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PONTINI, ALEX (2017) APPLICAZIONE SPERIMENTALE DI VASI UMANI DECELLULARIZZATI DI PICCOLO CALIBRO. [Tesi di dottorato]

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Abstract (inglese)

Tissue engineering mean a wide field of science application in searching the substitusion by different process of damaged human tissue. The main intent is to obtain scaffold that can perfectly mimic the structural and functional characteristics of damaged tissues or organs. In the field of vascular graft numerous studies were conducted to obtain suitable tissue that could supplant the use of autologous bypassor even the littlest vessel employed in microsurgery. Over the years, various solutions have been proposed. The use of resorbable or non-resorbable vascular synthetic conduits, associated or not to stem cells and growth factors, small-caliber decellularized vessels and scaffold-free methods have been described in the literature. First, our study has produced experimental animal models in which a section of the rabbit femoral vessels was replaced by small caliber (inner diameter < 6 mm) human decellularized vessel.

Materials and methods


Two arteries and two human veins of small caliber (< 6 mm) obtained by upper arm lesions not suitable for surgical procedure were withdrawn and subjected to a process of decellularization according to a modified Sheridan’s protocol. The effectiveness of the process was shown in vitro with histological, immunohistochemical, ultrastructural, and qualitative and quantitative analysis of DNA. Mechanical testing and functional ecodoppler in vivo were also performed. Decellularized vessels were implanted in vivo in rabbit femoral vessels. The patency was verified, in vivo, at time 0, 10 and 20 minutes later. After 30 days for vein implants and 10 and 40 days for artery implants, the rabbits were sacrificed. The patency of grafts, the inflammatory process and re-endothelialization was tested with histological and immunophenotypical analysis. At the same time we analyzed also synthetic vessel as control , after two weeks of implantation.

Results


The various assays of decellularized vessels, in vitro, demonstrated the success of the process. The cellular component was eliminated without altering the composition and three-dimensional structure of extra cellular matrix. It was also confirmed by mechanical testing. The scaffolds were implanted successfully in all models and the patency was maintained until animals’ sacrifice, as also documented by ecocolordoppler evaluation. The histological and immunophenotypical analysis of taken scaffolds showed a minimal inflammatory process, the maintenance of the extracellular matrix structure and the wall and luminal surface re-cellularization. The syntethic vessel demonstrate complete thrombosis.

Conclusions


The adjustments made to the decellularization protocol proposed by Sheridan helped to keep acceptable length of the process maintaining the same effectiveness. Non-use of endothelial stem cells reduced costs even if, for longer grafts the use of cells to improve biocompatibility seems to be necessary.

The histological studies on withdrawn grafts have demonstrated a minimal presence of inflammatory processes, a complete patent in two weeks till eight weeks and the presence of a process of re-cellularization. The excellent biocompatibility of implanted scaffolds suggest the possibility of a subsequent application in clinical microvascular field and, in the future, for the establishment of a tissue bank to find substitutes with suitable caliber and length. Additional animal testing, with long time survival, mechanical and histological analysis are definitely essential to determine the effectiveness of these implants and think of a possible clinical trials.

Abstract (italiano)

Riassunto




Ingegneria tissutale è il termine generico con cui si individua un vasto campo di studio e di applicazioni sperimentali ed industriali volti a ricreare in laboratorio tessuti biointegrabili nell’uomo allo scopo di sostituirne o riprodurne le funzioni in ambito di chirurgia rigenerativa. Una delle vie maggiormente utilizzate a tale fine è la realizzazione di un supporto ottenuto secondo diversi metodi e definito scaffold in grado di mimare perfettamente le caratteristiche sia strutturali che funzionali di tessuti od organi danneggiati, favorendo al contempo l’invasione cellulare e la sua integrazione nell’organismo ricevente. Nel campo dei tessuti vascolari, numerosi studi sono stati condotti al fine di produrre dei validi sostituti capaci in futuro di soppiantare l’utilizzo di bypass autologhi, non sempre disponibili e che comportano un danno secondario a volte non trascurabile. Ancora maggiore è l’esigenza nelle procedure microvascolari con vasi di piccole dimensioni. Negli anni, sono state ideati e realizzati condotti vascolari di materiale sintetico, riassorbibili o non riassorbibili, associati o meno a cellule staminali e fattori di crescita, scaffold ricavati da processi di decellularizzazione di vasi di piccolo calibro di origine animale e condotti vascolari realizzati con metodi scaffold free. Il presente studio ha prodotto dei modelli sperimentali animali in cui tratti di vasi femorali di coniglio sono stati sostituiti, in vivo, con scaffold ricavati da un processo di decellularizzazione di vasi umani di piccolo calibro (diametro interno < 6mm).

