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Finotti, Luca (2014) Progettazione e sviluppo di scaffolds naturali per la rigenerazione ossea. [Tesi di dottorato]

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Abstract (inglese)

For many years, the regenerative medicine has been using the biometarials like grafts to promote healing of bone tissue.
A heterologous graft should not evoke any inflammatory response where it is inserted and its antigenicity is eliminated with a decellularization technique .
The aim of this study was to provide a protocol creating a bovine xenograft which easily employed in medicine field for repairing bone defects. For this purpose, the decellularization protocols were applied on 26 rats' femur by means of freezing /disfreezing. The effect of the decellularization in vitro was tested by means of MTT and the light microscope (SEM).
The bovine xenograft obtained was inserted surgically in maxillary sinus of 30 sheeps to test the ability of regeneration of biomaterial in vivo.
The surgical intervention was carried out bilaterally: one side was experimental side (xenograft) and the other side was control side ( Bio-oss®,Geistlich Pharma AG Wohlhusen, Switzerland).
The study subjects were divided in 3 groups .Each group was consisting of 10 animals. They were euthanazied on 15, 30, 60 days from the surgical intervention, respectively.
Collected bone samples were decalcified, fixed and colored with hematoxylin-eosin and subsequently, they were evaluated under optical microscope.
The MTT tests showed that the duration of freezing at -20°C for 7 days and e -80°C for another 7 days, followed by disfreezing through thermal shock was more efficient for correct decellularization of the bovine extracellular matrix.
The RT-PCR results indicate that the bovine xenograft has the optimum osteoinductive competence.
In vivo tests showed that the biometarial already present for 15 days, has promoted the capacity of osteoconductive and osteoinductive with absence of inflammatory response.
The “gold standard” in medicine manifests the excellent osteoconductive and osteoinductive capacity at 60 days.
In conclusion, it can be confirmed that the protocol of decellularization by means of freezing /disfreezing, allows producing an ideal bone replacement for regeneration of bone defects in medicine field.

Abstract (italiano)

La medicina rigenerativa impiega ormai da molti anni biomateriali in grado di promuovere ed accelerare la guarigione del tessuto osseo in cui vengono innestati.
Un innesto osseo eterologo non dovrebbe evocare nessuna risposta infiammatoria nel sito in cui viene inserito. L’antigenicità del tessuto osseo eterologo viene eliminata grazie a tecniche di decellularizzazione ed il protocollo di decellularizzazione impiegato influisce in modo determinante sulle caratteristiche del biomateriale stesso.
L'obiettivo di questo studio è stato quello di mettere a punto un protocollo per la creazione di uno xenograft bovino di facile impiego in campo medico per la riparazione di difetti ossei.
A questo scopo, sono stati applicati protocolli di decellularizzazione mediante congelamento/scongelamento su 26 femori murini; l'effettiva decellularizzazione in vitro è stata valuta tramite test MTT e mediante microscopia elettronica (SEM).
Il protocollo di decellularizzazione più efficace è stato applicato in un secondo momento a tessuto osseo bovino.
Successivamente sono state validate in vitro le capacità osteoinduttive dello xenograft bovino mediante l'impiego di cellule staminali.
Lo xenograft bovino così ottenuto è stato innestato chirurgicamente nel seno mascellare di 30 pecore per testare in vivo le capacità rigenerative del biomateriale. L'intervento chirurgico è stato eseguito bilateralmente: un lato test (xenograft) e un lato controllo (Bio-oss®,Geistlich Pharma AG Wohlhusen, Switzerland).
I soggetti sono stati suddivisi in 3 gruppi da 10 animali ciascuno ed eutanasizzati a 15, 30 e 60 giorni dall’intervento chirurgico.
I campioni d’osso prelevati sono stati decalcificati, fissati, colorati con ematossilina ed eosina e successivamente valutati al microscopio ottico.
I test MTT hanno dimostrato che i tempi di congelamento a -20°C per 7 giorni e -80°C per altri 7 giorni, seguite da scongelamento tramite shock termico, sono più efficaci per una corretta decellularizzazione della matrice extracellulare bovina.
I risultati della RT-PCR indicano che lo xenograft bovino ha ottime capacità osteoinduttive.
I test in vivo hanno dimostrato che il biomateriale già a 15 giorni presenta: elevata capacità osteoconduttiva ed osteoinduttiva; assenza di risposta infiammatoria. A 30 e 60 giorni queste caratteristiche non subiscono variazioni significative.
Il gold standard in campo medico presenta ottime capacità osteoconduttive ed osteoinduttive a 60 giorni.
In conclusione si può affermare che il protocollo di decellularizzazione mediante congelamento/scongelamento da noi sviluppato consente di produrre un sostituto osseo ideale per la rigenerazione di difetti ossei in campo medico; il biomateriale testato promuove la neoangiogenesi e l'osteogenesi a soli 15 giorni dall'innesto.

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Tipo di EPrint:Tesi di dottorato
Relatore:Isola, Maurizio
Dottorato (corsi e scuole):Ciclo 25 > Scuole 25 > SCIENZE VETERINARIE > SCIENZE CLINICHE VETERINARIE
Data di deposito della tesi:28 Gennaio 2014
Anno di Pubblicazione:28 Gennaio 2014
Parole chiave (italiano / inglese):Decellularizzazione/Decellularization, Scaffold osseo/Bone scaffold, Xenograft bovino/Bovine xenograft, Pecora/Sheep, Cellule staminali/Stem cells
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > VET/09 Clinica chirurgica veterinaria
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Animale, Produzioni e Salute
Codice ID:6486
Depositato il:13 Nov 2014 11:05
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