesh+conjugated+with+an+MSC+affinity+peptide+for+MSC+homing+in+vivo.">Polycaprolactone electrospun mesh conjugated with an MSC affinity peptide for MSC homing in vivo.</a> Biomaterials. 33 (12), 3375-3387 (2012).</li><li>Tillman, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+stability+of+electrospun+polycaprolactone-collagen+scaffolds+in+vascular+reconstruction.">The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction.</a> Biomaterials. 30 (4), 583-588 (2009).</li><li>Wise, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multilayered+synthetic+human+elastin/polycaprolactone+hybrid+vascular+graft+with+tailored+mechanical+properties.">A multilayered synthetic human elastin/polycaprolactone hybrid vascular graft with tailored mechanical properties.</a> Acta Biomater. 7 (1), 295-303 (2011).</li><li>Vargel, I., Korkusuz, P., Mencelo&#287;lu, Y. Z., Pi&#351;kin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+performance+of+antibiotic+embedded+electrospun+PCL+membranes+for+prevention+of+abdominal+adhesions.">In vivo performance of antibiotic embedded electrospun PCL membranes for prevention of abdominal adhesions.</a> J Biomed Mater Res B Appl Biomater. 81 (2), 530-543 (2007).</li><li>Cao, H., McHugh, K., Chew, S. Y., Anderson, 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=The+topographical+effect+of+electrospun+nanofibrous+scaffolds+on+the+in+vivo+and+in+vitro+foreign+body+reaction.">The topographical effect of electrospun nanofibrous scaffolds on the in vivo and in vitro foreign body reaction.</a> J Biomed Mater Res A. 93 (3), 1151-1159 (2010).</li><li>Joshi, V. S., Lei, N. Y., Walthers, C. M., Wu, B., Dunn, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroporosity+enhances+vascularization+of+electrospun+scaffolds.">Macroporosity enhances vascularization of electrospun scaffolds.</a> J Surg Res. 183 (1), 18-26 (2013).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+poly(epsilon-caprolactone)+microfiber+and+multilayer+nanofiber/microfiber+scaffolds:+characterization+of+scaffolds+and+measurement+of+cellular+infiltration.">Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.</a> Biomacromolecules. 7 (10), 2796-2805 (2006).</li><li>Vaz, C. M., van Tuijl, S., Bouten, C. V., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+scaffolds+for+blood+vessel+tissue+engineering+using+a+multi-layering+electrospinning+technique.">Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique.</a> Acta Biomater. 1 (5), 575-582 (2005).</li><li>Kawamura, S., Ying, L., Kim, H. J., Dynybil, C., Rodeo, 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=Macrophages+accumulate+in+the+early+phase+of+tendon-bone+healing.">Macrophages accumulate in the early phase of tendon-bone healing.</a> J Orthop Res. 23 (6), 1425-1432 (2005).</li><li>Hays, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+macrophages+in+early+healing+of+a+tendon+graft+in+a+bone+tunnel.">The role of macrophages in early healing of a tendon graft in a bone tunnel.</a> J Bone Joint Surg Am. 90 (3), 565-579 (2008).</li><li>Dagher, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+modulates+macrophage+accumulation+in+tendon-bone+healing.">Immobilization modulates macrophage accumulation in tendon-bone healing.</a> Clin Orthop Relat Res. 467 (1), 281-287 (2009).</li><li>Bedi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+early+and+delayed+mechanical+loading+on+tendon-to-bone+healing+after+anterior+cruciate+ligament+reconstruction.">Effect of early and delayed mechanical loading on tendon-to-bone healing after anterior cruciate ligament reconstruction.</a> J Bone Joint Surg Am. 92 (14), 2387-2401 (2010).</li><li>Bedi, A., Kawamura, S., Ying, L., Rodeo, 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=Differences+in+tendon+graft+healing+between+the+intra-articular+and+extra-articular+ends+of+a+bone+tunnel.">Differences in tendon graft healing between the intra-articular and extra-articular ends of a bone tunnel.</a> HSS J. 5 (1), 51-57 (2009).</li><li>Fu, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+graft+tensioning+on+mechanical+restoration+in+a+rat+model+of+anterior+cruciate+ligament+reconstruction+using+free+tendon+graft.">Effect of graft tensioning on mechanical restoration in a rat model of anterior cruciate ligament reconstruction using free tendon graft.</a> Knee Surg Sports Traumatol Arthrosc. 21 (5), 1226-1233 (2013).