Cardiovascular diseases, responsible for approximately 18 million annual deaths worldwide, stand as the foremost cause of mortality. Within this category, aortic aneurysms and aortic syndromes, like aortic dissection, significantly impact major human blood vessels. Despite extensive research efforts in the cardiovascular field, therapeutic options for vascular disease remain limited. Since the 1970s, surgical interventions have predominantly relied on synthetic vascular grafts composed of materials like polyethylene- terephthalate (PET or Dacron®), polytetrafluoroethylene (PTFE), or expanded polytetrafluoroethylene (ePTFE). These synthetic grafts exhibit distinct mechanical properties compared to natural vessels, leading to compliance mismatches and non-physiological stresses at suture lines, resulting in complications like pseudo- aneurysms and intimal hyperplasia. Furthermore, their hydrophobic nature impedes the recreation of the necessary aqueous cellular environment and necessitates anticoagulant therapy due to inherent thrombogenicity. Although homografts were once considered an alternative, they were mostly abandoned due to issues such as immunogenicity, preservation challenges, delayed degradation, and aneurysm formation. However, they have been reintroduced in specific clinical situations, particularly in cases of aortic graft infection. In 1986, Weinberg and Bell introduced the concept of tissue- engineered blood vessels as an alternative to synthetic grafts, paving the way for innovative fabrication techniques such as electrospinning, cell-sheet engineering, tubular mold casting, and decellularization of xenogeneic vessels. These approaches entail complex, multi-step processes often involving manual cell seeding, resulting in uneven cell distribution within the construct. A breakthrough in this field is 3D bioprinting, which allows for the creation of engineered vascular grafts in a single step by extruding hydrogels embedded with cells, providing precise, reproducible, and controlled layer-by-layer deposition of different cell types. Various bioinks, including those based on natural materials like agarose, fibrinogen, xanthan gum, alginate, and gelatin, offer a conducive aqueous matrix to support regenerative approaches that are not possible with PET or PTFE grafts. Functionalized hydrogels and synthetic biomaterials like GelMA, PEGDA, and Pluronic F127 have also been widely used, ensuring low inter-batch variability and high reproducibility. However, the key challenge lies in creating a self-supporting, tubular hierarchical structure matching the dimensions of native large vessels. To address this, different materials have been explored as support structures in the bioprinting process, involving strategies such as using a support bath, synthetic materials to support cell-laden hydrogels, and rotating mandrel-aided bioprinting. Nevertheless, these methods still entail multi-step processes, including support material removal that can impact structural stability and cell viability. The objective of this study is to bioprint a self-supporting 3D multi-layered construct that replicates the hierarchical and chemical structure and functionality of the native vascular extracellular matrix (ECM). For this purpose, it has been developed a bioink comprised of natural polymers, enriched with decellularized extracellular matrix (dECM), obtained from aorta of 6-months pigs, to recreate the extracellular environment. The inclusion of biochemical signals from ECM components is expected to play a pivotal role in promoting cell proliferation while reducing the risk of thrombosis and inflammation. The formulation of this novel dECM-based bioink is optimized for extrusion-based 3D bioprinting through both a priori and post-printing assessments. Additionally, an ad hoc crosslinking process for alginate is developed to enhance self- stability and shape fidelity of the 3D bioprinted construct. This bioink is designed to facilitate the culture, in bioreactor specifically designed and developed, of the bioprinted construct for several weeks while preserving support for embedded cells.
