Les auteurs tiennent à remercier le démarrage fonds et département de chirurgie subvention de l&#39;Université Southern Illinois, École de médecine de la recherche; et la subvention de la Fondation Memorial médicale.

1. Prélèvement chirurgical Remarque: Une approbation de l&#39;IRB a été obtenu pour recueillir le moignon du LCA lors de la chirurgie de reconstruction du LCA. Exonération de la CISR a été donné comme le moignon du LCA utilisés étaient généralement jeté comme un déchet opératoire. 20 patients ont été utilisés pour recueillir le moignon du LCA. <ol><li> Administrer une anesthésie générale et des antibiotiques préopératoires selon le protocole de l&#39;établissement pour le patient. </li><li> Mettez le garrot et gonfler 100 mg dessus la pression artérielle systolique. </li><li> Situer le patient en décubitus dorsal. Faire une incision antéro-latérale horizontale avec le genou en 30-45 ° de flexion pour un portail de 5 mm et insérer le trocart arthroscopique et appareil photo dans la poche suprapatellaire à 30 °. </li><li> Effectuez une arthroscopie diagnostique de routine. </li><li> Entrez un cran et débrider la synoviale membraneuse sur la rupture du LCA. </li><li> Utilisez un rasoir de 4,5 mm et ablateur pour identifier la souche d&#39;ACL. </li><li> Utilisez un bec de canard droite amer pour recueillir spé multiplecimens de l&#39;ACL moignon restant. </li><li> Maintenir l&#39;échantillon dans une solution saline normale dans un contenant stérile. </li><li> Envoyer les échantillons de pathologie pour le décodage et la livraison finale à l&#39;installation de laboratoire. </li><li> Récupérer des patients et effectuer les soins post-opératoires selon le protocole de l&#39;établissement. </li></ol> 2. Digestion du tissu et HACL Isolation <ol><li> Transférer le tissu de LCA humain reçue de la pathologie à une boîte de Pétri avec une solution saline stérile / PBS (solution saline tamponnée au phosphate). </li><li> Émincer le tissu à l&#39;aide de ciseaux stériles dans 1-2 mm 3 pièces et laver le tissu haché avec une solution saline au moins 3 fois. </li><li> Digérer le tissu haché avec 0,4% de collagénase en DMEM/F-12 (50/50, 1x) supplémenté avec 10% de sérum bovin fœtal (FBS) et 1% de pénicilline / streptomycine pour 4-6 heures à 37 ° C. </li><li> Centrifuger les cellules à 1000 g pendant 5 min, puis remettre en suspension les cellules dans le milieu. </li><li> Laver les cellules avec le milieu de 2 à 3 fois, puis sEED / culture en flacons T-25 pour 2-3 jours. </li></ol> 3. Expansion cellulaire, de congélation et de décongélation <ol><li> Utiliser un microscope optique pour visualiser les cellules cultivées pendant 2-3 jours dans un flacon T-25. </li><li> Maintenir les cellules à 37 ° C dans du DMEM/F-12 (50/50, 1x) supplémenté avec 10% de sérum bovin fœtal (FBS) et 1% de pénicilline / streptomycine (P / S). Note: Changer les médias lorsque les cellules montrent le respect et la morphologie standard. Utiliser un microscope optique pour observer les cellules. </li><li> Laver les cellules confluentes avec du PBS au jour 7 (~ 1,3 x 10 6 cellules / flacon). Ajouter la trypsine (0,5 g / L) et incuber les cellules à 37 ° C pendant 5 min. Ajouter des médias sur les cellules en suspension pour neutraliser la trypsine. Répartir le mélange médias cellule entre trois flacons T-25. </li><li> Laver les cellules confluentes avec du PBS, ajouter de la trypsine (0,5 g / L), et de les remettre en suspension dans un milieu de congélation contenant le DMSO, le FBS et le milieu complet (DMEM/F-12 Medium + 10% FBS + 1% P / S) dans l&#39; rapport 01:02:07. </li><li> Magasinles flacons cryogéniques à -80 ° C si les cellules seront utilisés dans un mois, et dans l&#39;azote liquide si les cellules seront conservées pendant des