</li><li>Fan, H., Liu, H., Wong, E. J., Toh, S. L., Goh, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+study+of+anterior+cruciate+ligament+regeneration+using+mesenchymal+stem+cells+and+silk+scaffold.">In vivo study of anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold.</a> Biomaterials. 29 (23), 3324-3337 (2008).</li><li>Landis, J. R., Koch, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+observer+agreement+for+categorical+data.">The measurement of observer agreement for categorical data.</a> Biometrics. 33 (1), 159-174 (1977).</li><li>Joshi, S. M., Mastrangelo, A. N., Magarian, E. M., Fleming, B. C., Murray, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-platelet+composite+enhances+biomechanical+and+histologic+healing+of+the+porcine+anterior+cruciate+ligament.">Collagen-platelet composite enhances biomechanical and histologic healing of the porcine anterior cruciate ligament.</a> Am J Sports Med. 37 (12), 2401-2410 (2009).</li><li>Leong, N. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+evaluation+of+heparin+mediated+growth+factor+release+from+tissue-engineered+constructs+for+anterior+cruciate+ligament+reconstruction.">In vitro and in vivo evaluation of heparin mediated growth factor release from tissue-engineered constructs for anterior cruciate ligament reconstruction.</a> J Orthop Res. 10, (2014).</li><li>Seo, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+cell+migration+and+angiogenesis+in+a+composite+silk+scaffold+for+tissue-engineered+ligaments.">Increase in cell migration and angiogenesis in a composite silk scaffold for tissue-engineered ligaments.</a> J Orthop Res. 27 (4), 495-503 (2009).</li><li>Freeman, J. W., Woods, M. D., Laurencin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+the+anterior+cruciate+ligament+using+a+braid-twist+scaffold+design.">Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design.</a> J Biomech. 40 (9), 2029-2036 (2007).</li><li>Bashur, C. A., Shaffer, R. D., Dahlgren, L. A., Guelcher, S. A., Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fiber+diameter+and+alignment+of+electrospun+polyurethane+meshes+on+mesenchymal+progenitor+cells.">Effect of fiber diameter and alignment of electrospun polyurethane meshes on mesenchymal progenitor cells.</a> Tissue Eng Part A. 15 (9), 2435-2445 (2009).</li><li>Dash, T. K., Konkimalla, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly-&#1108;-caprolactone+based+formulations+for+drug+delivery+and+tissue+engineering:+A+review.">Poly-&#1108;-caprolactone based formulations for drug delivery and tissue engineering: A review.</a> J Control Release. 158 (1), 15-33 (2012).</li><li>Yoshimoto, H., Shin, Y. M., Terai, H., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biodegradable+nanofiber+scaffold+by+electrospinning+and+its+potential+for+bone+tissue+engineering.">A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering.</a> Biomaterials. 24 (12), 2077-2082 (2003).</li><li>Xu, Y., Ao, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+biomechanical+studies+of+inter-strand+healing+in+four-strand+autograft+anterior+cruciate+ligament+reconstruction+in+a+rabbit+model.">Histological and biomechanical studies of inter-strand healing in four-strand autograft anterior cruciate ligament reconstruction in a rabbit model.</a> Knee Surg Sports Traumatol Arthrosc. 17 (7), 770-777 (2009).</li><li>Shino, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+the+anterior+cruciate+ligament+by+an+allogeneic+tendon+graft.+An+experimental+study+in+the+dog.">Replacement of the anterior cruciate ligament by an allogeneic tendon graft. An experimental study in the dog.</a> J Bone Joint Surg Br. 66 (5), 672-681 (1984).</li><li>Stasiak, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+device+to+apply+controlled+flexion+and+extension+to+the+rat+knee+following+anterior+cruciate+ligament+reconstruction.">A novel device to apply controlled flexion and extension to the rat knee following anterior cruciate ligament reconstruction.</a> J Biomech Eng. 134 (4), 041008 (2012).</li><li>Brophy, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+short-duration+low-magnitude+cyclic+loading+versus+immobilization+on+tendon-bone+healing+after+ACL+reconstruction+in+a+rat+model.">Effect of short-duration low-magnitude cyclic loading versus immobilization on tendon-bone healing after ACL reconstruction in a rat model.</a> J Bone Joint Surg Am. 93 (4), 381-393 (2011).</li></ol>

