Gli autori desiderano ringraziare Gabriel Arom e Michael Yeranosian per i loro contributi tecnici alla iterazioni precedenti di questo progetto. Questo progetto è stato finanziato dalla OREF Clinico Scientist Training Grant (NL), HH Lee Surgical Research Grant (NL), Veterans Administration BLR &amp; D Merit recensione 1 I01 BX00012601 (DM) e muscoloscheletrico Trapianto Foundation Young Investigator Award (FP).

NOTA: Tutti gli interventi chirurgici su animali sono stati approvati dal personale e veterinaria comitato uso locale prima di iniziare gli esperimenti. 1. Preparazione di elettrofilate policaprolattone Ponteggi <ol><li> Pesare e sciogliere estere grado medicale poli terminato (ε-caprolattone) in granuli in 1,1,1,3,3,3-esafluoro-2-propanolo per creare un 10% w / w soluzione del polimero PCL. Lasciate che il mescolare soluzione con un piatto mescolare per almeno 3 ore per garantire una soluzione omogenea. </li><li> Electrospin la soluzione PCL per creare un bracciale di fibre PCL altamente allineati per ponteggio fabbricazione. <ol><li> Preparare la configurazione electrospinning in cappa con il ventilatore sempre acceso. Questo consiste in una grande scatola acrilica che servirà come mezzo vuoto isolato per il processo electrospinning e ha punti di ingresso per la soluzione di fonte PCL azionato da una sorgente di tensione, la raccolta mandrino a motore, e una porta di vuoto. Pulire il Acrylibox c accuratamente con etanolo e coprire tutte le superfici con fogli parafilm per rimuovere eventuali impurità che possono compromettere la qualità del prodotto elettrofilate. </li><li> Caricare circa 3 ml della soluzione di cui sopra in una siringa da 10 ml con un ottuso chiuso 18 G, 1 ½ pollice ago. Rimuovere eventuali bolle d&#39;aria spingendo la siringa up. Soluzione serratura in una pompa a siringa programmabili. Inserire l&#39;ago attraverso un piccolo foro nella scatola acrilica lasciando circa ½ pollice dell&#39;ago all&#39;esterno della scatola acrilica per fissaggio del filo sorgente di tensione. </li><li> Utilizzare un 30 millimetri di rotazione mandrino del tornio, come il collettore per le fibre PCL altamente allineate; coprire il mandrino ermeticamente con una sottile striscia di lamina di alluminio. Bloccare il mandrino nel motore sul lato opposto della scatola circa 15 cm dalla siringa. </li><li> Inserire il tubo di plastica nella porta del vuoto e collegarlo alla fonte di vuoto cappa. Accendere la sorgente di vuoto e coprire la scatola acrilico con un coperchio. </li><li> Set la velocità di infusione della pompa a siringa programmabili a 2,5 ml / hr. Attivare il motore per azionare il mandrino a 3.450 rpm e collegare il cavo positivo della sorgente di tensione alla punta dell&#39;ago all&#39;esterno della scatola utilizzando un coccodrillo. </li><li> Una volta che l&#39;infusione della soluzione PCL è iniziata, attivare il generatore di tensione e regolato ad una tensione di funzionamento 20 kV. </li><li> Infondere la soluzione per 12 min per creare un bracciale omogenea da 0,5 ml della soluzione di PCL. NOTA: In media, ogni bracciale ha materiale elettrofilate sufficiente a creare due fogli sei nastri, che possono essere utilizzati per creare un totale di tre ponteggi a quattro strati. </li></ol></li><li> Il laser ha tagliato il bracciale PCL per formare più piccoli fogli su un VersaLaser Cutter 2.3 operato a livello basso-vuoto, 10,0 keV tensione atterraggio, 6,4 millimetri distanza di lavoro, e il diametro della sonda di 3.0. NOTA: In questo esempio, un disegno assistito da computer è stato utilizzato per istruire la taglierina per produrre più fogli di 1,5 mm x 35 mm x 150 micron con scaffolduniformemente distribuito 150 micron fori del diametro a 15% dell&#39;area dei pori. </li><li> Plasma etch i ponteggi PCL utilizzando un detergente al plasma per indurre idrofilia della superficie PCL con l&#39;accelerazione di ioni. Impostare il vuoto a 450 mTorr e il trattamento dei ponteggi per 30 secondi alla massima potenza (29,6 W). </li><li> Bagnare i ponteggi in etanolo al 70% in un ambiente sterile. </li><li> Rivestire le singole scaffold di collagene per facilitare l&#39;adesione cellulare e la proliferazione in vivo. <ol><li> Creare una soluzione di rivestimento di collagene diluendo un 8: soluzione sterile 2,5 su Purecol collagene 3 mg / ml soluzione standard, 10x PBS e 0,1 N NaOH 1: 1 9 in 1x PBS a 4 ° C. Mescolare accuratamente per garantire la soluzione di omogeneità. </li><li> Coat individuo 1,5 millimetri x 35 mm x 150 micron scaffold con una pellicola sottile strato di soluzione di collagene