durées plus longues. </li><li> Décongeler les flacons dans un bain d&#39;eau à 37 ° pour la réutilisation. Remarque: Généralement 90-95% de cellules survivent. </li></ol> 4. Fabrication de l&#39;ingénierie tissulaire 2-D Poly lactique-co-acide glycolique (PLAGA) Échafaudages <ol><li> Dissoudre 1 g de PLAGA dans 12 ml de dichlorométhane dans un flacon à scintillation de 20 ml et le vortex de la solution pendant 8 heures à une vitesse constante (800 rpm). Remarque: Description détaillée dans Gupta et al 15. </li><li> Transférer la solution dissoute dans un plat en verre de Pétri tapissée de papier de protection fluoré. </li><li> Placez la boîte de Pétri sous une hotte d&#39;aspiration pour 30 min. Laisser la boîte de Pétri à -20 ° C pendant une nuit, puis à température ambiante. Placer les boîtes de Pétri dans lyophilisateur pour assurer l&#39;évaporation complète du solvant pour obtenir des films minces de polymère. </li><li> Placez le fi polymèrelms dans un dessiccateur pendant 24 heures. </li><li> Couper les films de polymère en 12 mm de diamètre disques circulaires et les stocker dans un dessiccateur jusqu&#39;à ce que nécessaire. </li></ol> 5. Microscopie électronique à balayage (MEB) <ol><li> Laver les cellules (50 000 cellules / disque) ensemencées sur l&#39;EPTC de contrôle et l&#39;échafaud de PLAGA avec du PBS 7 jours après l&#39;ensemencement. </li><li> Utilisez 1,5% glutaraldéhyde dans un tampon cacodylate 0,1 M pour fixer les cellules et de 2,5% OSO4 dans 0,1 M de tampon cacodylate de post-fixation. </li><li> Laver les cellules fixées avec du tampon cacodylate 0,1 M. Sécher les cellules fixées à l&#39;aide de la déshydratation de série de l&#39;éthanol (50%, 70%, 80%, 90% et 100%) pendant 15 minutes chacun, et en outre à sec hexaméthyldisilazane (HMDS) pendant une nuit. </li><li> Purger la chambre de SEM système de revêtement par pulvérisation avec soupape de bouton et soulever la plaque supérieure. </li><li> Placer les échantillons séchés dans la chambre. Abaissez la plaque supérieure. </li><li> Mettez le bouton de commande de la pompe de commencer à évacuer la chambre. </li><li> Ouvrir la vanne de fuite de l&#39;argon. Attendezpour le vide pour tomber à 0,05 mbar. </li><li> Enduire les échantillons séchés avec une couche mince (0,13 nm) d&#39;or / palladium. </li><li> Retourner la soupape de fuite d&#39;argon à sa position fermée. </li><li> Mettez le bouton de réglage sur off. </li><li> Purger la chambre de soupape de bouton et soulever la plaque supérieure. </li><li> Retirer les échantillons et les stocker dans un dessiccateur pendant 24 heures. </li><li> Observer les échantillons au microscope électronique à balayage. </li></ol> 6. Immunofluorescence <ol><li> Laver les cellules (50 000 cellules / disque) ensemencées sur l&#39;EPTC de contrôle et l&#39;échafaud de PLAGA avec du PBS 7 jours après l&#39;ensemencement. </li><li> Fixer les cellules en utilisant 70% d&#39;éthanol (à froid) pendant 10 min. </li><li> Incuber les cellules fixées à la température ambiante avec 1% de sérum-albumine bovine (BSA) dans du PBS avec 0,05% de Triton X-100 pendant 20 min. </li><li> Immerger les échantillons dans 1% de Tween à température ambiante pendant 20 min. </li><li> Ajouter anticorps anti-β-actine monoclonal de souris (1:400) et incuber les cellules overnight à 4 ° C. </li><li> Laver les cellules avec 0,05% de Tween. Ajouter l&#39;anticorps secondaire (chèvre anti-souris F (ab &#39;) 2 d&#39;IgG conjugué fragment avec une sonde de fluorescence, 1:400) aux cellules et incuber pendant 1 heure à température ambiante. </li><li> Laver les cellules avec du PBS. Colorer les cellules de colorant nucléaire et monter en utilisant 80% de glycérol. </li><li> Respecter les cellules sous un microscope confocal. </li></ol>

