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Sfriso, Maria Martina (2014) Sviluppo di scaffolds biologici da omento e muscolo scheletrico mediante processi di decellularizzazione. [Ph.D. thesis]

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Abstract (english)

ABSTRACT
BACKGROUND
The availability of organs and tissues, obtained in laboratory from biological material, is one of the main demands in the event of loss of substance, due to congenital or post-traumatic defects. For this purpose, bioengineered tissues have been developed through decellularization and recellularization processes.

AIM OF THE STUDY
The objectives of this study were:
• Development of a new type of biological scaffold derived from omentum that preserves the structural integrity of the extracellular matrix and the vascular network of the native tissue even after a phase of decellularization.
• Development of a further biological scaffold from skeletal muscle, derived from the decellularization of the rectus muscle of the rabbit, for the reconstruction of a defect in the abdominal wall.

MATERIAL AND METHODS
As regards the first part of the experiment, samples of omentum, from rat and human, were decellularized through physical reactions (freezing/thawing and mechanical agitation), chemical (EDTA and isopropanol) and enzymatic (trypsin, lipases and endonucleases) methods involving the removal of cells and their lipid content.
Samples were analyzed at various stages by histological staining (hematoxylin and eosin, azan Mallory, van Gieson, PAS, Sudan, Oil Red), immunohistochemical reactions (anti- CD31, -CD34, α-smooth muscle actin) and stained with DAPI. Furthermore, evaluations about the quantitative concentration of genomic DNA at the end of decellularization were carried out with a spectrophotometer.
Attempts have been made to recellularize scaffold by cells of the stromal vascular fraction (SVF), isolated from samples of human lipoaspirate.
It has, also, developed an additional scaffolds by decellularization of samples of the rectus muscle of the rabbit. Samples were subjected to physical (freezing/thawing), chemical (EDTA and Triton X -100) and enzymatic (DNase) methods and results were analyzed by histological staining (hematoxylin and eosin, azan Mallory, van Gieson).
Thereafter, the scaffold was implanted in a rabbit receiver in correspondence with a surgical defect in the abdominal wall with loss of substance. After three weeks, the implant was studied by ultrasound examination in vivo and, after sacrifice of the animal, it was analyzed with the histological stainings mentioned above.

RESULTS
Histological stainings confirmed the effectiveness of the decellularization process with the total loss of its lipid component of the omentum. This led to the creation of an acellular scaffold in which the three-dimensional organization of the collagen, elastic, and reticular fibers was preserved. Also, the preservation of the vascular network was highlighted.
Preliminary studies have shown, moreover, the possibility of recellularization of the scaffold through the introduction of SVF cells.
Regarding the second part of the project, it was shown that the muscle-derived scaffold has maintained the integrity of the extracellular matrix and the structure of the vascular channels, in the removal of cellular elements.
Following implantation in the receiving rabbit, a solution of continuity with the adjacent tissues has been highlighted, by ultrasound in vivo, with the absence of herniation .
After the sacrifice of the animal, histological analysis of the scaffold showed the presence of reparative tissue and new vascular channels in the seat of the abdominal defect.
Future perspectives will be to recellularize the scaffold with progenitor cells to promote muscle regeneration.

CONCLUSIONS
We obtained a new type of acellular biological structure resulting from the omentum, through the application of the process of decellularization, which allows to minimize possible alterations of the extracellular matrix during the removal of the cells.
The scaffold was preliminarily recellularized with cells of the stromal vascular fraction, but further experiments will focus on the further evolution of in vitro recellularization and in vivo implantation.
The decellularization of the muscle tissue and its implantation in vivo has allowed to identify a biological structure capable of offering a valid alternative to the materials currently used for the defects repair in the abdominal wall.
Also in this direction we will evaluate in the future the possibility of recellularization of the scaffold with progenitor cells.

Abstract (italian)

RIASSUNTO
PRESUPPOSTI DELLO STUDIO
La disponibilità di organi e tessuti ottenuti in laboratorio da materiale biologico è, ad oggi, una delle maggiori richieste nei casi di perdita di sostanza a causa di difetti congeniti o post-traumatici. A fronte di ciò, si è cercato di realizzare dei tessuti bioingegnerizzati mediante l’applicazione di processi di decellularizzazione e successiva ricellularizzazione.

SCOPO DELLO STUDIO
Gli obiettivi del presente lavoro sono stati i seguenti:
• Sviluppare un nuovo tipo di scaffold biologico derivato dall'omento che conservi l’integrità strutturale della matrice extracellulare e della rete vascolare del tessuto nativo anche dopo una fase di decellularizzazione.
• Sviluppare un ulteriore scaffold biologico da muscolo scheletrico, derivato dalla decellularizzazione del muscolo retto del coniglio, per la ricostruzione di un difetto della parete addominale.

