Valvulopathy is defined as any disease affecting the heart valves. In the case of valvulopathy, the only effective treatment is the replacement of the heart valve with a prosthesis. Annually, the number of patients requiring surgery is steadily increasing, and it is estimated that the number of global implants will triple by 2050 compared to now. Current surgical therapy uses mechanical or biological valve replacements, which frequently lead to reoperations or complications for patients. These types of valve substitutes lack two fundamental characteristics for long-term durability: growth capacity and regenerative potential. Regenerative Medicine, through a multidisciplinary approach including Tissue Engineering principles, aims to regenerate and reproduce all the characteristics of the native tissue by combining scaffolds and cells (xenogeneic or autologous) to produce a valve replacement that can self-repair, remodel, and adapt to the host. Although, to date, the efficacy of decellularized homografts has already been evaluated in two clinical trials, ESPOIR and ARISE, one of the main limitations associated with this type of valve replacement is the rate of in situ recellularization. This thesis aims to evaluate the cell-substrate interactions to investigate the possibility of in vitro recellularization of decellularized heart valves, in order to improve their repopulation and subsequent remodelling. The thesis work was carried out at the Department of Thoracic Surgery at the Leiden University Medical Center (LUMC, The Netherlands), within the DRAPH project: Decellularization and Recellularization of Aortic and Pulmonary Heart valves. The thesis work can be divided into two parts: the first focuses on the decellularization protocol of the heart valves to obtain a suitable substrate/scaffold, and the second concerns the attempt at their recellularization. A heart valve decellularization protocol using Sodium Dodecyl Sulfate (SDS) and Sodium Deoxycholate (SDC) was performed. The decellularized biological scaffold was biologically validated by DNA extraction and quantification, histological and immunofluorescence staining. To achieve recellularization, Valvular Interstitial Cells (VICs) and Endothelial Colony Forming Cells (ECFCs) were isolated respectively from porcine hearts and blood. They were phenotypically characterized by western blot and immunocytochemistry before being seeded onto the decellularized heart valve under static culture conditions without any chemical conditioning or mechanical stimulation. The ability to recellularize in vitro was assessed by Hematoxylin & Eosin and immunohistochemistry staining. The Decellularization protocol produced decellularized heart valves that respect the minimum criteria for clinical application. ECFCs showed the ability to interact with the decellularized substrate, forming a continuous monolayer on the leaflet surface, and showed partial recellularization of the vessel wall. VICs, on the other hand, did not show the ability to infiltrate the tissue or interact with the decellularized heart valve surface. Consequently, challenges remain in optimizing the in vitro recellularization of decellularized heart valves, including the choice of phenotypically appropriate cells and the mode of recellularization.
Per valvulopatia si intende qualsiasi malattia che colpisce le valvole cardiache. In caso di valvulopatia, l’unico trattamento efficace è la sostituzione della valvola cardiaca con una protesi. Ogni anno, il numero di pazienti che necessita di un intervento chirurgico è in costante aumento e si stima che il numero di impianti globali entro il 2050 sarà triplicato rispetto ad oggi. L’attuale terapia chirurgica utilizza sostituti valvolari meccanici o biologici, che frequentemente portano i pazienti al re-intervento o all’insorgenza di complicanze. Queste tipologie di sostituti valvolari mancano di due caratteristiche fondamentali per una durata a lungo termine: capacità di crescita e potenziale rigenerativo. La Medicina Rigenerativa, mediante un approccio multidisciplinare che include i principi dell’ingegneria tissutale, mira a rigenerare e riprodurre tutte le caratteristiche del tessuto nativo mediante la combinazione di scaffold e cellule (xenogeniche o autologhe) per produrre un sostituto valvolare che abbia la capacità di “autoripararsi”, rimodellarsi e adattarsi all’ospite. Sebbene, ad oggi, si stia già valutando l’efficacia di valvole cardiache decellularizzate in due clinical trials, ESPOIR e ARISE, una delle principali limitazioni associata a questa tipologia di sostituto valvolare è il tasso di recellularizzazione in situ. Il presente lavoro di tesi ha l’obiettivo di valutare l’interazione cellula-substrato per indagare la possibilità di recellularizzazione in vitro delle valvole cardiache decellularizzate al fine di migliorare il loro ripopolamento e il successivo rimodellamento. Il lavoro di tesi è stato svolto presso il dipartimento di cardiochirurgia toracica del Leiden University Medical Center (LUMC, Paesi Bassi) all’interno del progetto DRAPH: Decellularization and Recellularization of Aortic and Pulmonary Heart valves. Il lavoro di tesi può essere diviso in due parti: la prima incentrata sul protocollo di decellularizzazione delle valvole