sopra. Lasciare asciugare per 24 ore in un ambiente sterile. </li></ol></li><li> Utilizzando 5-0 suture Vicryl, impilare e apporre quattro singoli 1,5 millimetri x 35 mm x 150 micron ponteggiutilizzando un Krackow punto di creare un finale spessore di 0,6 mm, a più livelli, impalcatura di collagene rivestite che è pronto per l&#39;impianto. </li></ol> 2. Rat Chirurgia Protocol <ol><li> Indurre l&#39;anestesia mettendo topo in camera di inalazione e la consegna 2% isoflurano con 2 L / min di ossigeno. Confermare il ratto sia adeguatamente anestetizzato applicando una pressione Hind piede e valutare per qualsiasi risposta. </li><li> Sul tavolo posteriore non sterile, iniettare per via sottocutanea 25 mg / kg ampicillina e 0,03 mg / kg buprenorfina. </li><li> Applicare pomata oftalmica per gli occhi. Agganciare la pelliccia dal arti posteriori operativo e preparare il sito chirurgico con tre scrub alternati di clorexidina e il 70% di etanolo. </li><li> Trasferire il ratto al tavolo operatorio, su un tappetino riscaldato per prevenire l&#39;ipotermia. Ogiva sicura, e mantenere l&#39;anestesia attraverso la procedura con il 2% isoflurano in 2 L / min di ossigeno, cono consegnato. Telo in modo sterile, lasciando l&#39;arto operativa esposta. </li><li&gt; Prima di iniziare l&#39;intervento chirurgico, prima confermare adeguata anesthetization dalla mancanza di una risposta Hind pressione del piede. </li><li> Effettuare una 2 cm un&#39;incisione mediale verticale al ginocchio, centrato a livello della rotula. Ritrarre la pelle lateralmente fino a quando l&#39;incisione è centrata sopra il ginocchio. </li><li> Utilizzare un bisturi per fare una artrotomia mediale pararotulea tagliando semplicemente mediale alla rotula e si estende prossimalmente al livello della giunzione muscolo del quadricipite e distalmente al livello del tendine rotuleo sulla tubercule tibiale. Fare attenzione a non tagliare i rotulei o quadricipite tendini. </li><li> Rotula uscita lateralmente da una incisione verticale 1 cm attraverso l&#39;capsula ginocchio appena lateralmente al tendine rotuleo. </li><li> Assicurarsi che il ginocchio è esteso. Prendete un paio di forbici fini e passare sotto la rotula da laterale a mediale. Stendere le forbici un paio di volte in modo che il meccanismo estensore può essere tradotto in entrambi i lati. </li><li> Mentre flettendo tegli ginocchio, tradurre la rotula lateralmente per esporre l&#39;interno del ginocchio. Assicurare la visualizzazione della gola intercondiloidea e condili femorali. Usando un bisturi, transetto l&#39;ACL e PCL nella tacca. </li><li> Carico trapano elettrico con un k-fili 1,6 mm. Posizionare punta k-wire sull&#39;origine ACL nella gola intercondiloidea. Praticare superolaterally e visualizzare il punto di uscita sulla parte laterale del femore, rimuovere qualsiasi tessuto molle, se necessario, con un bisturi. Passare il filo k in e fuori un paio di volte a garantire libero il passaggio per l&#39;innesto. </li><li> Posizionare il filo k sull&#39;impronta ACL sul piatto tibiale. Praticare anterolaterally e visualizzare il punto di uscita sulla tibia prossimale antero-laterale. Utilizzare bisturi per cancellare tessuti molli come necessario in modo che il punto in cui il filo k esce tibia è completamente visualizzato. </li><li> Passare un ago accorciato Keith (lungo idealmente non più di 2 centimetri) attraverso il tunnel osso femorale. Infilare le due estremità di sutura da un&#39;estremità dell&#39;innesto attraverso l&#39;occhio del Keith ago. Utilizzare l&#39;ago per tirare una estremità dell&#39;innesto attraverso il tunnel femorale. </li><li> Ripetere la fase prima di passare l&#39;altra estremità dell&#39;innesto attraverso il tunnel tibiale. </li><li> Utilizzare 4-0 Vicryl sutura per fissare la fine femorale dell&#39;innesto di periostio o di altri tessuti molli circostanti con un punto figura di otto. Tensione manualmente l&#39;innesto con il ginocchio in estensione. Fissare la fine tibiale dell&#39;innesto a periostio o altri tessuti molli circostanti con un punto figura di otto. </li><li> Utilizzare una forbice per tagliare l&#39;eccesso innesto su entrambe le estremità, lasciando 1-2 mm su ciascuna estremità passato il punto figura di otto. </li><li> Estendere il ginocchio e ridurre la rotula. Utilizzando 4-0 Vicryl, posizionare un singolo punto figura-di-otto per chiudere la capsula articolare mediale, impedendo sublussazione laterale della rotula. </li><li> Chiudere la pelle con un running sottocuticolare 5-0 monocryl o Vicryl sutura, con attenzione a non suturare il muscolo sottostante