The authors declare that they have no competing financial interests.

ACL blessures sont une condition commune en chirurgie orthopédique de sport, avec des options limitées pour la reconstruction à l&#39;heure actuelle. Afin de développer un substitut d&#39;ingénierie tissulaire appropriée pour l&#39;ACL qui permettra la régénération in vivo, un modèle animal approprié est nécessaire. Dans cette étude, la fabrication d&#39;un greffon conçu biodégradable est décrite, de même que son implantation in vivo en utilisant un modèle de reconstruction du LCA rep...

Rupture du ligament croisé antérieur (LCA) est une de la plupart des lésions ligamentaires du genou communes 1. Parce que la rupture de l&#39;ACL est incapable de guérir sans intervention chirurgicale, les limitations dans les activités de la vie quotidienne ainsi que la participation à des activités sportives en voiture plus de 175 000 patients de subir une chirurgie chaque année 2, avec un coût estimé à un milliard de dollars par an 3. Actuellement, soit autogreffe ou allogreffe tendon est utilisé pour la reconstruction du ligament. Bien que les taux de réussite élevés peuvent être atteints à la fois avec autogreffe et allogreffe de remplacement, des complications graves sont associés à ces options de reconstruction 4. autogreffe de tissu est associé à morbidité du site donneur et est limité dans l&#39;approvisionnement, en particulier en cas de re-rupture ou blessures multi-ligamentaire. D&#39;autre part, le tissu d&#39;allogreffe est liée à l&#39;intégration de la greffe retardée, la réponse inflammatoire indésirable, risque infectieux théorique, et limité suppLy 5. Greffons non dégradables synthétiques ont été développés dans les années 1970 et 1980, mais ont été entravées par une rupture prématurée de greffe, les réactions de corps étranger, une ostéolyse et la synovite 6. À la suite de ces graves préoccupations, il n&#39;y a actuellement aucun greffes synthétiques disponibles pour une utilisation clinique aux États-Unis. En raison de ces limitations avec des options de greffons existants et aux développements récents de la biologie, l&#39;ingénierie et la médecine régénérative, il ya eu beaucoup d&#39;intérêt dans une solution de l&#39;ingénierie tissulaire pour ACL greffage. Stratégies d&#39;ingénierie tissulaire actuelles emploient des matériaux biologiques et synthétiques dégradables pour permettre la croissance du tissu hôte tout en évitant les contraintes liées à l&#39;implantation permanente matière synthétique 7. La polycaprolactone (PCL) est un polymère biodégradable qui est approuvé par la FDA pour un certain nombre d&#39;applications médicales, y compris l&#39;adhérence barrière et Pansement 8, qui a été used dans une grande variété d&#39;applications, notamment vasculaire, os, du cartilage, des nerfs, la peau, et de l&#39;ingénierie de tissu oesophagien 5,9-16. Biocompatibilité favorable, relativement longue in vivo de demi-vie, une résistance mécanique suffisante et une élasticité élevée contribue à la popularité de ce polymère dans l&#39;ingénierie tissulaire. Dans un modèle de rongeur de la cicatrisation des plaies, implanté PCL électrofilé se est révélée être non immunogène et à intégrer dans le tissu local sans effets indésirables 13. Une image SEM de électrofilé PCL est illustrée à la figure 1. Avec actuelle FDA normes de réglementation, l&#39;efficacité et la sécurité dans les deux modèles petits et grands animaux qui serait nécessaire pour une PCL ou tout autre greffe ACL conçu pour se déplacer dans les essais cliniques aux États-Unis. En outre, des conditions in vivo peuvent souvent augmenter les propriétés d&#39;un tissu greffé LCA in vitro d&#39;ingénierie. Un modèle de rat de reconstruction du LCA autologue avec fléchisseur digitorum tendon du long a été décrit précédemment, dans lequel la LCA natif a été sectionné, du fémur et du tibia tunnels ont été forés, et la greffe a été adopté et maintenu en place avec une suture 17-22. Dans cet article, nous allons décrire une modification de ce modèle pour l&#39;évaluation des remplacements ACL ingénierie plutôt que pour la reconstruction basée autogreffe (Figure 2). Bien que de nombreux modèles animaux existent pour l&#39;ingénierie tissulaire de ligament, le rat est avantageuse par rapport à de plus grands modèles pour un certain nombre de raisons. Ces avantages comprennent plus facile élevage et la manipulation, moins de considérations éthiques, et de 17,23 coût réduit. En outre, le modèle de rat a été largement utilisé comme modèle pour la régénération des tissus orthopédiques, y compris du cartilage, des tendons, os et l&#39;ingénierie tissulaire 24. En particulier, les rats nude athymiques ont été choisis en raison de leur manque de réponse immunitaire à médiation cellulaire 25, permettant l&#39;implantation éventuelle ocellules du donneur xénogéniques f dans ce modèle pour améliorer encore la greffe d&#39;ingénierie à l&#39;avenir. Dans le présent document des méthodes, nous décrivons la fabrication et l&#39;implantation chirurgicale d&#39;un acellulaire, greffé de polymère biodégradable dans un modèle de rat athymique de reconstruction du LCA.