<ol>
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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

L&#39;objectif principal de cette étude d&#39;échafaudage hACL/2D était d&#39;utiliser les cellules obtenues dans un patch pour augmenter la réparation primaire de larmes partielles du LCA. Traitement non chirurgical des ruptures du LCA partielles peut inclure une courte période d&#39;immobilisation, des renforts, un programme de réhabilitation progressive et régulière des évaluations de suivi 13,16,17. Cependant, de nombreuses études montrent que le traitement conservateur chez les athlètes a ét?...

Le ligament croisé antérieur (LCA) est un ligament intra-articulaire souvent blessée du genou. Environ 200 000 (ACL) blessures sont signalés chaque année aux États-Unis. Plus de 75% des patients souffrant de blessures ACL opter pour la chirurgie reconstructive orthopédique 1,2,3,4. Une intervention chirurgicale est souvent indiquée en raison d&#39;un potentiel de guérison intrinsèquement mauvaise 3. Reconstruction du LCA est généralement réalisé au moyen d&#39;une autogreffe ou allogreffe tendon. Autogreffe et allogreffe représentent l&#39;étalon-or pour la reconstruction car ils offrent des taux de réussite élevés, et la réparation de suture primaire, l&#39;autre option de traitement, a montré des taux allant jusqu&#39;à 94% 5,6,7 d&#39;échec. Ruptures partielles du LCA représentent 10% à 28% de toutes les ruptures du LCA 8. Dans une étude prospective, Noyes et al. Estime que 50% des patients atteints de ruptures partielles affectant plus de la moitié de la LCA a progressé à compléter ACL insuffisance après non-traitement chirurgical 9. D&#39;autres études font état ​​d&#39;instabilité persistante et la fonction ont diminué de moins de 30% des patients capables de retourner dans leur pré-blessure niveau d&#39;activité 9,10,11,12,13. Les options de traitement sont limitées et comprennent modalités conservatrices, retrait thermique de rester ACL, ou une reconstruction du LCA. Récemment, il ya eu un intérêt accru dans la réparation primaire augmentée. Ces techniques utilisent des produits biologiques pour améliorer la réparation de suture primaire 14. Des recherches récentes ont tenté de récolter mésenchymateuses comme HACL dérivée de cellules souches de contourner les limites de greffe, 31,32, mais la validité et l&#39;efficacité de ces cellules est encore inconnue. La source des cellules idéales pour des applications d&#39;ingénierie tissulaire semble être non mesechymal HACL dérivé des cellules de fibroblastes. La recherche actuelle se concentre sur l&#39;identification d&#39;un matériau de matrice approprié et la source de la cellule de l&#39;échafaudage d&#39;ingénierie. C&#39;est la procédure standard pour une rupture du LCA à être jetés comme sudéchets rgical cours de la chirurgie de reconstruction, cependant, ce ligament endommagé peut être une source de qualité pour l&#39;acquisition de cellules nécessaires pour développer et renforcer un tissu idéal conçu remplacement ACL. Notre laboratoire a mis au point un protocole pour l&#39;expansion in vitro de ces cellules dérivées de HACL récoltés. Utilisation d&#39;une matrice 2D conçu infusé avec des cellules HACL dérivés, nous avons conçu un patch qui pourrait augmenter partielle réparation du LCA et de renforcer les ligaments déchirés.

