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Danesin, Roberta (2011) Cellule staminali e matrici biomimetiche nanostrutturate per la medicina rigenerativa. [Tesi di dottorato]

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Abstract (inglese)

The aim of the first part of this research project was to improve the knowledge about the existence and location of adult stem cells in the adrenal gland.
Although some experiments might suggest that undifferentiated cells derived from the external periphery of cortical zone, the origin of the regenerating cells remains ambiguous, and there are not currently known surface markers for defining these cells.
Two distinct cellular populations, from the capsular zone and from the inner part of the adrenal gland, were isolated and investigated in vitro in order to study their phenotype, their proliferative potential and plasticity.
The second research project aims to design scaffolds for bone tissue engineering.
The development of novel scaffolds for bone tissue engineering is very complex, because ideal support for cellular colonization would possess the same structural and functional characteristics of ECM.
In this work, the creation of hybrid scaffolds, mixing a very well known biocompatible synthetic polymer, poly(ε-caprolactones), with different self-assembling peptides, is presented.
The microfibrous structure of the scaffold is assured by the electrospinning process, whereas the nanofibrous structure is produces by the self-assembling process of peptides.
We prepared six different scaffolds adding six different peptides to poly(ε-caprolactones). These peptides were synthesized by solid phase strategy, and one sequence was prepared condensing a RGD motif to a self-assembling sequence.
The characterization of the different scaffolds was carried out through SEM morphological analysis, FT-IR spectroscopy and contact angle measurements.
All prepared scaffold exhibit interwoven nanofibers comparable to the ECM ones.
FTIR investigations showed that self-assembling peptides incorporated in the PCL nanofibers retain the -sheet conformation, and that an incubation with saline buffered solution can increase the percentage of this structure in the RGD containing peptide.
The enrichment with peptides improves the wettability of the polymer scaffold adding an important requirement for better cellular adhesion.
The biological assay showed that the presence of self-assembling peptides into the scaffold increases cellular adhesion, the calcium amount and the gene expression of some proteins important for osteoblast.

Abstract (italiano)

Premessa
Il lavoro di ricerca svolto nel triennio di dottorato si è focalizzato su due progetti.
Il primo ha riguardato l’individuazione di cellule staminali nel surrene di ratto, al fine di poterle isolare e caratterizzare.
Nel secondo progetto ci si è occupati della progettazione e realizzazione di matrici biomimetiche nanofibrose per la rigenerazione del tessuto osseo.

RIASSUNTO
Il primo progetto di ricerca ha cercato di chiarire le conoscenze attuali riguardo l’esistenza di cellule staminali adulte residenti nel tessuto surrenale. Sebbene alcune evidenze sperimentali suggeriscano l’esistenza di tali cellule nella parte esterna della corticale surrenale, al riguardo non c’è ancora una teoria riconosciuta in modo unanime: non è stata identificata la loro zona d’origine, né tantomeno sono stati individuati dei marker caratteristici che permettano di isolarle.
L’individuazione di tali cellule potrebbe trovare applicazione nella cura di malattie surrenali, ad esempio nell’ipocorticosurrenalismo permetterebbe di evitare la terapia ormonale a vita e quindi, rappresenterebbe la terapia d’elezione.
Il progetto si è quindi prefisso come obiettivi di identificare e isolare una popolazione di cellule staminali all’interno della ghiandola surrenale di ratto.
Tramite analisi immunoistochimica e di immunofluorescenza sono state ricercate: a) zone cellulari BrdU+ (ratti trattati alla nascita con BrdU), a ciclo cellulare lento; b) marker di staminalità CD105, CD90 e c-kit.
Dai surreni di ratto sono state estratte due sottopopolazioni di cellule, capsulari e della parte interna: queste sono state coltivate in vitro, osservate e immunoseparate per CD105 e CD90. Infine, è stata testata la capacità differenziativa delle colture sia in senso osteogenico che adipogenico.

Nel secondo progetto sono stati ideati e prodotti scaffold da impiegare nell’ambito dell’ingegneria del tessuto osseo. La progettazione di uno scaffold per l’ingegneria tessutale risulta essere alquanto complessa visto che un supporto ideale per la colonizzazione cellulare dovrebbe possedere le caratteristiche, strutturali e funzionali, della matrice extracellulare. Lo scaffold ideato ha natura ibrida essendo composto di un polimero di sintesi biodegradabile quale il poli(ε-caprolattone) e di peptidi auto-assemblanti. La struttura fibrosa dello scaffold su scala micrometrica è stata assicurata mediante un processo di elettrofilatura. D’altro canto, è noto che i peptidi auto-assemblanti formano spontaneamente matrici tridimensionali con fibre nanometriche estremamente gradite a differenti tipi di cellule, incluse quelle del tessuto osseo. Sono stati ottenuti sei differenti tipi di scaffold utilizzando sei diversi peptidi, ottenuti per sintesi su fase solida, tra i quali una sequenza auto-assemblante coniugata ad un motivo adesivo RGD. Le matrici sono state estesamente caratterizzate mediante analisi di spettroscopia elettronica a scansione, spettroscopia all’infrarosso in trasformata di Fourier e valutazione dell’angolo di contatto. Le matrici risultano essere composte da un intreccio di fibre di dimensioni comparabili a quelle della matrice extracellulare; inoltre, è stato possibile confermare che la struttura β-sheet, che è alla base dell’auto-aggregazione, è presente nelle matrici e viene incrementata, nel caso del peptide auto-assemblante con motivo RGD, dal pre-trattamento con soluzione salina. E’ stato inoltre dimostrato come l’arricchimento con piccole percentuali (5%) di peptidi dello scaffold in poli(ε-caprolattone) produca un grado di bagnabilità notevolmente superiore e quindi crei i presupposti per una maggior colonizzazione della matrice da parte delle cellule. I saggi biologici, eseguiti su tutti gli scaffold, hanno permesso di dimostrare che la presenza di sequenze auto-assemblanti incrementa in modo significativo l’adesione cellulare, la produzione di calcio e l’espressione di geni che codificano per proteine importanti per gli osteoblasti.

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Tipo di EPrint:Tesi di dottorato
Relatore:Conconi, Maria Teresa
Correlatore:Dettin, Monica
Dottorato (corsi e scuole):Ciclo 23 > Scuole per il 23simo ciclo > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > INGEGNERIA DEI TESSUTI E DEI TRAPIANTI
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:31 Gennaio 2011
Parole chiave (italiano / inglese):adrenal gland, stem cells, self-assembling peptides, electrospinning, scaffold, osteoblast, poly-caprolactones
Settori scientifico-disciplinari MIUR:Area 03 - Scienze chimiche > CHIM/06 Chimica organica
Area 05 - Scienze biologiche > BIO/16 Anatomia umana
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Farmaceutiche
Codice ID:3373
Depositato il:19 Lug 2011 17:15
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