Colorectal cancer (CRC) is the second leading cause of cancer death worldwide according to Global Cancer Statistics in 2018. It is characterized by an extensive intra- and inter- patient heterogeneity, which causes a high therapy failure rate. Therefore, understanding the mechanisms underlying CRC could significantly improve therapies’ outcomes. 2D cancer models, even if currently widely used for cancer research, distinguishes from in vivo at least in protein and gene expression, morphology, proliferation, viability and disposition. 3D in vitro models are more suitable as they can more closely resemble physiological conditions. There are many strategies to create 3D models. Among them, 3D bioprinting is one of the most promising as it can allow researchers to fabricate simplified tissue models as well as more complex scaffolds. In cancer research, Matrigel® is the most used matrix thanks to its peculiar biochemical composition. However, it cannot be easily bioprinted pure due to its complex rheology. Therefore, it is often mixed with other bioinks to tune mechanical and biochemical properties, even if they are not the optimal choice from the biological point of view. To tackle this problem, in our thesis, we have designed a 3D printed coaxial nozzle to fabricate fibres having a cell-encapsulating hydrogel core and an outer supporting layer. The main goal is to maintain cells in the most suitable microenvironment for cell growth, while having a second layer for mechanical support. As a proof-of-concept, we have used both low- and high-viscosity hydrogels for printing the core fibre, while different combinations of alginate and alginate blends have been tested as shell material. Moreover, to improve structural integrity and degradation properties, several crosslinking protocols have been evaluated. Additionally, to better evaluate the extrusion process and the shear stresses created at the nozzle, experimental data were coupled with computational fluid dynamics (CFD) simulations. By looking at the results, we were able to produce complex structures with good coaxiality that can withstand a week in culture conditions. Moreover, the volumetric controlled over the extrusion process makes the system suitable also for Matrigel® bioprinting, which could have a significant impact in developing novel models for cancer research.
Il cancro al Colon retto rappresenta la seconda causa di morte in tutto il mondo. Esso è caratterizzato da una notevole eterogeneità genetica sia inter-paziente che intra-paziente, che determina un’alta incidenza di fallimenti delle terapie. Una maggiore comprensione dei meccanismi alla base di questa patologia permetterebbe di ottenere terapie più efficaci. I modelli in vitro 2D, anche se ancora ampiamente utilizzati nella ricerca contro il cancro, differiscono dalla condizione presente in vivo in termini di espressione proteica e genetica, morfologia, proliferazione, vitalità e organizzazione cellulare. I modelli di coltura 3D sono più adatti in quanto permettono di riprodurre meglio la condizione fisiologica. Tra le diverse strategie di fabbricazione, il 3D Bioprinting risulta uno dei più promettenti in quanto permette di fabbricare modelli di tessuti biologici di complessità strutturale variabile. Nella ricerca contro il cancro, il Matrigel® è la matrice più utilizzata per le colture cellulari grazie alla sua particolare composizione chimica. Tuttavia, è difficilmente utilizzabile allo stato puro come materiale per 3D-Bioprinting a causa delle complesse proprietà reologiche. Per questo motivo viene solitamente miscelato con altri materiali. Per superare questo ostacolo, nella presente tesi è stato progetto un ago coassiale ottenuto con 3D printing al fine di fabbricare fibre uno strato interno adatto per la coltura cellulare, e uno strato guaina per il supporto meccanico. L’obiettivo è permettere la coltura cellulare in condizioni ottimali, relegando al materiale esterno il ruolo di principale supporto meccanico. Per validare il sistema, sono stati impiegati materiali sia a bassa che ad alta viscosità per occupare lo strato interno della fibra, mentre materiali a base di Alginato e suoi blend sono stati testati e ottimizzati come materiale esterno. Inoltre, per ottimizzare le proprietà strutturali dei costrutti e le loro proprietà di degradazione in ambiente fisiologico, sono stati valutati diversi protocolli di reticolazione dell’Alginato. Per analizzare il processo di estrusione e l’insorgenza di sforzi di taglio nell’ago coassiale, i dati sperimentali sono stati accoppiati con simulazioni di fluido dinamica computazionale (CFD). I risultati ottenuti dimostrano come sia possibile ottenere strutture complesse con una buona coassialità, capaci di resistere alla degradazione in condizioni fisiologiche per circa una settimana. Inoltre, il processo di estrusione controllato in volume rende il presente sistema di 3D Bioprinting utilizzabile anche per il bioprinting di Matrigel®, che potrebbe avere portare ad un significativo miglioramento nello sviluppo di nuovi modelli tumorali.
Design of a coaxial nozzle for extrusion bioprinting of large scale constructs for long-term cell cultures
BIANCHI, ROMEO;BASHA, MELISSA
2020/2021
Abstract
Colorectal cancer (CRC) is the second leading cause of cancer death worldwide according to Global Cancer Statistics in 2018. It is characterized by an extensive intra- and inter- patient heterogeneity, which causes a high therapy failure rate. Therefore, understanding the mechanisms underlying CRC could significantly improve therapies’ outcomes. 2D cancer models, even if currently widely used for cancer research, distinguishes from in vivo at least in protein and gene expression, morphology, proliferation, viability and disposition. 3D in vitro models are more suitable as they can more closely resemble physiological conditions. There are many strategies to create 3D models. Among them, 3D bioprinting is one of the most promising as it can allow researchers to fabricate simplified tissue models as well as more complex scaffolds. In cancer research, Matrigel® is the most used matrix thanks to its peculiar biochemical composition. However, it cannot be easily bioprinted pure due to its complex rheology. Therefore, it is often mixed with other bioinks to tune mechanical and biochemical properties, even if they are not the optimal choice from the biological point of view. To tackle this problem, in our thesis, we have designed a 3D printed coaxial nozzle to fabricate fibres having a cell-encapsulating hydrogel core and an outer supporting layer. The main goal is to maintain cells in the most suitable microenvironment for cell growth, while having a second layer for mechanical support. As a proof-of-concept, we have used both low- and high-viscosity hydrogels for printing the core fibre, while different combinations of alginate and alginate blends have been tested as shell material. Moreover, to improve structural integrity and degradation properties, several crosslinking protocols have been evaluated. Additionally, to better evaluate the extrusion process and the shear stresses created at the nozzle, experimental data were coupled with computational fluid dynamics (CFD) simulations. By looking at the results, we were able to produce complex structures with good coaxiality that can withstand a week in culture conditions. Moreover, the volumetric controlled over the extrusion process makes the system suitable also for Matrigel® bioprinting, which could have a significant impact in developing novel models for cancer research.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/186602