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			<td height="21" style="height:21px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:507px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri-dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">08-757-103C</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP3994</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Collagenase</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">17018-029</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">DMEM/F-12</td>
			<td style="width:139px;">Cellgro</td>
			<td style="width:143px;">10-092-CV</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">10082</td>
			<td>Store at -800C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-602E</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Tubes- 15 ml</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T-25 flasks</td>
			<td style="width:139px;">BD Falcon</td>
			<td style="width:143px;">3013</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Trypsin-Versene mixture</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-161E</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP231-100</td>
			<td>Combustible liquid. Can cause skin, eye and respiratory tract irritation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryogenic vials</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430489</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">PLAGA</td>
			<td style="width:139px;">Purac Biomaterials</td>
			<td style="width:143px;">Purasorb PLG8523</td>
			<td>Store at -800C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCPS disks</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">12-545-82</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Dichloromethane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC36423-0010</td>
			<td>Possible cancer hazard. Store in a dry, cool place.</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Bytac paper</td>
			<td style="width:139px;">Saint gobin performance plastics</td>
			<td style="width:143px;">1420652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintillation vial</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>03-339-21G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A7906</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">mouse Anti-&#946;-actin antibody (10 Ab)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A5441</td>
			<td>Store at -200C</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">goat-anti-mouse antibody (20 Ab)</td>
			<td style="width:139px;">Cell Signaling</td>
			<td style="width:143px;">4408</td>
			<td>Store at -200C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Hoechst dye</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">14530</td>
			<td>Store at -200C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP229-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP2547-1</td>
			<td>Toxic by inhalation and if swallowed. Causes burns by all exposure routes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexamethyldisilazane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC43085-1000</td>
			<td>Flammable liquid and vapor. Causes burns by all exposure routes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile Scissors</td>
			<td style="width:139px;">McKesson</td>
			<td style="width:143px;">25-716</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:139px;">Eppendorf</td>
			<td style="width:143px;">5804R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light Microscope</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:143px;">CK40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water Bath</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:143px;">2845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:139px;">Labnet&#160;</td>
			<td style="width:143px;">VX-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Petri plates</td>
			<td style="width:139px;">fisher Scientific</td>
			<td style="width:143px;">S31473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acu-Punch</td>
			<td style="width:139px;">Acuderm Inc.</td>
			<td style="width:143px;">P1225</td>
			<td>Acu-Punch was used to cut 12mm disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cacodylate buffer</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">97068</td>
			<td>Flammable liquid, carcinogen and irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium tetraoxide</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">201030</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">T8787</td>
			<td>Harmful if swallowed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:139px;">Decon Labs</td>
			<td style="width:143px;">2705</td>
			<td>Keep away from heat, sparks, flame and other form of ignition.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">General/regional Anesthesia</td>
			<td style="width:139px;">Amphastar pharmaceuticals</td>
			<td style="width:143px;"></td>
			<td>1% Lidocaine, 0.25% Bupivacaine,Anesthetic agents for induction and maintenance</td>
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			<td height="21" style="height:21px;width:216px;">Antibiotics</td>
			<td style="width:139px;">Hospira</td>
			<td>0409-0805-01</td>
			<td>Ancef 1g i.v.</td>
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			<td height="21" style="height:21px;width:216px;">Arthroscopy trocar</td>
			<td style="width:139px;">Smith and Nephew</td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arthroscopy Camera</td>
			<td style="width:139px;">Smith and Nephew</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arthroscopic grasper and bitter</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4.5mm shaver</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Interference Screws</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td>stainless steel screws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sputter Coater</td>
			<td style="width:139px;">Polaron</td>
			<td style="width:143px;">E5400</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications

