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Sgrò, Alberto (2018) Tissue engineering for the surgical tratment of muscle defects: application on animal model of congenital diaphragmatic hernia and skeletal volume muscle loss. [Ph.D. thesis]

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Abstract (english)

Background. Repair of skeletal muscle loss due to trauma, surgical resection or malformations represent a challenge for clinicians. Several attempts to create a bioscaffold to substitute skeletal muscle have been done but no satisfying results were obtained due to lack in regeneration process and functionality of repaired tissue. Some studies on tissue engineering investigated the application of decellularized extracellular matrix (ECM) derived from skeletal muscle observing positive effect towards regeneration. It is becoming relevant the role of tissue-specificity in the field of tissue engineering. This study aims to compare the regenerative effect of both tissue-specific and no tissue-specific scaffolds when applied in a volume of volume muscle loss. Muscle regeneration and macrophagic response are investigated.

Material and Methods. Decellularized extracellular scaffold from murine skin, intestine and rhabdomyosarcoma (ARMS) were obtained using a detergent-enzymatic protocol. Scaffolds’ characteristics were investigated. Wild type mice were used as animal model for in vivo implantation on diaphragm and tibialis anterioris muscles. Samples were obtained at sequential timepoints and analysed with Histology, DNA quantification techniques, Immunofluorescence, Real-time PCR.

Results. Decellularized ECM scaffold were obtained from each tissue. Moreover, their ECM maintained ultrastructure and composition. Implantation in vivo showed a regeneration of new, centre nucleated myofibers when muscle scaffold was used. No significant regeneration was observed with other scaffolds. With muscle implants, macrophagic response was present and characterized by organized distribution of cells.

Conclusions. The decellularization protocol used in this study demonstrated to be effective in maintaining ECM properties even if in absence of cells. Pro-regenerative results obtained only with implantation of muscle-derived scaffolds underline the importance of tissue-specificity in order to obtain the ideal material to repair muscular defects.

Abstract (italian)

Premesse. Il trattamento della perdita di sostanza muscolare dovuta a traumi, resezioni chirurgiche o malformazioni rappresenta ancora una sfida in ambito medico. In passato sono stati creati diversi bioscaffold che potessero sostituire il tessuto muscolare ma i risultati sono stati poco soddisfacenti a causa del mancato stimolo alla rigenerazione tissutale e del mancato recupero funzionale. Alcuni studi hanno hanno esaminato le potenzialità rigenerative di bioscaffold derivati da matrice extracellulare di muscolo scheletrico. In ambito di ingegneria tissutale risulta sempre più importante la specificità tissutale dello scaffold. Questo studio mette a confronto il potenziale rigenerativo di scaffold tessuto-specifici e non in un modello di perdita di sostanza muscolare. In particolare vengono studiati i meccanismi di rigenerazione muscolare e la risposta macrofagica.

Materiali e Metodi. Utilizzando un protocollo di decellularizzazione detergente-enzimatico, sono stati ottenuti da modello murino scaffold di matrice extracellulare di cute, intestino, rabdomiosarcoma. Di tali scaffold sono state studiate le caratteristiche intrinseche. Come modello animale è stato utilizzato il topo wild type. Gli scaffold sono stati impiantati chirurgicamente a livello del diaframma e del muscolo tibiale anteriore. I campioni, prelevati a timepoints diversi, sono stati esaminati con istologia, quantificazione del DNA, Immunofluorescenza, Real-Time PCR.

Risultati. E' stato possibile ottenere scaffold di matrice extracellulare decellularizzata da ciascun tessuto esaminato. La struttura e la composizione della matrice extracellulare è stata preservata nonostante il trattamento di decellularizzazione. L'applicazione in vivo di scaffold derivati da muscolo ha indotto la rigenerazione di nuove fibre muscolari centro-nucleate. L'applicazione in vivo degli scaffold derivati dagli atri tessuti non ha condotto a rigenerazione tissutale. Una volta applicato lo scaffold derivato dal muscolo la risposta macrofagica è stata significativa e caratterizzata da una distribuzione regolare delle cellule.

Conclusioni- Il protocollo di decellularizzazione utilizzato in questo studio è risultato efficace nell'ottenere matrici extracellulari decellularizzate pur preservando le caratteristiche della matrice stessa. Lo stimolo rigenerativo ottenuto solamente mediante impianto di matrice muscolare sottolinea l'importanza della specificità tissutale nell'ottica di ottenere un valido sostituto in caso di danno con perdita di sostanza.

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EPrint type:Ph.D. thesis
Tutor:Gamba, Piergiorgio
Ph.D. course:Ciclo 30 > Corsi 30 > MEDICINA DELLO SVILUPPO E SCIENZE DELLA PROGRAMMAZIONE SANITARIA
Data di deposito della tesi:08 January 2018
Anno di Pubblicazione:08 January 2018
Key Words:Regenerative medicine. Tissue Engineering. Skeletal Muscle. Extracellular Matrix. Volume Muscle Loss. Medicina Rigenerativa. Ingegneria Tissutale. Muscolo scheletrico. Matrice Extracellulare
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:10570
Depositato il:25 Oct 2018 16:58
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