<table border="0" cellpadding="0" cellspacing="0" width="888">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:77px;">Catalog Number</td>
			<td style="width:317px;">Comments/Description</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Medical grade ester terminated poly (&#949;-caprolactone), granule form (MW = 110,000)</td>
			<td style="width:147px;">Lactel Absorbable Polymers</td>
			<td style="width:77px;"></td>
			<td style="width:317px;">Custom synthesized polymer to desired molecular weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>105228</td>
			<td style="width:317px;">Solvent for PCL polymer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">18G x 1 1/2&#34;bevel needle</td>
			<td style="width:147px;">BD Medical</td>
			<td>305196</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Remote Infuse/Withdraw Programmable Syringe Pump</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td style="width:77px;">702101</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">VersaLaser VLS2.30 Laser Engraver</td>
			<td style="width:147px;">Microgeo USA</td>
			<td style="width:77px;">VLS2.30</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Expanded Plasma Cleaner 115V</td>
			<td style="width:147px;">Harrick Plasma</td>
			<td style="width:77px;">PDC-001</td>
			<td style="width:317px;">Plasma etch just prior to collagen coating for surface modification</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">PureCol&#160; Collagen Standard Solution, 3 mg/ml</td>
			<td style="width:147px;">Advanced Biomatrix</td>
			<td style="width:77px;">5015-A</td>
			<td style="width:317px;">Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1N NaOH 1:9 in 1x PBS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Suture, 5-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1086471</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture, 4-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">6540072</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Sharp-pointed Dissecting Scissors (Straight; 4.5 inch)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:77px;">8940</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Buphrenorphine hydrochloride</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:77px;">B9275</td>
			<td style="width:317px;">Use 0.03 mg/kg for both intra- and post-operatively for pain control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Ampicillin, injectable</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1185678</td>
			<td style="width:317px;">Use 25 mg/kg subcutaneously during the procedure</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">K-wire, 1.6 mm</td>
			<td style="width:147px;">Spectrum Surgical</td>
			<td style="width:77px;">SI040062</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Keith Needle, Straight 1 1/2&#34;</td>
			<td style="width:147px;">Delasco Dermatology Lab &#38; Supply</td>
			<td style="width:77px;">KE-112</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Immunocal Decalcifying Solution</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>NC9491030</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td>
			<td style="width:147px;">US Dental Depot</td>
			<td style="width:77px;">OPTICRYL 100410&#160;</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:347px;">Instron Model 5564 Tensile Testing Machine</td>
			<td style="width:147px;">Instron</td>
			<td style="width:77px;">5564</td>
			<td style="width:317px;">Any comparable tensile testing apparatus is suitable</td>
		</tr>
	</tbody>
</table>

athymic rat model for evaluation of engineered anterior cruciate ligament grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Dans notre expérience de 92 chirurgies de rat par un seul chirurgien, le temps moyen de fonctionnement de l&#39;incision à l&#39;achèvement de la plaie était de 16,9 min, avec un écart type de 4,7 min. Au moment du sacrifice, les rats pesaient 356 ± 23 g. Tous les rats ont toléré la chirurgie bien, et ont connu aucune complication. Immédiatement après la chirurgie, les rats ont été notés à porter le poids sur l&#39;extrémité opérationnelle, mais présentaient une légère claudication. D&#39;une semaine...

Watch this Scientific Journal Video about Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts at JoVE.com

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Athymiques Rat Modèle d&#39;évaluation des Engineered ligament croisé antérieur greffes

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

athymic-rat-model-for-evaluation-engineered-anterior-cruciate

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Bioengineering

10.3K vues.  University of California Los Angeles. Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Vidéo: Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Méthodologie élastographie par résonance magnétique pour l&#39;évaluation des tissus Conçu croissance Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

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Bio-ingénierie

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Extraction chirurgicale, isolement et<em&gt; In vitro</em&gt; Extension de antérieure humaine Cruciate cellules du ligament dérivés pour les applications d&#39;ingénierie tissulaire

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Technique chirurgicale pour l&#39;implantation de l&#39;ingénierie tissulaire vasculaires greffes et suivantes<em&gt; In Vivo</em&gt; Surveillance

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Génération et greffage des navires par ingénierie tissulaire dans un modèle de souris

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Production, la caractérisation et utilisations potentielles d&#39;un humain de l&#39;oesophage muqueux modèle 3D de l&#39;ingénierie tissulaire

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Modèle de polarisée Neural Tissue base-Silk-collagène ingénierie 3D

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

des applications de<em&gt; In Vivo</em&gt; Tests fonctionnels du Rat jambier antérieur pour l&#39;évaluation de l&#39;ingénierie tissulaire Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

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.

Mise à l&#39;échelle de l&#39;ingénierie vasculaire Greffes Utilisation 3D Printed Guides et Méthode d&#39;empilage Anneau

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

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Traitement des constructions de ligament avec un sérum conditionné par l&#39;exercice: un modèle d&#39;ingénierie des tissus de traduction

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Utilisation du haut débit MICROBIOREACTEUR système automatisé pour la Production d’IgG1 de modèle dans les cellules CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...