o avere alcun sutura visibile una volta che la pelle è chiuso. </li><li> Injeratti ct sottocutanea con buprenorfina ogni 12 h per un totale di tre giorni dopo l&#39;intervento. Controllare sito chirurgico per qualsiasi drenaggio o deiscenza della ferita al momento dell&#39;iniezione. Zoppicando e qualche gonfiore sono normali nei primi giorni dopo l&#39;intervento chirurgico, ma prontamente risolvere eventuali problemi post-operatori, in collaborazione con il personale veterinario. L&#39;animale può essere di ritorno per l&#39;edilizia sociale a 2 settimane dopo l&#39;intervento, quando incisioni chirurgiche sono completamente guariti. </li></ol> 3. Raccolta dati Protocol <ol><li> Al momento del sacrificio, asphyxiate ratti singolarmente in una camera chiusa CO 2 seguito da toracotomia. </li><li> Raccolto entrambi gli arti chirurgicamente ricostruite e controlaterale separando in dell&#39;anca. <ol><li> Per gli arti ricostruiti, rimuovere tutti i tessuti molli, incluso il legamento crociato posteriore ed i resti del chirurgicamente perturbato ACL native, dalla dissezione multa per isolare solo il femore, la tibia, e l&#39;innesto. </li> &lt;li&gt; Per gli arti controlaterali, rimuovere tutti i tessuti molli tranne il ACL native come pure il femore e tibia mediante dissezione fine. </li><li> Utilizzare un utensile rotante come Dremel, per rimuovere tutto ma ¾ a 1 cm di osso da ciascuna estremità del complesso femore-graft-tibia. </li><li> Durante questo processo e per tutto il test biomeccanici, regolarmente e frequentemente spruzzare le regioni legamentose con soluzione fisiologica per prevenire l&#39;essiccamento del ginocchio raccolto che possono alterare i risultati falsamente. </li></ol></li><li> Per l&#39;analisi istologica, risolvere ogni ginocchio singolarmente in soluzione paraformaldeide 4% a 25 ° C per 48 ore. Successivamente, immergere il ginocchio in una soluzione di reagente per Immunocal completa decalcificazione; questo processo dipende dal contenuto calcifici del campione e può richiedere fino a cinque giorni. Controllare singoli campioni al giorno per valutare i progressi come decalcificazione incompleta può abbassare la qualità del campione. Una volta completata, eseguire sezionamento, il montaggio di diapositive, e la colorazione, se lo desideri. </li><li> Perform test biomeccanici per valutare la capacità funzionale del legamento ingegneria tessutale. <ol><li> Fissare il femore e tibia avvolgendo separatamente 28 G filo d&#39;acciaio galvanizzato intorno alla epifisi di ogni osso. Questo per evitare che i dati di test biomeccanici imprecisi di rottura per trazione prematura del campione all&#39;osso piuttosto che al legamento di interesse. </li><li> Pot femore in una miscela di cemento polimetilmetacrilato (PMMA) dell&#39;osso. Per fare questo, miscelare i due componenti del cemento e utilizzare immediatamente la miscela viscosa per fissare femore nel metallo, che stringe completamente i diafisi dell&#39;osso nel piatto cementato con l&#39;epifisi e legamento divisoria sporgenti liberamente. Lasciare spontanea polimerizzazione radicalica per trasformare gradualmente i componenti viscosi misti ad un materiale pastoso e infine in una matrice solida indurita. NOTA: Questo processo richiede diversi minuti e può essere controllata valutandone manualmente la temperatura di un bolo realizzatoil cemento residuo; la temperatura deve aumentare temporaneamente durante la reazione di polimerizzazione esotermica e regrediscono a RT dopo la solidificazione del materiale. </li><li> Ripetere la stessa procedura di cui sopra per cementare tibia, tranne mantenendo il legamento del ginocchio a 20 ° di flessione per prove meccaniche ideale. </li><li> Montare il complesso femore-graft-tibia cementata su un apparecchio per prove di trazione, e preparati a carico di registrazione e lo spostamento in funzione del tempo dall&#39;inizio della tensione al fallimento. In questo esempio, abbiamo utilizzato un modello di Instron 5564 con una cella di carico 1 kN. </li><li> Pre-tensionamento dell&#39;innesto a 2 N ad una velocità di rampa di 0,5 N / min e quindi verificare l&#39;innesto al fallimento ad una velocità di deformazione di 0,5 mm / sec. Durante il processo, assicurarsi che il legamento non riesce a metà sostanza e che il femore e tibia osseo sono sicuri e non prematuramente in mancanza, che potrebbe erroneamente valutare le proprietà biomeccaniche del legamento testato. </li><li> Usare il carico-spostamento generatacurve per calcolare il carico di rottura e la rigidità del legamento testato. </li></ol></li></ol>