cardiache per ottenere uno scaffold/substrato adatto, la seconda riguarda il tentativo di recellularizzazione delle stesse. È stato eseguito un protocollo di decellularizzazione della valvola cardiaca mediante l’uso di Sodio Dodecilsolfato (SDS) e Sodio Desossicolato (SDC). Lo scaffold biologico decellularizzato è stato validato biologicamente mediante l’estrazione e quantificazione del DNA, e mediante colorazioni istologiche e di immunofluorescenza. Per ottenere la recellularizzazione, sono state isolate Cellule Interstiziali Valvolari (VICs) e Cellule Endoteliali Formanti Colonie (ECFCs) rispettivamente da cuore e sangue suino. Sono state caratterizzate fenotipicamente mediante western blot e immunocitochimica, prima di essere seminate sulla valvola cardiaca decellularizzata, in condizioni di coltura statica senza nessun tipo di condizionamento chimico o stimolo meccanico. La capacità di recellularizzazione in vitro è stata valutata mediante le colorazioni di Ematossilina & Eosina e immunoistochimica. Il protocollo di decellularizzazione ha prodotto valvole cardiache decellularizzate che rispettano i criteri minimi per l’applicazione in ambito clinico. Le ECFCs hanno mostrato la capacità di interagire con il substrato decellularizzato; sono in grado di formare uno strato continuo sulla superficie delle valvole cardiache e di recellularizzare in modo parziale il tessuto vascolare. Le VICs, invece, non hanno mostrato la capacità di infiltrarsi nel tessuto né di interagire con la superficie della valvola cardiaca decellularizzata. Di conseguenza, rimangono ancora delle sfide da superare per ottimizzare la recellularizzazione in vitro delle valvole cardiache decellularizzate, che vanno dalla scelta delle cellule fenotipicamente appropriate alle modalità di recellularizzazione.
Interazioni Cellula-Substrato nel Ripopolamento di Valvole Cardiache Decellularizzate
AVOLA, AMANDA
2022/2023
Abstract
Valvulopathy is defined as any disease affecting the heart valves. In the case of valvulopathy, the only effective treatment is the replacement of the heart valve with a prosthesis. Annually, the number of patients requiring surgery is steadily increasing, and it is estimated that the number of global implants will triple by 2050 compared to now. Current surgical therapy uses mechanical or biological valve replacements, which frequently lead to reoperations or complications for patients. These types of valve substitutes lack two fundamental characteristics for long-term durability: growth capacity and regenerative potential. Regenerative Medicine, through a multidisciplinary approach including Tissue Engineering principles, aims to regenerate and reproduce all the characteristics of the native tissue by combining scaffolds and cells (xenogeneic or autologous) to produce a valve replacement that can self-repair, remodel, and adapt to the host. Although, to date, the efficacy of decellularized homografts has already been evaluated in two clinical trials, ESPOIR and ARISE, one of the main limitations associated with this type of valve replacement is the rate of in situ recellularization. This thesis aims to evaluate the cell-substrate interactions to investigate the possibility of in vitro recellularization of decellularized heart valves, in order to improve their repopulation and subsequent remodelling. The thesis work was carried out at the Department of Thoracic Surgery at the Leiden University Medical Center (LUMC, The Netherlands), within the DRAPH project: Decellularization and Recellularization of Aortic and Pulmonary Heart valves. The thesis work can be divided into two parts: the first focuses on the decellularization protocol of the heart valves to obtain a suitable substrate/scaffold, and the second concerns the attempt at their recellularization. A heart valve decellularization protocol using Sodium Dodecyl Sulfate (SDS) and Sodium Deoxycholate (SDC) was performed. The decellularized biological scaffold was biologically validated by DNA extraction and quantification, histological and immunofluorescence staining. To achieve recellularization, Valvular Interstitial Cells (VICs) and Endothelial Colony Forming Cells (ECFCs) were isolated respectively from porcine hearts and blood. They were phenotypically characterized by western blot and immunocytochemistry before being seeded onto the decellularized heart valve under static culture conditions without any chemical conditioning or mechanical stimulation. The ability to recellularize in vitro was assessed by Hematoxylin & Eosin and immunohistochemistry staining. The Decellularization protocol produced decellularized heart valves that respect the minimum criteria for clinical application. ECFCs showed the ability to interact with the decellularized substrate, forming a continuous monolayer on the leaflet surface, and showed partial recellularization of the vessel wall. VICs, on the other hand, did not show the ability to infiltrate the tissue or interact with the decellularized heart valve surface. Consequently, challenges remain in optimizing the in vitro recellularization of decellularized heart valves, including the choice of phenotypically appropriate cells and the mode of recellularization.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/16459