Le malattie cardiovascolari, responsabili di circa 18 milioni di morti annuali nel mondo, rappresentano la principale causa di mortalità. All'interno di questa categoria, gli aneurismi aortici e le sindromi aortiche, come la dissezione aortica, hanno un impatto significativo sui principali vasi sanguigni. Nonostante gli sforzi di ricerca estensivi nel campo cardiovascolare, le opzioni terapeutiche per le malattie vascolari rimangono limitate. Dal 1970, le procedure chirurgiche hanno prevalentemente fatto affidamento su innesti vascolari sintetici composti da materiali come polietilentereftalato (PET o Dacron®), politetrafluoroetilene (PTFE) o politetrafluoroetilene espanso (ePTFE). Questi innesti sintetici presentano proprietà meccaniche distinte rispetto ai vasi sanguigni naturali, portando a discrepanze di compliance e stress non fisiologici alle linee di sutura, con complicanze come pseudoaneurismi e iperplasia intima. Inoltre, la loro natura idrofobica impedisce la ricreazione dell'ambiente cellulare acquoso necessario e rende necessaria la terapia anticoagulante a causa della trombogenicità intrinseca. Anche se gli omografti sono stati considerati un'alternativa, sono stati in gran parte abbandonati a causa di problemi come l'immunogenicità, le sfide di conservazione, la degradazione ritardata e la formazione di aneurismi. Tuttavia, sono stati reintrodotti in specifiche situazioni cliniche, in particolare nei casi di infezione degli innesti aortici. Nel 1986, Weinberg e Bell introdussero il concetto di vasi sanguigni ingegnerizzati come alternativa agli innesti sintetici, aprendo la strada a tecniche innovative di fabbricazione come l'elettrofilatura, l'ingegneria di fogli cellulari, la colata di stampi tubolari e la decellularizzazione dei vasi xenogenici. Questi approcci comportano processi complessi e multistep spesso che coinvolgono la semina manuale delle cellule, con una distribuzione cellulare non uniforme all'interno del costrutto. Una svolta in questo campo è la biostampa 3D, che consente la creazione di innesti vascolari ingegnerizzati in un unico passaggio attraverso l'estrusione di idrogeli incorporati con cellule, fornendo una deposizione precisa, riproducibile e controllata strato per strato di diversi tipi cellulari. Vari bioinchiostri, compresi quelli basati su materiali naturali come agarosio, fibrinogeno, gomma di xanthan, alginato e gelatina, offrono una matrice acquosa favorevole per supportare approcci rigenerativi che non sono possibili con gli innesti PET o PTFE. Idrogeli funzionalizzati e biomateriali sintetici come GelMA, PEGDA e Pluronic F127 sono stati ampiamente utilizzati, garantendo una bassa variabilità intra-batch e una elevata riproducibilità. Tuttavia, la sfida principale sta nel creare una struttura autoportante e gerarchica tubolare che corrisponda alle dimensioni dei vasi sanguigni grandi nativi. Per affrontare questo problema, sono stati esplorati diversi materiali come strutture di supporto nel processo di biostampa, coinvolgendo strategie come l'uso di un bagno di supporto, materiali sintetici per supportare gli idrogeli caricati di cellule e la biostampa assistita da mandrini rotanti. Tuttavia, questi metodi comportano comunque processi multistep, inclusa la rimozione del materiale di supporto che può influenzare la stabilità strutturale e la vitalità cellulare. L'obiettivo di questo studio è biostampare un costrutto 3D autoportante e multistrato che replichi la struttura gerarchica e chimica e la funzionalità della matrice extracellulare vascolare nativa (ECM). A questo scopo, è stato sviluppato un bioinchiostro composto da polimeri naturali, arricchito con matrice extracellulare decellularizzata (dECM), ottenuta dall'aorta di maiali di 6 mesi, per ricreare l'ambiente extracellulare. L'inclusione di segnali biochimici dai componenti della ECM gioca un ruolo fondamentale nella promozione della proliferazione cellulare riducendo il rischio di trombosi e infiammazione. La formulazione di questo nuovo bioinchiostro basato su dECM è ottimizzata per la biostampa 3D basata sull'estrusione attraverso valutazioni sia a priori che a posteriori della stampa. Inoltre, è stato sviluppato un processo di reticolazione ad hoc per l'alginato per migliorare l'autostabilità e la fedeltà di forma del costrutto biostampato 3D. Questo bioinchiostro è progettato per facilitare la coltura, in un bioreattore specificamente progettato e sviluppato, del costrutto biostampato per diverse settimane preservando il supporto per le cellule incorporate.
