The corneal endothelium (CE) is the innermost layer of the cornea that regulates the stromal hydration state required to maintain corneal transparency. During adulthood, the endothelial cell density (ECD) decreases by 0.6% each year. As human corneal endothelial cells (hCECs) do not proliferate, the loss of aging-induced hCECs is compensated by migration and enlargement of neighbouring cells. When ECD falls below a threshold of 500 cells/mm2, by aging or trauma/disease, the endothelium does not have enough pumping power to guarantee a correct corneal hydration, leading to oedema, corneal opacity, and visual impairment. Corneal transplantation, with related problems, is the only efficient treatment for corneal endothelial diseases up to date. However, the worldwide donor corneas shortage is increasingly becoming a non-negligible issue, with only 1 cornea available for 70 needed. This has led to investigate alternative strategies for treating corneal endothelial diseases, such as tissue engineering approaches. Corneal endothelial tissue engineering is an emerging therapeutic approach that involves the use of hCECs combined with a biomaterial to create tissue engineered grafts for transplantation. Our research group has previously demonstrated that proper binding of an RGD (Arg-Gly-Asp) peptide to the chitin scaffold guaranteed maintenance of human corneal epithelial cells behaviour. Scaffold characteristics were optimised to produce a substrate with biomechanical properties resembling the human corneal stroma for transparency, architecture, stiffness, and mechanical strength. In this research project, our aim is to investigate the use of this functionalized biological scaffold as a potential substrate also for hCECs adhesion and expansion. The experiments were carried out in order to obtain a functional tissue engineered endothelial graft and, from a future perspective, a three-dimensional human cornea with all its layers (epithelium + stroma + endothelium). If successful, this elegant approach has the potential to increase access to corneal therapy by treating multiple patients. However, CE tissue engineering is a major challenge for several reasons: a) the hCECs have a low natural proliferative capacity that must be finely stimulated in vitro with an appropriate mitogen-rich medium; b) during in vitro expansion, hCECs undergo premature senescence (particularly in cultures derived from older donors) and phenotypic transformation to a mesenchymal phenotype, so-called Endothelial-Mesenchymal Transition (EnMT), which must be prevented c) few specific molecular markers define the quality of cultured hCECs, which are needed to control their physiological cell functions; d) finally, to develop a tailored engineered corneal endothelium, a substrate material that is able to create a favourable microenvironment for hCECs activity has not been yet developed. Thus, in this research project we analysed some challenges faced with hCECs in terms of (I) optimization of hCECs culture techniques (Chapter I), (II) identification of specific hCECs functional markers (Chapter II), (III) prevention of EnMT which leads to a cellular trans-differentiation towards a myofibroblastic phenotype causing a cellular loss of function (Chapter III), and (IV) analysis of the identified scaffold to make bioengineered corneal endothelial grafts (Chapter IV).

L'endotelio corneale regola lo stato di idratazione stromale necessario per la trasparenza corneale. In età adulta, la densità cellulare endoteliale (ECD) diminuisce annualmente dello 0,6%. Poiché le cellule endoteliali corneali umane hanno ridotta capacità proliferativa in vivo, la loro perdita è compensata dalla migrazione e allargamento delle cellule vicine. Quando l' ECD scende al di sotto del valore soglia di 500 cellule/mm2, in seguito a invecchiamento o trauma o una condizione patologica, l'endotelio non è in grado di garantire una corretta idratazione corneale, causando edema, opacità corneale e disturbi visivi. Il trapianto corneale, con le relative limitazioni, ad oggi è l'unico trattamento efficiente per le malattie endoteliali corneali. Tuttavia, la carenza mondiale di cornee donatrici sta diventando sempre più un problema non trascurabile, con solo 1 cornea disponibile ogni 70 cornee richieste. Questo ha indotto a sviluppare strategie alternative per il trattamento di malattie endoteliali corneali, tra cui gli approcci di ingegneria tissutale. L'ingegneria tissutale è un approccio terapeutico emergente che combina l'utilizzo di cellule endoteliali corneali con l’utilizzo di un appropriato biomateriale per la coltura ed il trapianto di queste cellule. Il nostro gruppo di ricerca ha precedentemente dimostrato che il legame di un decapeptide contenente il motivo peptidico RGD (Arg-Gly-Asp) allo scaffold di chitina garantisce il mantenimento del comportamento delle cellule epiteliali corneali umane. Le caratteristiche dello scaffold sono state ottimizzate per produrre un substrato con proprietà biomeccaniche simili allo stroma corneale umano per trasparenza, architettura, rigidità e resistenza meccanica. In questo progetto di ricerca, il nostro obiettivo è quello di studiare l'utilizzo della chitina funzionalizzata con l’ RGD come potenziale substrato anche per l'adesione e l'espansione delle cellule corneali endoteliali umane. Gli esperimenti sono stati condotti al fine di ottenere un tessuto endoteliale ingegnerizzato e, in una prospettiva futura, una cornea umana tridimensionale con tutti i suoi strati (epitelio + stroma + endotelio). Tuttavia, l'ingegneria tissutale dell’ endotelio corneale è una sfida complessa per diversi motivi: a) le cellule corneali endoteliali hanno una bassa capacità proliferativa che deve essere stimolata finemente in vitro con terreni di coltura appropriati; b) durante la coltura in vitro, le cellule corneali endoteliali vanno incontro a senescenza prematura (in particolare nelle colture cellulari derivate da donatori più anziani) e a una trasformazione fenotipica assumendo un fenotipo mesenchimale, la cosiddetta transizione endoteliale-mesenchimale; e) pochi marcatori molecolari specifici definiscono la qualità delle cellule corneali endoteliali, necessari per controllare le loro funzioni fisiologiche cellulari; d) infine, per l’approccio di ingegneria tissutale dell’ endotelio corneale, non è stato ancora sviluppato un biomateriale in grado di creare un microambiente favorevole all'attività delle cellule corneali endoteliali. Per questo motivo, in questo progetto di ricerca abbiamo affrontato alcune sfide che rendono difficile l’utilizzo delle cellule corneali endoteliali, in termini di (I) ottimizzazione della tecnica di coltura delle cellule corneali endoteliali umane (Capitolo I), (II) identificazione di marcatori funzionali specifici delle cellule corneali endoteliali (Capitolo II), (III) prevenzione della transizione endotelio-mesenchimale che induce ad un trans-differenziamento cellulare verso un fenotipo mio-fibroblastico che causa una perdita della funzione cellulare (Capitolo III) e (IV) analisi dello scaffold selezionato per coltivare le cellule corneali endoteliali (Capitolo IV).