MATERIALI E METODI
Per quanto riguarda la prima parte sperimentale, campioni di omento, di ratto e di uomo, sono stati decellularizzati mediante l'impiego di una serie di reazioni fisiche (congelamento/scongelamento ed agitazione meccanica), chimiche (EDTA e isopropanolo) ed enzimatiche (tripsina, endonucleasi e lipasi) che comportano la rimozione delle cellule e della loro componente lipidica. I campioni sono stati analizzati nelle varie fasi mediante colorazioni istologiche (ematossilina ed eosina, azan Mallory, van Gieson, PAS, Sudan, Oil Red), reazioni immunoistochimiche (anti-CD31, -CD34, -α actina del muscolo liscio) e colorazione con DAPI. Inoltre, sono state effettuate valutazioni quantitative allo spettrofotometro della concentrazione di DNA genomico al termine della decellularizzazione.
Sono stati effettuati tentativi di ricellularizzazione dello scaffold mediante cellule della frazione stromale vascolare (SVF), isolate da campioni di lipoaspirato umano.
È stato altresì sviluppato un ulteriore scaffold mediante decellularizzazione di campioni del muscolo retto di coniglio. Questi sono stati sottoposti a reazioni fisiche (congelamento/scongelamento), chimiche (EDTA e Triton X-100) ed enzimatiche (DNasi) ed i risultati sono stati analizzati mediante colorazioni istologiche (ematossilina ed eosina, azan Mallory, van Gieson).
In seguito, lo scaffold è stato impiantato a tutto spessore in un coniglio ricevente in corrispondenza di un difetto della parete addominale con perdita di sostanza prodotto chirurgicamente. Dopo tre settimane, l’impianto è stato studiato mediante esame ecografico in vivo e, dopo sacrificio dell’animale, è stato analizzato mediante le colorazioni istologiche di cui sopra.

RISULTATI
Le colorazioni istologiche hanno confermato l'efficacia del procedimento di decellularizzazione con la perdita totale della componente lipidica propria dell'omento. Ciò ha portato alla realizzazione di uno scaffold acellulare nel quale è stata mantenuta l’organizzazione tridimensionale delle fibre collagene, elastiche e reticolari. Inoltre è stata evidenziata la preservazione della rete di canali vascolari.
Studi preliminari hanno dimostrato, inoltre, la possibilità di ricellularizzare lo scaffold mediante introduzione di cellule appartenente alla SVF.
Riguardo la seconda parte del progetto, è stato dimostrato che lo scaffold di derivazione muscolare ha mantenuto l'integrità della matrice extracellulare e della struttura dei canali vascolari, di cui rimangono apprezzabili i diversi strati che li compongono.
A seguito dell’impianto nel coniglio ricevente è stata evidenziata, mediante ecografia in vivo, l’integrazione dello scaffold con i tessuti adiacenti con buone proprietà meccaniche.
Dopo il sacrificio dell’animale l’analisi istologica dello scaffold ha delineato la presenza di tessuto riparativo e di nuovi canali vascolari nella sede del difetto addominale. Obiettivo futuro sarà quello di ricellularizzare lo scaffold con cellule progenitrici muscolari per favorire la rigenerazione in senso muscolare dello scaffold stesso.

CONCLUSIONI
Abbiamo ottenuto un nuovo tipo di struttura acellulare biologica derivante dall'omento, mediante l'applicazione del processo di decellularizzazione, il quale permette di minimizzare il più possibile le alterazioni della matrice extracellulare durante la rimozione delle cellule. In via preliminare lo scaffold è stato ricellularizzato con cellule appartenenti alla frazione stromale vascolare ma ulteriori esperimenti si focalizzeranno sul potenziamento della ricellularizzazione in vitro e su impianto in vivo.
La decellularizzazione del tessuto muscolare ed il suo impianto in vivo ha permesso di identificare una struttura biologica in grado di offrire una valida alternativa ai materiali attualmente impiegati per la riparazione dei difetti di parete addominale. Anche in questa direzione verrà valutata in futuro la possibilità di ricellularizzare lo scaffold con cellule progenitrici.

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EPrint type:Ph.D. thesis
Tutor:De Caro, Raffaele
Ph.D. course:Ciclo 26 > Scuole 26 > BIOMEDICINA
Data di deposito della tesi:29 January 2014
Anno di Pubblicazione:29 January 2014
Key Words:decellularization, scaffolds
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/16 Anatomia umana
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Molecolare
Codice ID:6574
Depositato il:04 Nov 2014 13:51
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