Biofabrication of a self-supporting multi-layered 3D bioprinted construct to regenerate biologically functional tissues
POTERE, FEDERICA
2023/2024
Abstract
Cardiovascular diseases, responsible for approximately 18 million annual deaths worldwide, stand as the foremost cause of mortality. Within this category, aortic aneurysms and aortic syndromes, like aortic dissection, significantly impact major human blood vessels. Despite extensive research efforts in the cardiovascular field, therapeutic options for vascular disease remain limited. Since the 1970s, surgical interventions have predominantly relied on synthetic vascular grafts composed of materials like polyethylene- terephthalate (PET or Dacron®), polytetrafluoroethylene (PTFE), or expanded polytetrafluoroethylene (ePTFE). These synthetic grafts exhibit distinct mechanical properties compared to natural vessels, leading to compliance mismatches and non-physiological stresses at suture lines, resulting in complications like pseudo- aneurysms and intimal hyperplasia. Furthermore, their hydrophobic nature impedes the recreation of the necessary aqueous cellular environment and necessitates anticoagulant therapy due to inherent thrombogenicity. Although homografts were once considered an alternative, they were mostly abandoned due to issues such as immunogenicity, preservation challenges, delayed degradation, and aneurysm formation. However, they have been reintroduced in specific clinical situations, particularly in cases of aortic graft infection. In 1986, Weinberg and Bell introduced the concept of tissue- engineered blood vessels as an alternative to synthetic grafts, paving the way for innovative fabrication techniques such as electrospinning, cell-sheet engineering, tubular mold casting, and decellularization of xenogeneic vessels. These approaches entail complex, multi-step processes often involving manual cell seeding, resulting in uneven cell distribution within the construct. A breakthrough in this field is 3D bioprinting, which allows for the creation of engineered vascular grafts in a single step by extruding hydrogels embedded with cells, providing precise, reproducible, and controlled layer-by-layer deposition of different cell types. Various bioinks, including those based on natural materials like agarose, fibrinogen, xanthan gum, alginate, and gelatin, offer a conducive aqueous matrix to support regenerative approaches that are not possible with PET or PTFE grafts. Functionalized hydrogels and synthetic biomaterials like GelMA, PEGDA, and Pluronic F127 have also been widely used, ensuring low inter-batch variability and high reproducibility. However, the key challenge lies in creating a self-supporting, tubular hierarchical structure matching the dimensions of native large vessels. To address this, different materials have been explored as support structures in the bioprinting process, involving strategies such as using a support bath, synthetic materials to support cell-laden hydrogels, and rotating mandrel-aided bioprinting. Nevertheless, these methods still entail multi-step processes, including support material removal that can impact structural stability and cell viability. The objective of this study is to bioprint a self-supporting 3D multi-layered construct that replicates the hierarchical and chemical structure and functionality of the native vascular extracellular matrix (ECM). For this purpose, it has been developed a bioink comprised of natural polymers, enriched with decellularized extracellular matrix (dECM), obtained from aorta of 6-months pigs, to recreate the extracellular environment. The inclusion of biochemical signals from ECM components is expected to play a pivotal role in promoting cell proliferation while reducing the risk of thrombosis and inflammation. The formulation of this novel dECM-based bioink is optimized for extrusion-based 3D bioprinting through both a priori and post-printing assessments. Additionally, an ad hoc crosslinking process for alginate is developed to enhance self- stability and shape fidelity of the 3D bioprinted construct. This bioink is designed to facilitate the culture, in bioreactor specifically designed and developed, of the bioprinted construct for several weeks while preserving support for embedded cells.| File | Dimensione | Formato | |
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Descrizione: Tesi dottorato: Biofabrication of a self-supporting multi-layered 3D bioprinted construct to regenerate biologically functional tissues
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https://hdl.handle.net/10589/220412