Endotelio corneale umano: dagli studi in vitro all’applicazione dell’ ingegneria tissutale / Alessia Merra , 2023 May 23. 35. ciclo, Anno Accademico 2021/2022.

Endotelio corneale umano: dagli studi in vitro all’applicazione dell’ ingegneria tissutale

MERRA, ALESSIA
2023

Abstract

The corneal endothelium (CE) is the innermost layer of the cornea that regulates the stromal hydration state required to maintain corneal transparency. During adulthood, the endothelial cell density (ECD) decreases by 0.6% each year. As human corneal endothelial cells (hCECs) do not proliferate, the loss of aging-induced hCECs is compensated by migration and enlargement of neighbouring cells. When ECD falls below a threshold of 500 cells/mm2, by aging or trauma/disease, the endothelium does not have enough pumping power to guarantee a correct corneal hydration, leading to oedema, corneal opacity, and visual impairment. Corneal transplantation, with related problems, is the only efficient treatment for corneal endothelial diseases up to date. However, the worldwide donor corneas shortage is increasingly becoming a non-negligible issue, with only 1 cornea available for 70 needed. This has led to investigate alternative strategies for treating corneal endothelial diseases, such as tissue engineering approaches. Corneal endothelial tissue engineering is an emerging therapeutic approach that involves the use of hCECs combined with a biomaterial to create tissue engineered grafts for transplantation. Our research group has previously demonstrated that proper binding of an RGD (Arg-Gly-Asp) peptide to the chitin scaffold guaranteed maintenance of human corneal epithelial cells behaviour. Scaffold characteristics were optimised to produce a substrate with biomechanical properties resembling the human corneal stroma for transparency, architecture, stiffness, and mechanical strength. In this research project, our aim is to investigate the use of this functionalized biological scaffold as a potential substrate also for hCECs adhesion and expansion. The experiments were carried out in order to obtain a functional tissue engineered endothelial graft and, from a future perspective, a three-dimensional human cornea with all its layers (epithelium + stroma + endothelium). If successful, this elegant approach has the potential to increase access to corneal therapy by treating multiple patients. However, CE tissue engineering is a major challenge for several reasons: a) the hCECs have a low natural proliferative capacity that must be finely stimulated in vitro with an appropriate mitogen-rich medium; b) during in vitro expansion, hCECs undergo premature senescence (particularly in cultures derived from older donors) and phenotypic transformation to a mesenchymal phenotype, so-called Endothelial-Mesenchymal Transition (EnMT), which must be prevented c) few specific molecular markers define the quality of cultured hCECs, which are needed to control their physiological cell functions; d) finally, to develop a tailored engineered corneal endothelium, a substrate material that is able to create a favourable microenvironment for hCECs activity has not been yet developed. Thus, in this research project we analysed some challenges faced with hCECs in terms of (I) optimization of hCECs culture techniques (Chapter I), (II) identification of specific hCECs functional markers (Chapter II), (III) prevention of EnMT which leads to a cellular trans-differentiation towards a myofibroblastic phenotype causing a cellular loss of function (Chapter III), and (IV) analysis of the identified scaffold to make bioengineered corneal endothelial grafts (Chapter IV).
Human corneal endothelium: from in vitro studies to tissue engineering application
23-mag-2023
PELLEGRINI, Graziella
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