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Milan, Anna (2016) L'ingegneria tissutale e l'uso delle matrici decellularizzate nella terapia sostitutiva. [Tesi di dottorato]

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Abstract (inglese)

ABSTRACT
BACKGROUND: Oesophageal Atresia (OA) is a congenital defect that occurs during gestation and prevents the correct oesophagus development in 1 every 3000-4500 ¬births. Tissue Engineering could represent a therapeutic alternative for the most severe cases, where oesophageal replacement becomes necessary. Decellularised matrices are the ideal option because they are derived from tissue-specific extracellular-matrix (ECM) after removing all the cells therefore avoiding the risk of incompatibility and rejection. However, appropriate preservation may significantly affect scaffold behaviour.
AIM: here we aim to create a decellularised oesophageal scaffold in a large animal and to establish an innovative method of scaffold storing.
METHODS: rabbit oesophagi were decellularised using a detergent-enzymatic treatment (DET) and evaluated at 2 and 4 weeks, 3 and 6 months of storage. Four storage methods were compared: SCM (slow cooling in medium with 10% DMSO at -1°C/min, then stored in liquid nitrogen), SF (snap-freezing in liquid nitrogen, then stored in -80°C), FD (freeze-drying, then stored in -20°C) and 4C (phosphate-buffered saline solution at 4°C). Structural and functional analyses were performed prior to and after each storage condition.
RESULTS: efficient decellularisation with preservation of the ECM was achieved after 2 DET cycles as evidenced by histology and DNA quantification. Only the SCM method maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, all other methodologies failed long-term preservation of the original structure. In particular, SF-oesophagi displayed irreversible tissue collapse, FD-samples were impossible to rehydrate 3 and 6 months post-storage and the 4C option led to a progressive distortion of the tissue architecture.
DISCUSSION: scaffolds for oesophageal tissue engineering can be obtained in a large animal using a combination of detergent and enzymatic agents. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe for the first time that slow cooling in a DMSO/medium solution and liquid nitrogen leads to reliable long-term storage of decellularised scaffolds.

Abstract (italiano)

RIASSUNTO
INTRODUZIONE: vi sono patologie nelle quali è necessario procedere a sostituzione totale o parziale dell'esofago. Tra di esse vi è l'atresia esofagea, una malattia dello sviluppo esofageo che colpisce 1 ogni 3000-4500 neonati. Tra le terapie emergenti ed innovative per il trattamento delle forme più gravi si propone l'uso di matrici decellularizzate create mediante tecniche di Tissue Engineering. Queste strutture rappresenterebbero la scelta ideale, in quanto esse mantengono le caratteristiche della matrice extracellulare (ECM) dell'esofago nativo, ma sono private della componente cellulare abbattendo il rischio di incompatibilità e di rigetto. Tuttavia va identificata un'adeguata strategia di conservazione delle matrici prodotte in laboratorio per garantirne la preservazione in modo da renderle disponibili e utilizzabili nella pratica clinica.
SCOPI: identificare un metodo efficace di produzione e conservazione delle matrici esofagee decellularizzate in un modello di grande animale.
MATERIALI E METODI: esofagi di coniglio sono stati prelevati e decellularizzati utilizzando un metodo che combina l'uso di detergenti ed enzimi (DET). Le matrici sono state analizzare dopo 2-4 settimane e 3-6 mesi dopo averle conservate in 4 modi differenti: SCM (congelate lentamente in medium e conservate in azoto liquido); SF (snap-frozen in azoto liquido e conservate a -80°C), FD (freeze-dryed e poi conservate a -20°C); 4C (in PBS a 4°C). Analisi di struttura e meccanica sono state effettuate ad ognuno dei 4 time-points.
RISULTATI: abbiamo raggiunto una decellularizzazione efficace con preservazione dell'ECM dopo 2 cicli DET (come mostrato dalla quantificazione del DNA e dall'istologia). Solo il metodo SCM preserva le caratteristiche strutturali e meccaniche della matrice fino all'ultimo time point dei 6 mesi. Gli altri metodi non sono efficaci, in particolar modo SF porta a collasso della micro-architettura tissutale, i campioni in FD non possono essere reidratati dopo 3 o 6 mesi e la conservazione a 4°C porta a progressiva distorsione delle strutture.
DISCUSSIONE: è possibile creare matrici esofagee decellularizzate utilizzando la combinazione di detergenti ed agenti enzimatici. Un metodo di conservazione efficace permette di preservare la matrice esofagea decellularizzata rendendola uno strumento concretamente utilizzabile nella terapia sostitutiva dell'esofago. Abbiamo illustrato come il metodo SCM sia il migliore a tale scopo.

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Tipo di EPrint:Tesi di dottorato
Relatore:De Coppi, Paolo
Dottorato (corsi e scuole):Ciclo 28 > Scuole 28 > MEDICINA DELLO SVILUPPO E SCIENZE DELLA PROGRAMMAZIONE SANITARIA > EMATO-ONCOLOGIA, GENETICA, MALATTIE RARE E MEDICINA PREDITTIVA
Data di deposito della tesi:26 Gennaio 2016
Anno di Pubblicazione:2016
Parole chiave (italiano / inglese):scaffold, decellularisation, tissue engineering
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/20 Chirurgia pediatrica e infantile
Struttura di riferimento:Dipartimenti > Dipartimento di Salute della Donna e del Bambino
Codice ID:9137
Depositato il:20 Ott 2016 10:00
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