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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.

Lesioni ACL sono una condizione comune in chirurgia ortopedica sportiva, con opzioni limitate per la ricostruzione, al momento presente. Per sviluppare un sostituto tissutale appropriate per LCA che permetterà la rigenerazione in vivo, è necessario un modello animale adatto. In questo studio, la realizzazione di un innesto ingegnerizzato biodegradabile è descritto, così come il suo impianto in vivo utilizzando un modello riproducibile di ricostruzione del LCA in ratti atimici. Questo modello può e...

Rottura del legamento crociato anteriore (LCA) è uno dei più comuni lesioni legamentose del ginocchio 1. Perché la rottura del LCA è in grado di guarire senza intervento chirurgico, le limitazioni nelle attività della vita quotidiana e la partecipazione a sport di auto oltre 175.000 pazienti di sottoporsi ad intervento chirurgico ogni anno 2, con un costo stimato di un miliardo di dollari l&#39;anno 3. Attualmente, sia autologo o allogenico tendine viene utilizzato per la ricostruzione del legamento. Anche se gli alti tassi di successo possono essere realizzati sia con autologo e allogenico sostituzione, gravi complicanze sono associate con queste opzioni di ricostruzione 4. Tessuto autologo è associata a morbilità del sito donatore ed è limitata dell&#39;offerta, in particolare nei casi di re-rotture o lesioni multi-legamentosa. D&#39;altra parte, il tessuto allotrapianto è collegata con l&#39;integrazione dell&#39;innesto ritardato, risposta infiammatoria avversa, rischio teorico infettiva, e supp limitataly 5. Sintetici innesti non degradabili sono stati sviluppati negli anni 1970 e 1980, ma sono stati ostacolati dalla rottura prematura trapianto, reazioni del corpo estraneo, osteolisi, e sinovite 6. Come risultato di questi gravi problemi, non esistono attualmente innesti sintetici disponibili per uso clinico negli Stati Uniti. A causa di queste limitazioni con opzioni innesto esistenti e ai recenti sviluppi nel campo della biologia, ingegneria e medicina rigenerativa, c&#39;è stato un grande interesse per una soluzione di ingegneria tessutale per ACL innesto. Strategie di ingegneria tissutale attuali impiegano materiali biologici e sintetici degradabili per consentire di crescita interna del tessuto ospite, evitando le limitazioni associate con materiale sintetico permanente dell&#39;impianto 7. Policaprolattone (PCL) è un polimero biodegradabile che è approvato dalla FDA per un certo numero di applicazioni mediche, tra cui barriera adesione e medicazione 8, che è stato used in una vasta gamma di applicazioni tra cui vascolare, osso, cartilagine, nervi, pelle, e l&#39;ingegneria dei tessuti dell&#39;esofago 5,9-16. Biocompatibilità favorevole, relativamente lunga in-vivo emivita, un&#39;adeguata resistenza meccanica, ed elevata elasticità contribuisce alla popolarità di questo polimero in ingegneria dei tessuti. In un modello di roditore di guarigione delle ferite, impiantato PCL elettrofilate ha dimostrato di essere non-immunogenico e integrare nel tessuto locale, senza reazioni avverse 13. L&#39;immagine SEM di elettrofilate PCL è mostrato in Figura 1. Con corrente FDA regolamentazione norme, l&#39;efficacia e la sicurezza in entrambi i modelli piccoli e grandi animali sarebbe richiesto per un PCL o qualsiasi altro progettato innesto ACL a muoversi in studi clinici negli Stati Uniti. Inoltre, le condizioni in vivo spesso può aumentare le proprietà di un trapianto di tessuto ACL in vitro di ingegneria. Un modello di ratto di ricostruzione del LCA autologo con flessore digitorum lungo del tendine è stato descritto in precedenza, in cui l&#39;ACL nativo è stato reciso, femore e tibia tunnel sono stati perforati e l&#39;innesto è stato approvato e fissato in posizione con sutura 17-22. In questo articolo, descriveremo una modifica di