Materiali e metodi


Sono state prelevate due arterie e due vene umane di piccolo calibro (DI < 6 mm) ottenute da tessuti umani vivi di arto superiore ,previo consenso informato, ove non era possibile procedere a loro reimpianto e sottoposte ad un processo di decellularizzazione secondo un adattamento del protocollo di Sheridan. L’efficacia del processo è stata dimostrata, in vitro, con analisi istologiche, immunoistochimiche, ultrastrutturali, e analisi qualitative e quantitative del DNA. Abbiamo altresì condotto analisi meccaniche e valutazioni ecocolorDoppler in vivo della funzionalità. I vasi decellularizzati sono stati impiantati, in vasi femorali di conigli adulti realizzando un anastomosi termino-terminale. a pervietà è stata verificata, in vivo, al tempo 0, dopo 10 e dopo 20 minuti. Le cavie sono state sacrificate a 30 giorni ( impianti venosi) e 10 e 40 giorni (impianti arteriosi) , è stata verificata la pervietà degli innesti prelevati e sono state eseguite analisi istologiche e immunofenotipiche per rilevare il processo infiammatorio e di ricellularizzazione. Un controllo è stato rappresentato dall'impianto di sostituti vascolari sintetici pro arteria con sacrificio a 2 settimane.


Risultati


Le varie analisi in vitro dei vasi decellularizzati hanno dimostrato la totale riuscita del processo. La componente cellulare è stata eliminata senza alterare la composizione e la struttura tridimensionale della matrice extra cellulare, come dimostrato anche da test meccanici. Gli scaffold sono stati impiantati con successo e in tutti i modelli la pervietà è stata mantenuta fino al sacrificio degli animali. L’analisi Doppler ha ulteriormente confermato la sovrapponibilità con i tessuti originali ed il mantenimento del flusso senza evidenza di trombosi. Le analisi istologiche e immunofenotipiche degli scaffold prelevati hanno evidenziato un minimo processo infiammatorio, il mantenimento della struttura della matrice extracellulare e la ricellularizzazione sia della parete che della superficie luminale vascolare. Gli impianti sintetici hanno dimostrato invece una completa trombizzazione.



Conclusioni


Gli adattamenti apportati al protocollo di decellularizzazione proposto da Sheridan su animale si sono dimostrati parimenti efficaci sul tessuto umano, procedendo , anzi, ad una migliore qualità nella decellularizzazione che ha favorito l’integrazione in vivo. Grazie al mancato utilizzo di cellule staminali endoteliali i costi del processo risultano estremamente ridotti, seppure l’efficacia sia dimostrata per brevi tratti e ,per favorire l’integrazione di vasi di maggiore lunghezza ( come nei peduncoli micorvascolari dei lembi) il ricorso al potenziamento cellulare rimane una strada necessaria. Le analisi istologiche sui prelievi degli innesti hanno dimostrato una minima presenza di processi infiammatori, una completa pervietà sino a 6 settimane con un processo di neoendotelizzazione arteriosa.

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Tipo di EPrint:Tesi di dottorato
Relatore:BASSETTO, FRANCO
Dottorato (corsi e scuole):Ciclo 29 > Corsi 29 > BIOMEDICINA
Data di deposito della tesi:06 Febbraio 2017
Anno di Pubblicazione:06 Febbraio 2017
Parole chiave (italiano / inglese):decellularizzazione/decellularization - vasi/vessel- microchirurgia/microsurgery - bioingegneria/bioengineering
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/19 Chirurgia plastica
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Molecolare
Codice ID:10385
Depositato il:24 Nov 2017 09:52
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