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Trkov, Sasa (2009) Micropatterned 3D Hydrogel System to Study Endothelial-Mesenchymal Stem Cell Interactions. [Tesi di dottorato]

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Abstract (inglese)

The creation of functional vascularized tissue-engineered substitutes of clinically relevant size is the current challenge in the field of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell type and optimal conditions for engineered vascular networks have to be determined.

In this study, angiogenic behavior of multiple mesenchymal stem cell (MSC) sources was compared. In addition to bone marrow (BM)-derrived MSCs, two perivascular cell populations from umbilical cord (UC) vein and artery were isolated and identified as MSCs according to their phenotype and differentiation potential. A novel co-culture system was developed to study endothelial cell interactions with MSCs in a spatially controlled three-dimensional (3D) hydrogel model. Using a microfluidic based patterning technique it was possible to localize endothelial cells (HUVECs) and MSCs in parallel channels at varying relative distances (500, 1000 and 2000 µm) within a 3D fibrin hydrogel. The cell angiogenic behavior and intracellular interactions were observed in terms of directed migration of MSCs toward HUVECs and the creation of stable vascular network.

The monitoring and quantification of MSC sprouting toward HUVECs revealed different distance related response between the different cell sources. BM-MSCs demonstrated a strong distance-dependent sprouting, implying that the closest (500 µm) initial distance from HUVECs resulted in the highest migration. In contrast, UC vein derived MSCs migrated independently of the initial distance from HUVECs, while UC artery MSCs showed moderate dependence. In addition, it was shown that BM-MSCs greatly supported the formation of stable vascular network after two weeks of culture. The created network was composed of long, highly branched, interconnected tube-like structures, which were co-aligned with SMA-positive cells. This organization was not observed in the single cultures of BM-MSCs or HUVECs. In contrast to BM-MSCs, the other two MSC types did not exert any stabilizing effect on tubular structures.

The presented system is a novel approach that provides a simple and robust model of cell-cell communication based on varying distances without using complicated and expensive equipment. It can be used in a variety of applications and can assess important interactions between the different cell types.

Abstract (italiano)

Uno dei principali obiettivi della ricerca nel campo dell’ingegneria dei tessuti è la pre-vascolarizzazione dei tessuti ingegnerizzati, in quanto il trasporto dei nutrienti alle cellule è essenziale per sostenere la loro funzione in vivo. Nonostante sia provato che le cellule endoteliali insieme con le cellule del tessuto connettivo circondante sono i costituenti integrali per la formazione dei vasi sanguigni stabili, la sorgente cellulare più appropriata e le condizioni di cultura ottimali per ottenere reti vascolari ingegnerizzate devono ancora essere determinati. Sono stati individuati diversi tipi di cellule che possono costituire le pareti dei vasi sanguigni. In questo studio sono state utilizzate cellule staminali mesenchimali isolate da sorgenti diverse, come dalla vena e dall’arteria ombelicale, e sono state paragonate con quelle isolate dal midollo osseo. Le cellule isolate dalla vena e dall’arteria ombelicale hanno un fenotipo cellulare simile a quello delle cellule mesenchimali staminali derivate da midollo osseo e hanno il potenziale di differenziare in tessuto adiposo e osseo.

Nel nostro studio presentiamo un modello innovativo, basato sulla tecnologia del “micropattering”, ideale per studiare la co-cultura di cellule endoteliali e cellule mesenchimali derivanti da sorgenti diverse. La microtecnologia ha permesso di avere il controllo preciso del microambiente cellulare, sfruttando la possibilità di poter localizzare le cellule mesenchimali nei canali a varie distanze dalle cellule endoteliali dentro il sistema tridimensionale del gel di fibrina. Le distanze tra i canali erano 500, 1000 e 2000 µm. La ramificazione di cellule derivanti da sorgenti diverse è stata monitorizzata entro i primi tre giorni, a partire dall’incapsulazione, per dimostrare l’eventuale effetto chemotattico esistente tra i due diversi tipi cellulari con il variare della distanza tra di loro. La lunghezza dei singoli prolungamenti cellulari è stata misurata come la distanza di migrazione di cellule mesenchimali staminali verso le cellule endoteliali. I risultati ottenuti dimostrano che le cellule mesenchimali da midollo osseo e dalla arteria ombelicale rispondono in modo dipendente dalla distanza, quindi più piccola era la distanza tra le due sorgenti cellulari, maggiore era la migrazione delle cellule; mentre le cellule derivanti dalla vena ombelicale hanno migrato verso le cellule endoteliali in modo indipendente dalla distanza che c’era tra di loro.

Inoltre, le cellule endoteliali incapsulate nel gel tridimensionale sono capaci di creare delle reti vascolari formate da strutture tubolari, che sono stabilizzate in presenza delle cellule mesenchimali. Nel nostro studio, la formazione delle reti vascolari è dipendente dal sorgente di cellule staminali mesenchimali. Dopo due settimane di coltura, le cellule staminali mesenchimali dal midollo osseo hanno dimostrato la maggiore stabilizzazione delle reti vascolari, promuovendo strutture tubolari ben definite, lunghe e ramificate. Le cellule derivate dalla vena e dall’arteria ombelicale, invece, non hanno avuto lo stesso effetto sulle cellule endoteliali.

Il modello qui presentato è un approccio innovativo di studiare le comunicazioni tra diversi tipi cellulari, a varie distanze, senza l’uso di un’attrezzatura complicata e costosa. Questo stesso modello potrebbe essere usato in altre numerose applicazioni e ci permette di studiare le interazioni importanti tra tipi cellulari differenti variando delle condizioni del loro microambiente.

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Tipo di EPrint:Tesi di dottorato
Relatore:Parnigotto, Pier Paolo
Correlatore:Di Liddo , Rosa - Vunjak-Novakovic, Gordana
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > INGEGNERIA DEI TESSUTI E DEI TRAPIANTI
Data di deposito della tesi:02 Febbraio 2009
Anno di Pubblicazione:02 Febbraio 2009
Parole chiave (italiano / inglese):Mesenchymal stem cells, angiogenesis, micropatterning, 3D hydrogel model
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/13 Biologia applicata
Area 05 - Scienze biologiche > BIO/12 Biochimica clinica e biologia molecolare clinica
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Farmaceutiche
Codice ID:1919
Depositato il:02 Feb 2009
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