questo modello per la valutazione delle sostituzioni ACL ingegnerizzati piuttosto che per ricostruzione basata autograft-(Figura 2). Anche se esistono molti modelli animali per l&#39;ingegneria dei tessuti del legamento, il ratto è vantaggioso rispetto ai modelli più grandi per una serie di motivi. Questi vantaggi comprendono più facile allevamento e maneggevolezza, un minor numero di considerazioni etiche, e ridotto 17,23 costo. Inoltre, il modello di ratto è stato ampiamente utilizzato come modello per la rigenerazione tissutale ortopedici, tra cui la cartilagine, tendini, ossa e del tessuto ingegneria 24. In particolare, ratti nudi atimici sono stati scelti a causa della loro mancanza di cellula-mediata risposta immunitaria 25, consentendo l&#39;eventuale fissaggio ocellule del donatore xenogeniche f in questo modello per migliorare ulteriormente l&#39;innesto ingegnerizzato in futuro. In questo metodo di carta, descriviamo la fabbricazione e l&#39;impianto chirurgico di un acellulare, biodegradabile trapianto polimero in un modello di ratto atimico di ricostruzione del 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.

Nella nostra esperienza di 92 interventi chirurgici di ratto da un singolo chirurgo, tempo medio operatorio dall&#39;incisione al completamento della ferita era 16,9 min, con una deviazione standard di 4,7 min. Al momento del sacrificio, ratti pesati 356 ± 23 g. Tutti i ratti hanno tollerato la chirurgia bene, e con esperienza senza complicazioni. Subito dopo l&#39;intervento chirurgico, i ratti sono stati notati a sopportare il peso sull&#39;estremità operativa, ma hanno mostrato una leggera zoppia. Di una settimana ...

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.

Atimico Rat modello per la valutazione di Engineered legamento crociato anteriore Innesti

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

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Bioengineering

10.3K Views.  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.

Video: 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).

Metodologia Elastografia risonanza magnetica per la valutazione dei Tissue Engineered Construct crescita

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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Bioingegneria

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.

Recupero chirurgico, Isolamento e<em&gt; In vitro</em&gt; Ampliamento anteriore umana crociati Cells Legamento di derivazione per applicazioni di ingegneria tissutale

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.

Tecnica chirurgica per l&#39;impianto di ingegneria tessutale vascolari Innesti e successive<em&gt; In Vivo</em&gt; Monitoraggio

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.

Generazione e innesto delle navi tissutale in un modello di topo

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.

Produzione, caratterizzazione e possibilità di utilizzo di un 3D tissutale umano esofageo mucose Modello

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.

A base di seta-collagene Engineered Modello 3D del tessuto neurale polarizzata

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.

applicazioni di<em&gt; In Vivo</em&gt; Test funzionale del Topo tibiale anteriore per la valutazione di ingegneria tessutale muscolo scheletrico di riparazione

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.

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

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

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.

Trattamento delle strutture ligamentali con serum esercitato-condizionato: un modello di ingegneria tessuto transazionale

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.

Uso di High-Throughput Microbioreactor sistema automatizzato per la produzione del modello IgG1 in cellule 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 ...