Blessure à l&#39;ACL est un problème fréquemment rencontré chez les personnes actives. Même ruptures partielles de cette intra-articulaire ligament du genou conduire à des carences biomécaniques qui nuisent à la fonction et la stabilité. Les options actuelles pour le traitement des déchirures partielles du LCA vont de non fonctionnement, la gestion prudente de plusieurs options chirurgicales, telles que: modification thermique, mono-faisceau de réparation, reconstruction complète, et la reconstruction de la partie endommagée du ligament natif. Peu d&#39;études, le cas échéant, ont démontré une méthode unique pour la gestion soit toujours supérieure, et dans de nombreux cas, les patients continueront à démontrer l&#39;instabilité persistante et d&#39;autres comorbidités. Le but de cette étude est d&#39;identifier une source de cellules potentiel pour une utilisation dans le développement d&#39;un patch de l&#39;ingénierie tissulaire qui pourraient être mis en œuvre dans la réparation d&#39;un ACL partiellement déchiré. Un protocole selon l&#39;invention a été développé pour l&#39;expansion des cellules dérivées de patients subissant une reconstruction du LCA. Pour isoler les cellules, les tissus HACL hachée obtenu lors de la reconstruction du LCA a été digéré dans une solution de collagénase. L&#39;expansion a été réalisée en utilisant un milieu DMEM/F12 supplémenté avec 10% de sérum fœtal bovin (FBS) et 1% de pénicilline / streptomycine (P / S). Les cellules ont ensuite été stockés à -80 ° C ou dans l&#39;azote liquide dans un milieu de congélation constitué de DMSO, FBS et le moyen d&#39;expansion. Après décongélation, les cellules HACL dérivés ont ensuite été ensemencées sur un échafaudage de l&#39;ingénierie tissulaire, PLAGA (acide lactique-co-glycolique Poly) et le contrôle des tissus culture polystyrène (TCPS). Après 7 jours, SEM a été réalisée pour comparer l&#39;adhésion cellulaire à la PLAGA contre l&#39;EPTC de commande. La morphologie cellulaire a été évaluée à l&#39;aide d&#39;immunofluorescence. SEM (Scanning Electron Microscope) micrographies démontré que les cellules ont augmenté et respectées sur les deux surfaces PLAGA et EPTC et ont été confluentes sur les surfaces entières par jour 7. Immunofluorescence a montré normale, morphologique non sollicitéal motifs sur les deux surfaces. Cette technique est prometteuse pour les applications de ACL régénération et de reconstruction.

Le modèle de travail pour prélèvement chirurgical, la digestion du tissu et de l&#39;isolement de la personne humaine Ligament croisé antérieur (HACL) des cellules dérivées est illustrée à la figure 1. Les cellules ayant migré à partir des explants et adhéré aux flacons T-25. Ces cellules ont été cultivées pendant 3 jours, puis ont été visualisées sous un microscope optique (figure 2). Une monocouche confluente a été obtenu par jour 7. La présence de cellules saine...

Watch this Scientific Journal Video about Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications at JoVE.com

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

Pour les applications futures comme un patch pour réparer des déchirures partielles du ligament croisé antérieur (LCA), des cellules dérivées de LCA humain ont été isolés à partir de tissus obtenus au cours de procédures de reconstruction, d&#39;agrandissement in vitro et cultivés sur des échafaudages de l&#39;ingénierie tissulaire. L&#39;adhésion cellulaire et la morphologie ont ensuite été effectuées pour confirmer la biocompatibilité sur la surface de l&#39;échafaudage.

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 ...

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Research

JoVE Journal

Bioengineering

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

11.4K vues.  Southern Illinois University School of Medicine.  Pour les applications futures comme un patch pour réparer des déchirures partielles du ligament croisé antérieur (LCA), des cellules dérivées de LCA humain ont été isolés à partir de tissus obtenus au cours de procédures de reconstruction, d'agrandissement in vitro et cultivés sur des échafaudages de l'ingénierie tissulaire. L'adhésion cellulaire et la morphologie ont ensuite été effectuées pour confirmer la biocompatibilité sur la...

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

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

génie tissulaire d&#39;un être humain 3D<em&gt; In vitro</em&gt; Système de tumeur de test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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Bio-ingénierie

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.

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 ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Irritation des yeux Test (IET) pour l&#39;identification des dangers des produits chimiques irritants oculaires utilisant épithélial (RHCE) Tissue modèle reconstruit humain Cornea-like

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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 ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Faire fondre électrofilage écrit d’échafaudages Poly(ε-caprolactone) en trois dimensions avec des Morphologies contrôlables pour les Applications de génie tissulaire

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Isolement des cellules souches mésenchymateuses du périoste alvéolaire humaine et les effets de la vitamine D sur activité ostéogène du périoste dérivées des cellules

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Deux méthodes de DÉCELLULARISATION de tissus végétaux pour utilisations en génie tissulaire

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

Production de plasma autologue riche en plaquettes pour stimuler l’expansion in vitro des fibroblastes humains

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Plate-forme de culture d’organes du segment antérieur pour le suivi des blessures du globe ouvert et des performances thérapeutiques

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Tissu conjonctif humain dérivé de fibroblastes pour les applications de dépistage

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...